424: Inhibition, Na+/I- symporter (NIS)

Short Name: Inhibition, Na+/I- symporter (NIS)

AOPs Including This Key Event

Stressors

Biological Organization

Cell term

Apart from the human, functional NIS protein has been also identified in different species, including  the rat (Dai et al., 1996), the mouse (Perron et al., 2001), the pig (Selmi-Ruby et al., 2003), zebrafish (Thienpont et al., 2011) and in xenopus (amphibian)  (Lindenthal et al., 2009). Mouse and rat contain 618 amino acid residues, while the human and pig contain 643. There are several NIS variants that produce three active proteins in the pig due to alternative splicing at mRNA sites that are not present on the other species (Selmi-Ruby et al., 2003).

NIS orthologs are discussed in the review by Darrouzet's group ( Darrouzet et al., 2014). Interestingly, functional differences have been identified between mouse or rat NIS (mNIS or rNIS, respectively) and human NIS (hNIS). The rat and themouse orthologs were shown to accumulate radioisotopes more efficiently than the human protein (Dayem et al., 2008; Heltemes et al., 2003). The molecular basis of these functional differences could be helpful for further characterization of NIS. Zhang and collaborators showed that rNIS is localized in a higher proportion at the plasma membrane than hNIS and the N-terminal region up to putative TM7 appears to be involved in this difference (Zhang et al., 2005). These authors also reported differences in the kinetics of the Na+ binding, implicating the region spanning from TM4 to TM6 and Ser200 of hNIS. They, thus, proposed that this region could be involved in sodium binding (Zhang et al., 2005). In our laboratory, it was shown that the Vmax of the mouse protein is four times higher than the Vmax of the human protein when expressed in the same cell line (HEK-293) (Dayem et al., 2008; Darrouzet et al., 2014). The KmI value determined for hNIS (9.0 ± 0.8 μM) was significantly lower than the KmI for the mouse protein (26.4 ± 3.5 μM) whereas the KmNa values were not significantly different. Similarly to the rat protein, mNIS is predominantly localized in the plasma membrane whereas the human ortholog is detected intracellularly in 40% of the cells in which it is expressed (Darrouzet et al., 2014). However, the difference in the Vmax values does not only seem to be related to the higher intracellular localization of hNIS. Using chimeric proteins between human and mouse NIS, we showed that the N-terminal region up to TM8 is most probably involved in iodide binding, and that the region from TM5 to the C terminus could play an important role in targeting the protein to the plasma membrane (Dayem et al., 2008). One of the long-term goals of these studies is the engineering of a chimeric NIS protein most suitable for gene therapy, i.e. preserving regions responsible for the high turnover rate and the efficient plasma membrane localization of the mouse proteinwhile replacing the immunogenic extracellular regions with those of the human ortholog. The porcine NIS gene gives rise to splice variants leading to three active NIS proteins with differences in their C-terminal extremities [4]. However, it is not known if these differences lead to distinct properties (Darrouzet et al., 2014).

There is evidence that the MIE (NIS inhibition) is of relevance also for fish as an expression of the slc5a5 transcript (sodium/iodide co-transporter) has been described by various publications for the zebrafish embryo (see www.zfin.org). It has been demonstrated that NIS inhibitors in zebra fish lead also to a strong repression of thyroid hormone levels (Thienpont et al., 2011) and in xenopus (amphibian) to  inhibition of the iodide-induced current  (Lindenthal et al., 2009).

425: Decrease of Thyroidal iodide

Short Name: Thyroidal Iodide, Decreased

AOPs Including This Key Event

Biological Organization

Cell term

Organ term

Various species express functional NIS  encoded by the following genes: Human SLC5A5 (6528), Mouse Slc5a5 (114479), Rat Slc5a5 (114613), Zebrafish slc5a5 (561445), chicken SLC5A5 (431544), domestic cat SLC5A5 (101092587), dog SLC5A5 (484830), domestic guinea pig Slc5a5 (100714457), naked mole-rat Slc5a5 (101701995), cow SLC5A5 (505310), sheep SLC5A5 (101112315). The encoded protein is responsible for the uptake of iodine in tissues such as the thyroid and lactating breast tissue. The iodine taken up by the thyroid is incorporated into the metabolic regulators triiodothyronine (T3) and tetraiodothyronine (T4). Mutations in this gene are associated with thyroid dyshormonogenesis that significantly influences phenotypic expressions such as severity of hypothyroidism, goiter rates, and familial clustering demonstrating essentiality of NIS function to maintain TH status (Bakker et al., 2000; Spitzweg and Morris, 2010; Ramesh et al., 2016) . Animal studies have also proven that iodine normalizes elevated adrenal corticosteroid hormone secretion and has the ability to reverse the effects of hypothyroidism in the ovaries, testicles and thymus in thyroidectomized rats (Nolan et al., 2000).

277: Thyroid hormone synthesis, Decreased

Short Name: TH synthesis, Decreased

AOPs Including This Key Event

Stressors

Biological Organization

Cell term

Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across taxa, with in vivo evidence from humans, rats, amphibians, some fish specis, and birds, and in vitro evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Grave's disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong medical evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005).

281: Thyroxine (T4) in serum, Decreased

Short Name: T4 in serum, Decreased

AOPs Including This Key Event

Stressors

Biological Organization

Organ term

The overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebra fish (Thienpont et al., 2011), amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their existence and importance has also been described in many different animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such extrapolation regarding TH action across species should be done with caution.

With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000).  In contrast, in adult rats the majority of THs are bound to TTR. Thyroid binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes.

280: Thyroxine (T4) in neuronal tissue, Decreased

Short Name: T4 in neuronal tissue, Decreased

AOPs Including This Key Event

Stressors

Biological Organization

Organ term

THs are critical for normal brain development in most vertebrates, primarily documented empirically in mammalian species (Bernal, 2013).  However, there is compelling data that demonstrates the need for TH in brain development for many other taxa, including: birds, fish and frogs (Van Herck et al., 2013; Denver, 1998; Power et al., 2001). The most well known non-mammalian action of TH is to induce metamorphosis in amphibians and some fish species. However, there is a fundamental difference in the mechanisms by which T3 affects amphibian metamorphosis vs its role in mammalian brain development (Galton, 1983). In the rat, brain development proceeds, even if defective, despite the absence of TH. By contrast, TH administration to tadpoles induces early metamorphosis, whereas in its absence, tadpoles grow to extremely large size, but the metamorphosis program is never activated (Galton, 1983).

381: Reduced levels of BDNF

Short Name: BDNF, Reduced

AOPs Including This Key Event

Biological Organization

Cell term

BDNF plays a critical role in normal brain development in most vertebrates, primarily documented empirically in mammalian species. Klein et al. (2011) examined blood, serum, plasma and brain-tissue and measured BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method (Aid et al., 2007), whereas Trajkovska et al. 2007 determined BDNF levels in human blood.

There is compelling data that demonstrates the role  of  BDNF  in brain development for many other taxa, including fish where it acts as neurotrophic factor in controlling cell proliferation (D'Angelo L et al., 2014; Heinrich and Pagtakhan, 2004) and  birds where BDNF influences development of the brain area that involved in the song control (Brenowitz 2013) and  the addition of new neurons to a cortical nucleus in adults . In the Xenopus visual system, BDNF acts as neurotrophic factor that mediates synaptic differentiation and maturation of the retinotectal circuit through cell autonomous TrkB signaling on retinal ganglion cells (Sanchez et al., 2006; Marshak et al., 2007).

851: Decrease of GABAergic interneurons

Short Name: GABAergic interneurons, Decreased

AOPs Including This Key Event

Biological Organization

Cell term

GABAergic interneurons play a vital role in the wiring and circuitry of the developing nervous system of all organisms, both invertebrates and vertebrates (Hensch, 2005; Owens and Kriegstein, 2002; Wang et al., 2004). However, restricted expression of GABA in a considerable population of neurons is observed in the non-vertebrate animals. A nematode Caenorhabditis elegans has 302 neurons, among them, 26 cells are GABAergic (Sternberg and Horvitz, 1984; McIntire et al., 1993). Another nematode Ascaris has 26 GABAergic neurons (Obata, 2013). GAD, VGAT, GABA receptors and GABA-system-specific molecules are analogous to those of vertebrates. Except for one interneuron, GABAergic neurons are connected with muscle cells and exert direct inhibitory, sometimes excitatory, control on locomotion, defecation and foraging. The muscle innervation of both excitatory and inhibitory axons is maintained also in Crustacea (Obata, 2013).   

385: Decrease of synaptogenesis

Short Name: Synaptogenesis, Decreased

AOPs Including This Key Event

Biological Organization

Cell term

The mechanisms governing synapse formation is considered conserved among both vertebrates and invertebrates (Munno and Syed, 2003). Invertebrates have served as simple animal models to study synapse formation. Indeed, Colón-Ramos (2009) has recently reviewed the early developmental events that take place in the process of synaptogenesis pointing out the importance of this process in neural network formation and function. The experimental evaluation of synaptogenesis has been performed using invertebrates and in particular C. elegans and Drosophila as well as vertebrates (Colón-Ramos, 2009).

This vulnerable period of synaptogenesis appears to happen in different developmental stages across species. For example, in rodents primarily synaptogenesis occurs during the first two weeks after birth (Bai et al., 2013). For rhesus monkeys, this period ranges from approximately 115-day gestation up to PND 60 (Bai et al., 2013). In humans, it starts from the third trimester of pregnancy and continues 2-3 years following birth (Bai et al., 2013).

386: Decrease of neuronal network function

Short Name: Neuronal network function, Decreased

AOPs Including This Key Event

Biological Organization

Organ term

In vitro studies in brain slices applying electrophysiological techniques showed significant variability among species (immature rats, rabbits and kittens) related to synaptic latency, duration, amplitude and efficacy in spike initiation (reviewed in Erecinska et al., 2004).

341: Impairment, Learning and memory

Short Name: Impairment, Learning and memory

AOPs Including This Key Event

Biological Organization

Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).

Inhibition, Na+/I- symporter (NIS) leads to Thyroidal Iodide, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from in vitro works using rat follicular cells (Cianchetta et al., 2010; Waltz et al., 2010; Lecat-Guillet et al., 2007; 2008; Lecat-Guillet et al., 2008b), human in vitro cell models (Wen et al., 2016) and in vivo data (Arriagada et al. 2015), as well as Xenopus oocytes (Lindenthal et al., 2009) and Zebrafish (Thienpont et al., 2011).

How Does This Key Event Relationship Work

NIS is a membrane protein implicated in iodide uptake into the follicular cells of the thyroid. Other large anions can be also bound by NIS and inhibit accumulation of iodide into the thyroid by competing binding with iodide (Wolff, 1964).

Weight of Evidence

NIS is a membrane bound glycoprotein and its main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. It uses the sodium gradient generated by the Na+/K+ ATPase for the active transport of iodide into the thyrocytes (Eskandari et al., 1997). Extensive studies on NIS protein have identified 14 different mutations and each one of them is related to Iodine Transport Deficiencies (ITD) (reviewed in Spitzweg and Morris, 2010). Most of these mutations have been characterized and it is well known that they even lead to the synthesis of truncated protein (Pohlenz et al., 1997; Pohlenz et al., 1998), partial deletions (Kosugi et al., 2002; Tonacchera et al., 2003; Montanelli et al., 2009) or substitutions of amino acids (Matsuda and Kosugi, 1997; Kosugi et al., 1999; Szinnai et al., 2006) that eventually result in total or partial NIS dysfunction. While most of the NIS mutants have been further investigated and the functional relationship between the NIS dysfunction and ITD is well established (reviewed in Darrouzet et al., 2014; Portulano et al., 2014), the exact structural relationship between mutated NIS and ITD still needs to be elucidated and the molecular modelling of the protein would greatly benefit these studies.

Recent revision of the affinity constant for perchlorate binding to the NIS symporter based on in vitro and human in vivo data, performed by refitting published in vitro data, in which perchlorate-induced inhibition of iodide uptake via the NIS was measured, yielding a Michaelis-Menten kinetic constant (K_m) of 1.5 μm, showed that a 60% lower value for the Km, equal to 0.59 μm. Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg^-1 day^-1 (Schlosser PM, 2016).

Many studies have shown inhibition of radioactive iodide uptake by using different cell models and assays. However, there have been identified only few specific NIS inhibitors up to date, while all the others are thought to act through different inhibitory mechanisms. Monovalent anions, others than iodide, are also transported by NIS but Nitrate (NO3-), thiocyanate (SCN-), perchlorate (ClO-4), dysidenin and aryltrifluoroborates are of particular dietary and environmental importance (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006).

Recent revision of the affinity constant for perchlorate binding to the NIS symporter based on in vitro and human in vivo data, performed by refitting published in vitro data, in which perchlorate-induced inhibition of iodide uptake via the NIS was measured, yielding a Michaelis-Menten kinetic constant (K_m) of 1.5 μm, showed that a 60% lower value for the Km, equal to 0.59 μm. Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg^-1 day^-1 (Schlosser PM, 2016).

There are many studies showing the effect of inhibition of NIS on thyroidal iodide uptake:

- Cianchetta et al., 2010 For this study the rat FRTL5 thyroid cell line endogenously expressing NIS, and the monkey kidney fibroblast-like cells (COS-7) transfected with hNIS were used. NIS functionality was assessed with the use of the Yellow Fluorescent Protein (YFP) variant YFP-H148Q/I152L, a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Decrease of YFP-H148Q/I152L fluorescence in FRTL-5 cells occurs as a result of NIS-mediated uptake and binding to the intracellular fluorochrome (Rhoden et al., 2007). The biosensor was used to compare the kinetics of iodide and perchlorate transport by NIS, and to assess the ability of perchlorate to inhibit iodide transport. Additionally, perchlorate was shown to inhibit NIS function (competitive inhibition) by preventing iodide-induced changes in fluorescence of FRTL5 cells. Perchlorate caused a concentration-dependent inhibition of iodide uptake in the initial influx rate (IC50=1.6μM) and in the intracellular concentration of iodide (IC50=1.1μM). Also, both perchlorate and iodide (1–1000 μM) induced concentration-dependent decreases in YFP-H148Q/I152L fluorescence in COS-7 cells expressing hNIS, but had no effect (< 2%) in COS-7 cells lacking hNIS. Additionally, perchlorate induced a significantly smaller decrease in fluorescence (10.6% at 1 mM) than iodide (31.8% at 1 mM iodide). Thus, it was confirmed that the reduction of fluorescence was due to NIS-mediated anion transport into the cells, excluding non-specific effects.

- Tonacchera et al., 2004 Chinese hamster ovary (CHO) cell line had been stably transfected with human NIS and the measurement of iodide uptake was performed with the use of radioactive iodide uptake (RAIU) method. It was shown that the inhibition of iodide uptake was dose-dependent when using the known NIS inhibitors (ClO-4, NO3-, SCN-). Additionally, unlabeled I- (non ¹²⁵I) was used to investigate the inhibition level of radioiodide uptake and to compare it with the potency of the other monoions, which are known NIS inhibitors. The IC₅₀ values for the studied monoions were the following: ClO₄^-: IC₅₀ was 1.22 μΜ; SCN^-: IC₅₀ was 18.7 μΜ; NO₃^-: IC₅₀ was 293 μΜ; I^-: IC₅₀ was 36.6 μΜ. Finally, the present study investigated the joint effects of simultaneous exposure to multiple RAIU inhibitors, by generating multiple dose-response curves in the presence of fixed concentrations of inhibitors. The results of those experiments indicated a competition between the four anions with similar size for access to the binding sites of the NIS. The prediction model developed in this study, actually suggests that thyroidal iodide uptake is approximately proportional to iodide nutrition for any fixed inhibitor concentration, answering to the question whether dietary iodide can modulate the inhibitory effects of the known environmental goitrogens.

- Waltz et al., 2010 Measurement of iodide uptake was performed with a non-radioactive method. By using the rat thyroid low –serum 5 (FRTL5) cells, which endogenously express NIS, a spectrophotometric assay was developed and the iodide accumulation was determined based on the catalytic reduction of yellow cerium to colorless cerium in the presence of arsenious acid (Sandell-Kolthoff reaction). A dose-dependent inhibition of iodide uptake was shown. The IC50 values for the studied compounds were the following: Sodium perchlorate (NaClO4): IC50 was 0.1 μΜ Sodium thiocyanate (NaSCN): IC50 was 12 μΜ Sodium nitrate (NaNO3): IC50 was 800 μΜ Sodium Tetrafluoroborate (NaBF4): IC50 was 1.2 μΜ

- Lecat-Guillet et al., 2007; 2008a A fully automated radioiodide uptake assay was developed and some known NIS inhibitors were tested. A dose-dependent inhibition of iodide uptake was shown. The IC₅₀ values for the studied compounds were the following: Sodium perchlorate (NaClO₄): IC₅₀ was 1 μΜ; Sodium thiocyanate (NaSCN): IC₅₀ was 14 μΜ; Sodium nitrate (NaNO₃): IC₅₀ was 250 μΜ; Sodium Tetrafluoroborate (NaBF₄): IC₅₀ was 0.75 μΜ.  Additionally, a library of 17020 compounds was screened for the identification of new human NIS inhibitors. The identification was based on the magnitude of changes in iodide uptake using Human Embryonic Kidney 293 (HEK293) cells, stably transfected with the hNIS. The same experiments and with similar results were also performed in rat thyroid derived cells (FRTL5), which endogenously express NIS. Compounds that inhibited iodide uptake in a time-dependent manner were considered to act through direct NIS inhibition. In contrast, those compounds that had a delayed effect on iodide uptake were thought to act through a sodium gradient disruption system resulting in indirect inhibition of iodide transport. Perchlorate was used as a positive control in these experiments and, as expected, it blocked iodide uptake immediately and totally throughout the experiment. Dysidenin was also used as a control and the IC50 value identified was 2 μΜ. All the compounds that were used for these experiments were small drug-like molecules that have not been detected in the environment and they were named as ITBs (Iodide Transport Blockers).

- Lecat-Guillet et al., 2008b With the same fully automated radioiodide uptake assay, as described above, new NIS inhibitors were also identified. The organotrifluoroborate (BF3−) was found to inhibit iodide uptake with an IC50 value of 0.4 μM using rat-derived thyroid cells (FRTL5). The biological activity is rationalized by the presence of the ion BF3− as a minimal binding motif for substrate recognition at the iodide binding site.

- Lindenthal et al., 2009 With the use of a patch-clamp technique an analysis of the NIS inhibitors identified by Lecat-Guillet et al., 2008 (named ITB-1 to ITB-10 for "Iodide Transport Blockers") was evaluated in Xenopus oocytes expressing NIS to further assess the inhibitory effect of those molecules specifically on NIS activity. Four of those molecules (ITB-3, ITB-9, ITB-5 and ITB-4) were identified as the most potent, non-competitive NIS inhibitors. The effects of dysidenin were also analyzed with the same technique, as it had been reported to be a specific inhibitor of NIS (Vroye et al., 1998). It was found that dysidenin (50 μM) induced a rapid and reversible inhibition of the iodide (about 40%) of induced current in mNIS-expressing oocytes, but did not evoke any currents in the absence of iodide, suggesting that this effect was due to the inhibition of NIS activity.

- Greer et al., 2002 In human studies, potassium perchlorate was used to predict inhibition of thyroidal iodide uptake by applying the RAIU method. Greer et al., tested body weight adjusted doses of potassium perchlorate and an assessment of RAIU uptake was performed on day 2 and day 14 of treatment and 24 h following treatment termination (on day 15). The NOEL value for inhibition of thyroidal uptake was 0.007 mg/kg-day, while the true NEL value was estimated to be 0.0052 and 0.0064 mg/kg-day. According to the dose-response inhibition of iodide uptake the maximum percentage of iodide inhibition at the doses of 0.0052 and 0.0064 mg/kg-day is 8.3-9.5%, which is physiologically insignificant for a person with dietary sufficient iodine intake.

- Wen et al., 2016 By using human MCF-7 cells, a breast adenocarcinoma cell line, which express inducible NIS in the presence of all-trans retinoic acid (ATRA) it has been shown that inhibition of sterol regulatory element-binding proteins (SREBP) maturation by treatment with 25-hydroxycholesterol (5 µM) for 48 hr reduced ATRA (1 µM)-induced mRNA concentration of NIS and decreased iodide uptake by approximately 20%. This study showed for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells.

- Arriagada et al. 2015 This study showed that 2 hr or 5 hr exposure to excess I^- (100 μM) respectively in FRTL-5 cells and in ex-vivo rat thyroid gland (removed after single in vivo i.p. injection of 100 μg of I⁻ in 500 μL of distilled water, and analysis of ¹²⁵I thyroid uptake), induced inhibition of I- uptake through the NIS (~ 30% uptake inhibition after 5 hr in vivo), a process known as the Wolff-Chaikoff effect, which was not associated with a decrease of NIS expression or a change in NIS localization. Incubation of FRTL-5 cells with excess I- for 2 hr increased hydrogen peroxide generation. Also incubation with hydrogen peroxide (100 μM) decreased NIS-mediated I- transport, effect that was reverted by ROS scavengers.

The thyroid system is quite complex and therefore some inconsistent results have been produced by recent studies. For example, it has been observed in healthy volunteers that a 6-month exposure to perchlorate at doses up to 3 mg/d (low doses) had no effect on thyroid function, including inhibition of thyroid iodide uptake as well as serum levels of thyroid hormones, TSH, and Tg (Braverman et al., 2006). However, this study was limited by the small sample size and is obviously underpowered.

For this relationship there is not enough data linking quantitatively the inhibition of NIS with the amount of thyroidal uptake. The NIS inhibition is possible to be directly measured by using the fact that the simultaneous transport of 2 Na+ and 1 I- generates a current, which could be easily measured with electrophysiological methods (Eskandari et al., 1997) or with patch clamp techniques (Van Sande et al., 2003). However, the exact stoichiometry of the molecules that are transferred is not yet known, meaning that in some cases it cannot be detected. For example, perchlorate does not cause depolarization of the cellular membrane, as it is thought to be transferred in 1 to 1 stoichiometry with the Na+ (Van Sande et al., 2003). However, I^- uptake can also be measured in vivo, as shown in rats i.p. injected with 100 μg of I⁻ in 500 μL of distilled water (known to cause an inhibition of NIS- mediated I- transport), followed by analysis of radioactive ¹²⁵I thyroid uptake (Arriagada et al. 2015). Further studies are needed to support quantitative evaluation of this KER.

References

Arriagada AA, Albornoz E, Opazo MC, Becerra A, Vidal G, Fardella C, Michea L, Carrasco N, Simon F, Elorza AA, Bueno SM, Kalergis AM, Riedel CA. (2015). Excess iodide induces an acute inhibition of the sodium/iodide symporter in thyroid male rat cells by increasing reactive oxygen species. Endocrinology. Apr;156(4):1540-51.

Braverman LE, Pearce EN, He X, Pino S, Seeley M, Beck B, Magnani B, Blount BC, Firek A. (2006). Effects of six months of daily low-dose perchlorate exposure on thyroid function in healthy volunteers. J Clin Endocrinol Metab. 91:2721-2724.

Cianchetta S, di Bernardo J, Romeo G, Rhoden KJ (2010). Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L. Toxicol Appl Pharmacol. 243:372-380.

Darrouzet E, Lindenthal S, Marcellin D, Pellequer JL, Pourcher T. (2014). The sodium/iodide symporter: state of the art of its molecular characterization. Biocim Biophys Acta. 1838:244-253.

De Groef B, Decallonne BR, Van der Geyten S, Darras VM, Bouillon R. (2006). Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Europ J Endocr. 155:17-25.

Eskandari S, Loo DD, Dai G, Levy O, Wright M, Carrasco N. (1997). Thyroid Na+/I- symporter: mechanism, stoichiometry, and specificity. J Biol Chem 272: 27230-27238.

Greer MA, Goodman G, Pleus RC, Greer SE. (2002). Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans. Environm Health Persp. 110: 927-937.

Jones PA, Pendlington RU, Earl LK, Sharma RK, Barrat MD. (1996). In vitro investigations of the direct effects of complex anions on thyroidal iodide uptake: identification of novel inhibitors. Toxicol. In Vitro. 10: 149-160.

Kosugi S, Bhayana S, Dean HJ. (1999). A novel mutation in the sodium/iodide symporter gene in the largest family with iodide transport defect. J Clin Endocrinol Metab. 84: 3248-3253.

Kosugi S, Okamoto H, Tamada A, Sanchez-Franco F. (2002). A novel peculiar mutation in the sodium/iodide symporter gene in Spanish siblings with iodide transport defect. J Clin Endocrinol Metab. 87: 3830–3836.

Lecat-Guillet N, Merer G, Lopez R, Pourcher T, Rousseau B, Ambroise Y. (2008a). Small-molecule inhibitors of sodium iodide symporter function. Chembiochem 9:889–895.

Lecat-Guillet N, Ambroise Y. (2008b). Discovery of aryltrifluoroborates as potent sodium/iodide symporter (NIS) inhibitors. Chem Med Chem 3:1207–1209.

Lecat-Guillet N, Merer G, Lopez R, Pourcher T, Rousseau B, Ambroise Y. (2007). A 96-well automated radioiodide uptake assay for sodium/iodide symporter inhibitors. Assay Drug Dev Technol 5:535-540.

Lindenthal S, Lecat-Guillet N, Ondo-Mendez A, Ambroise Y, Rousseau B, Pourcher T. (2009). Characterization of small-molecule inhibitors of the sodium iodide symporter. J Endocrinol 200:357–365.

Matsuda A, Kosugi S. (1997). A homozygous missense mutation of the sodium/iodide symporter gene causing iodide transport defect. J Clin Endocrinol Metab. 82: 3966-3971.

Montanelli L, Agretti P, Marco G, Bagattini B, Ceccarelli C, Brozzi F, Lettiero T, Cerbone M, Vitti P, Salerno M, Pinchera A, Tonacchera M. (2009). Congenital hypothyroidism and late-onset goiter: identification and characterization of a novel mutation in the sodium/iodide symporter of the proband and family members. Thyroid 19: 1419-1425.

Pohlenz J, Rosenthal IM, Weiss RE, Jhiang SM, Burant C, Refetoff S. (1998). Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3′ splice site. J Clin Invest. 101:1028-1035.

Pohlenz J, Medeiros-Neto G, Gross JL, Silveiro SP, Knobel M, Refetoff S. (1997). Hypothyroidism in a Brazilian kindred due to iodide trapping defect caused by a homozygous mutation in the sodium/iodide symporter gene. Biochem Biophys Res Commun. 240: 488-491.

Portulano C, Paroder-Belenitsky M, Carrasco N. (2014). The Na+/I- symporter (NIS): Mechanism and medical impact. Endocr Rev. 35: 106-149.

Rhoden KJ, Cianchetta S, Stivani V, Portulano C, Galietta LJV, Romeo G. (2007). Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L. Am. J. Physiol., 292, pp. C814–C823.

Schlosser PM. (2016). Revision of the affinity constant for perchlorate binding to the sodium-iodide symporter based on in vitro and human in vivo data. J Appl Toxicol. Dec;36(12):1531-1535.

Spitzweg C, Morris JC. (2010). Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Mol Cell Endocrinol. 322: 56-63.

Szinnai G, Kosugi S, Derrien C, Lucidarme N, David V, Czernichow P, Polak M. (2006). Extending the clinical heterogeneity of iodide transport defect (ITD): a novel mutation R124H of the sodium/iodide symporter gene and review of genotype-phenotype correlations in ITD. J Clin Endocrinol Metab. 91: 1199–1204.

Thienpont B, Tingaud-Sequeira A, Prats E, Barat, C., Babin P.J, Raldua D, (2011). Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environ Sci Technol 45, 7525-7532.

Tonacchera M, Agretti P, de Marco G, Elisei R, Perri A, Ambrogini E, De Servi M, Ceccarelli C, Viacava P, Refetoff S, Panunzi C, Bitti ML, Vitti P, Chiovato L, Pinchera A. (2003). Congenital hypothyroidism due to a new deletion in the sodium/iodide symporter protein. Clin Endocrinol. 59: 500–506.

Tonacchera M, Pinchera A, Dimida A, Ferrarini E, Agretti P, Vitti P, Santini F, Crump K, Gibbs J. (2004). Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter. Thyroid. 14: 1012-1019.

Van Sande J, Massart C, Beauwens R, Schoutens A, Costagliola S, Dumont JE, Wolff J. (2003). Anion selectivity by the sodium iodide symporter. Endocrinology. 144: 247-252.

Vroye L, Beauwens R, Van Sande J, Daloze D, Braekman JC, Golstein PE. (1998). The Na+/I- co-transporter of th e thyroid: characterization of new inhibitors. Pflugers Archiv. 435:259-266.

Waltz F, Pillette L, Ambroise Y. (2010). A nonradioactive iodide uptake assay for sodium iodide symporter function. Anal Biochem. 396:91-95.

Wen G, Pachner LI, Gessner DK, Eder K, Ringseis R. (2016). Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells. J Dairy Sci. Nov;99(11):9211-9226.

Wolff J. (1964). Transport of iodide and other anions in the thyroid gland. Physiol Rev 44: 45-90.

Thyroidal Iodide, Decreased leads to TH synthesis, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from in vivo studies in rats (Wu F et al., 2012; Davidson et al., 1978), in vitro studies using thyroid follicular rat cells (Spitzweg et al., 1999; Sue et al., 2012) and porcine thyroid follicles (Sugawara et al., 1999), and human epidemiological studies (Steinmaus et al., 2016b; Horton et al., 2015; Brechner et al., 2000)

How Does This Key Event Relationship Work

Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized in the thyroid gland in the presence of functional NIS and thyroid peroxidase (TPO) as iodinated thyroglobulin (Tg), and stored in the colloid of thyroid follicles. NIS is a membrane bound glycoprotein whose main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. Extensive studies on NIS protein have identified 14 different mutations and each one of them is related to Iodine Transport Deficiencies (ITD) (Spitzweg and Morris, 2010). Once inside the follicular cells, the iodide diffuses to the apical membrane, where it is metabolically oxidized through the action of TPO to iodinium (I+), which in turn iodinates tyrosine residues of the Tg proteins in the follicle colloid. Therefore, NIS is essential for the synthesis of thyroid hormones (T3 and T4). TPO is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for TH synthesis (Taurog, 2005). Propylthiouracil (PTU) and methimazole (MMI), are thioureylene drugs that are known to inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978) and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound T3 and T4 (Taurog, 2005). PTU and MMI have been found to decrease also the expression of NIS mRNA and consequently iodide accumulation, as shown in FRTL-5 cells (Spitzweg et al. 1999).

Other compounds, such as triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) have been reported to decrease thyroid hormone (TH) levels by inducing an inhibition of NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis in rat thyroid follicular FRTL-5 cells, and on the activity of thyroid peroxidase (TPO), using rat thyroid microsomes (Wu Y et al. 2016).

Perchlorate, thiocyanate, nitrate, and iodide, which are competitive inhibitors of iodide uptake, have been shown to inhibit radioactive iodide uptake by NIS (Tonacchera et al. 2004), consequentially resulting in inhibition of TH synthesis. In particular, perchlorate blocks iodide uptake into the thyroid through NIS inhibition and decreases the production of TH (Steinmaus, 2016a). More recent evidence also suggests that young children, pregnant women, foetuses, and people co-exposed to similarly acting agents may be especially susceptible to perchlorate-induced toxicity (Steinmaus et al., 2016b).

Weight of Evidence

The association between these two KEs is strong, and supported by in vitro, in vivo and epidemiological studies. Blocking iodide uptake into the thyroid follicular cells as a consequence of NIS inhibition or functional impairment, leads to reduced TH synthesis. Compounds that have been shown to inhibit NIS function (e.g., perchlorate, thiocyanate, nitrate, and iodide), has also been proven to decrease TH synthesis by inducing a downregulation of TPO gene expression and/or increase of TSH level, which are both indicative of a reduce TH biosynthesis. TSH receptor controls transcription and posttranslational modification of NIS (Dai et al., 1996). Stimulation of TSH receptor increases T3 and T4 production and secretion (Szkudlinski et al., 2002). NIS gene expression is suppressed by growth factors such as IGF-1 and TGF-β (the latter is induced by the BRAF-V600E oncogene), which prevent NIS to localize in the basolateral membrane (Riesco-Eizaguirre et al., 2009). The BRAF-V600E oncogene is also associated with downregulation TSH receptor (Kleiman et al. 2013). Altogether these studies support the association between NIS inhibition-induced decreased iodide uptake (KE up) and reduced TH synthesis (KE down).

- Spitzweg et al., 1999: A 48 hr treatment of FRTL-5 cells with MMI (100 µM), PTU (100 µM), and potassium iodide (40 µM) induced ~ 50% decrease of NIS mRNA steady-state levels. Incubation with MMI and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%.

- Wu Y et al., 2016: This study showed that triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) induced a concentration-dependent inhibition of NIS-mediated iodide uptake. Moreover,  triclosan or triclocarban did not affect the expression of genes involved in TH synthesis (Slc5a5, TPO, and Tgo) or thyroid transcription factors (Pax8, Foxe1, and Nkx2-1), BDE-47 decreased the level of TPO, while BPA altered the expression of all six genes, as shown in rat thyroid follicular FRTL-5 cells. At the same time, triclosan and triclocarban also inhibited the activity of TPO at 166 and >300 μM, respectively. 

- Steinmaus et al., 2016b: In 1,880 pregnant women from San Diego County, California, during 2000–2003, it has been found that the presence of high level of perchlorate, thiocyanate, nitrate, and iodide in water supply induced a decrease of total thyroxine (T4) [regression coefficient (β) = –0.70; 95% CI: –1.06, –0.34], a decrease of free thyroxine (fT4) (β = –0.053; 95% CI: –0.092, –0.013), and an increase of thyroid-stimulating hormone (TSH), all indicators of reduced TH synthesis.

- Horton et al., 2015: in this study TSH levels measured in blood samples of 284 pregnant women at 12 (± 2.8) weeks gestation were found to positively correlate with the levels of urinary concentrations of perchlorate, nitrate and thiocyanate (NIS inhibitors), but perchlorate had the largest weight in the index, indicating the largest contribution to the weighted quantile sum regression. This indicates a perchlorate-dependent alteration of maternal thyroid function, through NIS inhibition.

- Brechner et al., 2000: Median newborn TSH levels in a city where drinking water supply was perchlorate-contaminated (from the Colorado River below Lake Mead) were significantly higher than those in a city totally supplied with non-perchlorate-contaminated drinking water, even after adjusting for factors known or suspected to elevate newborn TSH levels.

- Wu F et al., 2012: This study found that high dose perchlorate (520 mg/kg b.wt.) in Sprague-Dawley rats (28-day old) caused a decrease of Tg (~ 50% lower than control), and TPO (~ 45% lower than control) gene expression, indicative of reduced TH biosynthesis, together with a decrease of FT3 (~ 50% lower than control) and FT4 levels (~ 50% lower than control), and a remarkable increase of TSH levels (125% higher than control).

Several other studies have proven that NIS inhibitors lead to a decrease of thyroidal iodide uptake (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006; Waltz et al., 2010), leading to a reduction of TH synthesis.

Some studies have highlighted contradictory results in relation to response to chemicals. For instance, PTU and MMI have been shown to inhibit the activity of TPO in rats (Davidson et al., 1978), while inducing an increase of cellular TPO activity and TPO mRNA in cultured porcine thyroid follicles (Sugawara et al., 1999). PTU was also found to increase NIS gene expression, and the accumulation of ¹²⁵I, as shown in in rat thyroid FRTL-5 cells, while MMI had no effect (Sue et al., 2012).

Moreover, despite the well described effects of perchlorate, thiocyanate, nitrate, and iodide on iodide uptake into the thyroid, occupational and clinical dosing studies have not identified clear adverse effects, particularly in the case of perchlorate (Tarone et al. 2010). For instance, a longitudinal epidemiologic Chilean study found that there were no increases of thyroglobulin (Tg) or thyrotropin (TSH) levels, and no decreases of free T4 levels among either women during early pregnancy, late pregnancy, or the neonates at birth related to perchlorate in drinking water, suggesting that perchlorate in drinking water at 114 microg/L did not cause changes in neonatal thyroid function or fetal growth retardation (Téllez Téllez et al., 2005). Similarly, no associations between urine perchlorate concentrations and serum TSH or free T4 were found in individual euthyroid or hypothyroid/hypothyroxinemic cohorts of 261 hypothyroid/hypothyroxinemic and 526 euthyroid women from Turin and 374 hypothyroid/hypothyroxinemic and 480 euthyroid women from Cardiff (Pearce et al., 2010), suggesting that log perchlorate may not be a predictor of serum free T4 or TSH. However, it should be considered that these studies may be limited by short study durations, and the inclusion of mostly healthy adults (Steinmaus, 2016b).

In vitro and in vivo studies have specifically reported data supporting quantitative understanding of this KER.

- Spitzweg et al., 1999:  this study showed that inhibition of TH synthesis (induced by TPO specific inhibitors) decreases the expression of NIS. A 48 hr treatment of FRTL-5 cells with the TPO specific inhibitors MMI (100 µM), PTU (100 µM), and potassium iodide (40 µM), induced a ~ 50% decrease of NIS RNA steady-state levels. Incubation with MMI and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%.

- Wu F et al., 2012: An in vivo study found that high dose of NIS inhibitor perchlorate (520 mg/kg b.wt.) in Sprague-Dawley rats (28-day old) caused a decrease of Tg (~ 50% lower than control), and TPO (~ 45% lower than control) gene expression, indicative of reduced TH biosynthesis, together with a decrease of free T3 (~ 50% lower than control) and free T4 levels (~ 50% lower than control), and a remarkable increase of TSH levels (125% higher than control) (Wu F et al. 2012). Additional studies with quantitative data for this KER are also described in Empirical Support for Linkage. However, further studies are needed in order to drive global conclusions about the magnitude of iodide uptake inhibition required to impact TH synthesis.

References

Brechner RJ, Parkhurst GD, Humble WO, Brown MB, Herman WH. (2000). Ammonium perchlorate contamination of Colorado River drinking water is associated with abnormal thyroid function in newborns in Arizona. J Occup Environ Med. Aug;42(8):777-82.

Dai G, Levy O, Carrasco N. (1996). Cloning and characterization of the thyroid iodide transporter. Nature;379:458–460.

Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., and Kieffer, J.D. (1978). The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103:871–882.

De Groef B, Decallonne BR, Van der Geyten S, Darras VM, Bouillon R. (2006). Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Europ J Endocr. 155:17-25.

Horton MK, Blount BC, Valentin-Blasini L, Wapner R, Whyatt R, Gennings C, Factor-Litvak P. (2015). CO-occurring exposure to perchlorate, nitrate and thiocyanate alters thyroid function in healthy pregnant women. Environ Res. Nov;143(Pt A):1-9.

Jones PA, Pendlington RU, Earl LK, Sharma RK, Barrat MD. (1996). In vitro investigations of the direct effects of complex anions on thyroidal iodide uptake: identification of novel inhibitors. Toxicol. In Vitro. 10: 149-160.

Kleiman DA, Buitrago D, Crowley MJ, Beninato T, Veach AJ, Zanzonico PB, Jin M, Fahey TJ 3rd, Zarnegar R. (2013). Thyroid stimulating hormone increases iodine uptake by thyroid cancer cells during BRAF silencing. J Surg Res. Jun 1;182(1):85-93.

Pearce EN, Lazarus JH, Smyth PP, He X, Dall'amico D, Parkes AB, Burns R, Smith DF, Maina A, Bestwick JP, Jooman M, Leung AM, Braverman LE. (2010). Perchlorate and thiocyanate exposure and thyroid function in first-trimester pregnant women. J Clin Endocrinol Metab. Jul;95(7):3207-15.

Riesco-Eizaguirre G, Rodríguez I, De la Vieja A, Costamagna E, Carrasco N, Nistal M, Santisteban P. (2009). The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer. Cancer Res. Nov 1;69(21):8317-25.

Spitzweg C, Morris JC. (2010). Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Mol Cell Endocrinol. 322: 56-63.

Spitzweg C, Joba W, Morris JC, Heufelder AE. (1999). Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells. Thyroid. Aug;9(8):821-30.

Steinmaus CM. (2016a). Perchlorate in Water Supplies: Sources, Exposures, and Health Effects. Curr Environ Health Rep. Jun;3(2):136-43.

Steinmaus C, Pearl M, Kharrazi M, Blount BC, Miller MD, Pearce EN, Valentin-Blasini L, DeLorenze G, Hoofnagle AN, Liaw J. (2016b). Thyroid Hormones and Moderate Exposure to Perchlorate during Pregnancy in Women in Southern California. Environ Health Perspect. Jun;124(6):861-7.

Sue M, Akama T, Kawashima A, Nakamura H, Hara T, Tanigawa K, Wu H, Yoshihara A, Ishido Y, Hiroi N, Yoshino G, Kohn LD, Ishii N, Suzuki K.(2012). Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH. Thyroid. 2012 Aug;22(8):844-52.

Sugawara M, Sugawara Y, Wen K. (1999). Methimazole and propylthiouracil increase cellular thyroid peroxidase activity and thyroid peroxidase mRNA in cultured porcine thyroid follicles. Thyroid. May;9(5):513-8.

Szkudlinski MW, Fremont V, Ronin C, Weintraub BD. (2002). Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiological Reviews. 82 (2): 473–502.

Tarone RE, Lipworth L, McLaughlin JK. (2010). The epidemiology of environmental perchlorate exposure and thyroid function: a comprehensive review. J Occup Environ Med. Jun;52(6):653-60.

Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81.

Téllez Téllez R, Michaud Chacón P, Reyes Abarca C, Blount BC, Van Landingham CB, Crump KS, Gibbs JP. (2005). Long-term environmental exposure to perchlorate through drinking water and thyroid function during pregnancy and the neonatal period. Thyroid, Sep;15(9):963-75.

Tonacchera M, Pinchera A, Dimida A, Ferrarini E, Agretti P, Vitti P, Santini F, Crump K, Gibbs J. (2004). Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter. Thyroid, Dec;14(12):1012-9.

Waltz F, Pillette L, Ambroise Y. (2010). A nonradioactive iodide uptake assay for sodium iodide symporter function. Anal Biochem. 396:91-95.

Wu F, Zhou X, Zhang R, Pan M, Peng KL. (2012). The effects of ammonium perchlorate on thyroid homeostasis and thyroid-specific gene expression in rat. Environ Toxicol. Aug;27(8):445-52.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

TH synthesis, Decreased leads to T4 in serum, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

While the majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including PTU and MMI), some amphibian species (e.g., frog) (Hornung et al. 2010), and some avian species (e.g, chicken) (Van Herck et al. 2013). The following are samples from a large literature that supports this concept: Cooper et al. (1982; 1983); Hornung et al.(2010); Van Herck et al. (2013); Paul et al. (2013);  Alexander et al. (2017). Despite many physiological similarities, humans and rats exhibit significantly different susceptibilities to thyroid perturbation. For instance, dose-response data for changes in serum T3, T4, and TSH levels have been analysed from studies in humans, rats, mice, and rabbits. It was found that thyroid homeostasis in the rat appeared to be strikingly more sensitive to perchlorate (NIS inhibitor) than any of the other species. Therefore, data obtained from rat NIS studies should be critically evaluated for their relevance to humans (Lewandowski et al., 2004).

How Does This Key Event Relationship Work

Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).

NIS main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. Once inside the follicular cells, the iodide diffuses to the apical membrane, where it is metabolically oxidized through the action of TPO to iodinium (I⁺), which in turn iodinates tyrosine residues of the Tg proteins in the follicle colloid. Therefore, NIS and TPO are both essential for the synthesis of thyroid hormones (T3 and T4).

Compounds, such as triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) have been reported to decrease TH synthesis by inducing an inhibition of NIS-mediated iodide uptake and decreasing the expression of genes involved in TH synthesis, as shown in rat thyroid follicular FRTL-5 cells, and on the activity of TPO, using rat thyroid microsomes (Wu Y et al., 2016).

Perchlorate, thiocyanate, nitrate, and iodide, competitive inhibitors of iodide uptake, have been shown to inhibit radioactive iodide uptake by NIS (Tonacchera et al. 2004), consequentially causing inhibition of TH synthesis. In particular, perchlorate blocks iodide uptake into the thyroid and decreases the production of TH (Steinmaus, 2016a).

Weight of Evidence

The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).

The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.

There is limited direct evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development. Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. However, while empirical data is scarce, it is widely accepted dogma the TPO or NIS inhibition leads to declines in serum TH levels. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine (or NIS inhibition) will lead to decreased serum TH concentrations due to inadequate synthesis. Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008).

In Sui and Gilbert, 2003, developing rats were exposed in utero and postnatally to 0, 3, or 10 ppm propylthiouracil (PTU, TPO inhibitor), administered in the drinking water of dams from GD6 until PND30. Excitatory postsynaptic potentials and population spikes (indicative of neuronal network function) were recorded in area CA1 of hippocampal slices from offspring between PND21 and PND30. PTU caused at PND30 a decrease of maternal total T4 (43.9% with 3-ppm, and 65.0% with 10-ppm) compared to controls, with no change in total T3.  Maternal TSH was increased above control levels in a dose-dependent manner. In pups, total T4 was depressed (by 75%) in both treated groups relative to control pups, and total T3 was depressed (35.8% in the 3-ppm group and 66.5% in the 10-ppm group) relative to controls. TH insufficiency also compromised synaptic communication in area CA1 of developing rat hippocampus (involved in learning and memory).

Similarly, in Gilbert, 2004 developing rats were transiently exposed to PTU (0 or 15 ppm), through the drinking water of pregnant dams beginning on GD18 until PND21. This regimen markedly reduced circulating levels of TH in pups (T3: ∼50% lower than control at PND21; T4: T4 ∼40% below control levels). TH insufficiency also compromised synaptic function in the hippocampus and neuronal network function.

In Dong et al., 2005, dam rats were administered through gestation and lactation with either iodine-deficient diet or MMI (TPO inhibitor) added to drinking water. Exposure was terminated on PND30. Both treated groups showed lower concentrations of serum FT3 (~60% decrease on PND30) and FT4 (~80% decrease on PND30), and a reduction of LTP in hippocampus.

Similarly, other studies have shown associations between NIS inhibition and decreased TH serum levels (Dong et al., 2017; Calil-Silveira et al., 2016; Tang et al., 2013; Liu et al., 2012; Pearce et al., 2012).

Temporal Evidence: For the current AOP, the temporal nature of this is developmental (Seed et al., 2005). There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages. Good evidence for the temporal relationship comes from thyroid system modeling (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012).   

Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data are available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). These data were used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013), which support the role of NIS activity inhibition in relation to TH levels (T3 and T4) in serum.

There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO or NIS, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.

- Fisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat, which mimics NIS inhibition. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. In particular, HPT axis adaptations to dietary iodide intake in euthyroid (4.1-39 µg iodide/day) and iodide-deficient (0.31 and 1.2 µg iodide/day) conditions were evaluated. Alterations in T4 homeostasis were more apparent than for T3. In rat pups that were iodide deficient during gestation and lactation, decreases in serum T4 levels were associated with declines in TH levels in the fetal brain and a suppression of synaptic responses in the hippocampal region of the brain of the adult offspring (Gilbert et al., 2013).

There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO and has recently adapted for rat (Leonard et al., 2016). While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or thyroidal hormone synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme.

- Steinmaus et al., 2016b Authors of this study showed that the presence of high level of perchlorate, thiocyanate, nitrate, and iodide (NIS inhibitors) in water supply induced a decrease of total T4 [regression coefficient (β) = –0.70; 95% CI: –1.06, –0.34], a decrease of free thyroxine (fT4) (β = –0.053; 95% CI: –0.092, –0.013), and an increase of TSH, as shown in 1880 pregnant women from San Diego County, California, during 2000–2003.

- Wu F et al., 2012 Authors of this study found that high dose perchlorate (NIS inhibitor) (520 mg/kg body weight) in Sprague-Dawley rats (28-day old) caused a decrease of Tg (~ 50% lower than control), and TPO (~ 45% lower than control) gene expression, indicative of reduced TH biosynthesis, together with a decrease of free T3 (~ 50% lower than control) and free T4 levels (~ 50% lower than control), and a remarkable increase of TSH levels (125% higher than control).

- Dong et al., 2017 Wistar rats exposed to di-(2-ethylhexyl)phthalate (DEHP) (at 0, 150, 300, and 600 mg/kg/day for 3 and 6 months), a chemical known to elicit a reduction of serum TH levels, underwent decreased of serum TH levels (FT3, FT4 in the DEHP-dosed group were lower than those in the control (p < 0.05)). With the increase of DEHP-treated dose, serum total T3, total T4 and TSH level in all groups of different DEHP doses were lower than the control group at 6 months (p < 0.05), and treated rats presented also a general increase of  the expression of thyrotropin releasing hormone receptor (TRHr), Deiodinases 1 (D1), thyroid stimulating hormone beta (TSHβ), NIS, thyroid stimulating hormone receptor (TSHr), TPO, thyroid transcription factor 1 (TTF-1), and thyroglobulin (TG) genes. In particular, NIS protein, after 3 month exposure to middle (300 mg/kg/day) and high-dose (600 mg/kg/day) DEHP, resulted decreased (by ~ 30% with 300 mg/kg/day, and by ~ 85% with 600 mg/kg/day), whilst after 6 months a progressive protein increase was observed. Globally these data indicate that DEHP-dependent reduction of TH serum levels occurs via a strong perturbation of the HPT axis.

- Calil-Silveira et al., 2016 Male Wistar rats treated for two months with NaI (0.05% and 0.005%) or NaI⁺NaClO₄ (0.05%) (NIS inhibitors), underwent high levels of urine iodine, increased serum thyrotropin levels, slightly decreased serum TH levels, and a decreased expression of NIS, thyrotropin receptor, and TPO mRNA and protein, indicating a primary thyroid dysfunction.

- Tang et al., 2013 showed that 2,3',4,4',5-pentachlorobiphenyl (PCB118) (i.p. injected in male Wistar rats at 10, 100, or 1000 μg/kg/day, 5 days/week for 13 weeks) caused a progressive decrease of free T4, free T3 and TSH levels in serum (e.g., serum free T3, free T4 and TSH were reduced to 75%, 31% and 52%, respectively, at 1000 μg/kg/day PCB118), together with a downregulation of NIS (~ 30% at 1000 μg/kg/day PCB118) and Tg (~ 40% at 1000 μg/kg/day PCB118) mRNA expression. Moreover, PCB118 also led to histopathological deterioration of the thyroid (i.e., follicular hyperplasia and expansion).

- Liu et al., 2012 This in vivo study reported that PCB153 (i.p. injected in Sprague-Dawley rats at 0, 4, 16 and 32 mg/kg/day for 5 consecutive days) caused a decrease of NIS, TPO and Tg, deiodinases, the receptors (TSHr and TRHr), together with a decrease of serum total T4, total T3, and thyrotropin releasing hormone (TRH).

- Pearce et al., 2012 A cross-sectional study conducted on 134 first-trimester pregnant women from Athens, Greece, showed an inverse correlation between the level of urinary perchlorate (NIS inhibitor) and free T3 and free T4 plasma values.

References

Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Laurberg P, Lazarus JH, Mandel SJ, Peeters RP, Sullivan S.  2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017 Mar;27(3):315-389.

Antonica F, Kasprzyk DF, Opitz R, Iacovino M, Liao XH, Dumitrescu AM, Refetoff S, Peremans K, Manto M, Kyba M, Costagliola S.  Generation of functional thyroid from embryonic stem cells.  Nature. 2012 491(7422):66-71.

Atterwill CK, Fowler KF. A comparison of cultured rat FRTL-5 and porcine thyroid cells for predicting the thyroid toxicity of xenobiotics. Toxicol In Vitro. 1990. 4(4-5):369-74.

Braverman, L.E. and Utiger, R.D. (2012). Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text (10 ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp. 775-786. ISBN 978-1451120639.

Brown CG, Fowler KL, Nicholls PJ, Atterwill C. Assessment of thyrotoxicity using in vitro cell culture systems. Food Chem Toxicol. 1986 24(6-7):557-62.

Brucker-Davis F. Effects of environmental synthetic chemicals on thyroid function. Thyroid. 1998 8(9):827-56.

Calil-Silveira J, Serrano-Nascimento C, Laconca RC, Schmiedecke L, Salgueiro RB, Kondo AK, Nunes MT. (2016). Underlying Mechanisms of Pituitary-Thyroid Axis Function Disruption by Chronic Iodine Excess in Rats. Thyroid. Oct;26(10):1488-1498.

Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.

Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC.Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.

Degon, M., Chipkin, S.R., Hollot, C.V., Zoeller, R.T., and Chait, Y. (2008). A computational model of the human thyroid. Mathematical Biosciences 212, 22–53

Dong X, Dong J, Zhao Y, Guo J, Wang Z, Liu M, Zhang Y, Na X. (2017). Effects of Long-Term In Vivo Exposure to Di-2-Ethylhexylphthalate on Thyroid Hormones and the TSH/TSHR Signaling Pathways in Wistar Rats. Int J Environ Res Public Health. Jan 4;14(1). pii: E44.

Ekerot P, Ferguson D, Glämsta EL, Nilsson LB, Andersson H, Rosqvist S, Visser SA. Systems pharmacology modeling of drug-induced modulation of thyroid hormones in dogs and translation to human. Pharm Res. 2013 30(6):1513-24.

Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME.  Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 2013 132(1):75-86.

Gilbert ME. (2004). Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev Brain Res 148:11-18.

Gilbert ME, Hedge JM, Valentín-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW.  An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome.  Toxicol Sci. 2013 132(1):177-95.

Hornung MW, Degitz SJ, Korte LM, Olson JM, Kosian PA, Linnum AL, Tietge JE.Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci. 2010 118(1):42-51.

Hurley PM. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health Perspect. 1998 106(8):437-45.

King DB, May JD. Thyroidal influence on body growth. J Exp Zool. 1984 Dec;232(3):453-60.

Köhrle J. Environment and endocrinology: the case of thyroidology. Ann Endocrinol (Paris). 2008 69(2):116-22.

Leonard JA, Tan YM, Gilbert M, Isaacs K, El-Masri H. Estimating margin of exposure to thyroid peroxidase inhibitors using high-throughput in vitro data, high-throughput exposure modeling, and physiologically based pharmacokinetic/pharmacodynamic modeling. Toxicol Sci. 2016 151(1):57-70.

Lewandowski TA1, Seeley MR, Beck BD. (2004). Interspecies differences in susceptibility to perturbation of thyroid homeostasis: a case study with perchlorate. Regul Toxicol Pharmacol. Jun;39(3):348-62.

Liu C, Wang C, Yan M, Quan C, Zhou J, Yang K. (2012). PCB153 disrupts thyroid hormone homeostasis by affecting its biosynthesis, biotransformation, feedback regulation, and metabolism. Horm Metab Res. Sep;44(9):662-9.

Lu, M-H, and Anderson, RR. Thyroxine secretion rats during pregnancy in the rat.  Endo Res. 1994. 20(4):343-364.

Paul KB, Hedge JM, Macherla C, Filer DL, Burgess E, Simmons SO, Crofton KM, Hornung MW. Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology. 2013. 312:97-107.

Pearce EN, Alexiou M, Koukkou E, Braverman LE, He X, Ilias I, Alevizaki M, Markou KB. (2012). Perchlorate and thiocyanate exposure and thyroid function in first-trimester pregnant women from Greece. Clin Endocrinol (Oxf). Sep;77(3):471-4.

Steinmaus CM. (2016a). Perchlorate in Water Supplies: Sources, Exposures, and Health Effects. Curr Environ Health Rep. Jun;3(2):136-43.

Steinmaus C, Pearl M, Kharrazi M, Blount BC, Miller MD, Pearce EN, Valentin-Blasini L, DeLorenze G, Hoofnagle AN, Liaw J. (2016b). Thyroid Hormones and Moderate Exposure to Perchlorate during Pregnancy in Women in Southern California. Environ Health Perspect. Jun;124(6):861-7.

Sui L, Gilbert ME. (2003). Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology 144:4195-4203.

Tang JM, Li W, Xie YC, Guo HW, Cheng P, Chen HH, Zheng XQ, Jiang L, Cui D, Liu Y, Ding GX, Duan Y. (2013). Morphological and functional deterioration of the rat thyroid following chronic exposure to low-dose PCB118. Exp Toxicol Pathol. Nov;65(7-8):989-94.

Tonacchera M, Pinchera A, Dimida A, Ferrarini E, Agretti P, Vitti P, Santini F, Crump K, Gibbs J. (2004). Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter. Thyroid, Dec;14(12):1012-9.

Van Herck SL, Geysens S, Delbaere J, Darras VM.  Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol. 2013. 190:96-104.

Wu F, Zhou X, Zhang R, Pan M, Peng KL. (2012). The effects of ammonium perchlorate on thyroid homeostasis and thyroid-specific gene expression in rat. Environ Toxicol. Aug;27(8):445-52.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Zoeller, R. T., Tan, S. W., and Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology 37(1-2), 11-53.

T4 in serum, Decreased leads to T4 in neuronal tissue, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The majority of the information on this KER comes from in vivo studies with rodents (mainly MCT8 knock-out mice and thyroidectomized rats) and histopathological analyses of human brain tissues derived from patients affected by AHDS (Allan-Herndon-Dudley syndrome). The evolutionary conservation of the transport of TH from circulation to the developing brain suggests, with some uncertainty, that this KER is also applicable to other taxa, including birds, fish and frogs (Van Herck et al., 2013; Denver, 1998; Power et al., 2001).

How Does This Key Event Relationship Work

In mammals, thyroxine (T4) in brain tissue is derived almost entirely from the circulating pool of T4 in blood. Transfer of free T4 (and to a lesser extent, T3) from serum binding proteins (thyroid binding globulin (TBG), transthyretin (TTR) and albumin; see McLean et al., 2017, for a recent review) into the brain requires transport across the blood brain barrier (BBB) and/or indirect transport from the cerebral spinal fluid (CSF) into the brain through the blood-CSF-barrier.  The blood vessels in rodents and humans express the main T4 transporter, MCT8, (Roberts et al. 2008), as does the choroid plexus which also expresses TTR and secretes the protein into the CSF (Alshehri et al. 2015).

T4 entering the brain through the BBB is taken up into astrocytes via cell membrane iodothyronine transporters (e.g., organic anion-transporting polypeptides) (Visser et al., 2011). In astrocytes, T4 is then deiodinated by Type II deiodinase to triiodothyronine (T3) (St Germain and Galton, 1997), which is then transported via other iodothyronine transporters (e.g., monocarboxylate transporter) into neurons (Visser et al., 2011). While some circulating T3 may be taken up into brain tissue directly from blood (Dratman et al., 1991), the majority of neuronal T3 comes from deiodination of T4 in astrocytes. Decreases in circulating T4 will result in decreased brain T3 tissue concentrations. It is also known that Type II deiodinase can be up-regulated in response to decreased T4 concentrations to maintain tissue concentrations of T3 (Pedraza et al., 2007; Lavado-Autric et al., 2013; Morse et al., 1986), except in tanycytes of the paraventricular nucleus (Fekete and Lechan, 2014).

T4 and T3 are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into the blood. More detailed descriptions of this process can be found in reviews by Braverman 2012, Utiger 2006, and Zoeller et al., 2007.

Monocarboxylate transporter 8 (MCT8) is a specific human transporter for the T4 and T3 that allows their entry in the brain and other organs. Mutations in MCT8 (Allan-Herndon-Dudley syndrome, AHDS) lead to a severe form of X-linked truncal hypotonia, spasticity, mental retardation (or 'cretinism'), and are characterised by normal to high TSH, elevated plasma T3, low T4, and decreased TH signaling in discrete brain areas (McAninch and Bianco, 2014; Anık et al., 2014; López-Espíndola et al., 2014).

Other in vivo studies have proven direct associations between levels of serum T4 and the levels of TH-dependent signalling in the brain (i.e., brain TH levels). For instance, mice characterized by single MCT8 deficiency showed low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in both the hypothalamus and the cortex (Stohn et al., 2016). Analogously, another study reported that MCT8 knock-out mice were characterized by high serum T3, low serum T4, and decreased forebrain TH content (Müller et al., 2014). The hippocampus seems to be one of the brain regions mostly affected by low TH levels, as shown in thyroidectomized Wistar rats, which resulted affected by hippocampal hypothyroidism (da Conceição et al., 2016). These data altogether support direct associations between levels of serum T4 and levels of TH-dependent signalling in the brain (i.e., brain TH levels).

Weight of Evidence

The weight of evidence linking reductions in circulating serum TH and reduced brain concentrations of TH is moderate. Many studies support this basic linkage. However, there are compensatory mechanisms (e.g., upregulation of deiodinases, transporters) that may alter the relationship between hormones in the periphery and hormone concentrations in the brain. There is limited information available on the quantitative relationship between circulating levels of TH, these compensatory processes, and neuronal T4 concentrations, especially during development. Furthermore, in certain conditions, such as iodine deficiency, the decreases in circulating hormone might have greater impacts on tissue levels of TH (see for instance, Escobar del Rey, et al., 1989).

The biological relationship between these two KEs is strong as it is well accepted dogma within the scientific community. There is no doubt that decreased circulating T4 leads to declines in tissue concentrations of T4 and T3 in a variety of tissues, including brain. However, compensatory mechanisms (e.g., increased expression of Type 2 deiodinase) may differ during different life stages and across different tissues, especially in different brain regions.  Similarly, the degree to which serum TH must drop to overwhelm these compensatory responses has not been established.

Several studies have shown that tissue levels (including the brain) of TH are proportional to serum hormone level (Oppenheimer, 1983; Morreale de Escobar et al., 1987; 1990; Calvo et al., 1992; Porterfield and Hendrich, 1992, 1993; Broedel et al., 2003). In thyroidectomized rats, brain concentrations of T4 were decreased and Type II deiodinase (DII) activity was increased. Both brain T3 and T4 as well as DII activity returned to normal following infusion of T4 (Escobar-Morreale et al., 1995; 1997). Animals treated with PTU, MMI, or iodine deficiency during development demonstrate both lower serum and lower brain TH concentrations (Escobar-Morreale et al 1995; 1997; Taylor et al., 2008; Bastian et al., 2012; 2014; Gilbert et al., 2013).

Temporal Evidence: For the current AOP the temporal nature of this KER is described in the context of different developmental stages (Seed et al., 2005). While all brain regions will be impacted by changes in serum hormones, brain concentrations will be a function of development stage and brain region. Data are available from thyroid hormone replacement studies that demonstrate recovery of fetal brain T3 and T4 levels (following low iodine diets or MMI exposure) to control levels after maternal thyroid hormone replacement or iodine supplementation (e.g., Calvo et al.,1990;  Obregon et al., 1991). For example, Calvo et al. (1990) carried out a detailed study of the effects of TPO inhibition on serum and tissues levels of TH in gestating rats. Clear dose-dependent effects of T4 replacement, but not T3 replacement were seen in all maternal tissues. However, for fetal tissues, neither T4 nor T3, at any dose, could completely restore tissue TH levels to control levels.

Dose-Response Evidence:  There is good evidence, albeit from a limited number of studies of the correlative relationship between circulating thyroid hormone concentrations and brain tissue concentrations during fetal and early postnatal development following maternal iodine deficient diets or chemical treatments that depress serum THs (c.f., Calvo et al., 1990; Obregon et al., 1991; Morse et al.,1996)

Empirical support for the linkage comes from pathological analyses of human brain tissues and in vivo studies on MCT8 knock-out mice and thyroidectomized rats.

Four male patients affected by AHDS from two unrelated Turkish families (age from 1.5 to 11 years) presented high plasma T3, low plasma T4 and rT3, and normal/mildly elevated TSH levels. Functional analysis of a novel missense MCT8 mutation (p.G495A) revealed lowered TH transport (from 20 to 85%) depending on the cell/tissue context. Phenotypically, patients were characterized by severe psychomotor retardation, axial hypotonia, lack of speech, diminished muscle mass, increased muscle tone, hyperreflexia, myopathic facies (Anık et al., 2014).

Another study analyzed brain sections from a 30^th gestational week male fetus and an 11-year-old boy in comparison with  brain tissue from a 30^th and 28^th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy, as controls. The MCT8-deficient fetal cerebral cortex showed 50% reduction of TH (i.e., T4, T3, and rT3), while T3 and T4 levels were normal in the liver. This TH deficiency in the brain produced an expected increase in type 2 deiodinase and decrease in type 3 deiodinase mRNA expression. Also, MCT8-deficient fetus showed a delay in cortical and cerebellar development and myelination, loss of parvalbumin expression, abnormal calbindin-D28k content, impaired axonal maturation, and diminished biochemical differentiation of Purkinje cells. The 11-year-old boy displayed altered cerebellar structure, deficient myelination, deficient synaptophysin and parvalbumin expression, and abnormal calbindin-D28k expression (López-Espíndola et al., 2014).

Several in vivo studies have proven direct associations between levels of serum T4 and the levels of TH-dependent signalling in the brain (i.e., brain TH levels). For instance, mice characterized by single MCT8 deficiency showed low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in both the hypothalamus and the cortex (Stohn et al., 2016).

Analogously, another study reported that MCT8 knock-out mice were characterized by high serum T3, low serum T4, and decreased forebrain TH content, while TH concentrations in the liver, kidneys, and thyroid gland resulted increased (Müller et al., 2014).

The hippocampus, critical brain structure for cognitive, learning and memory processes, seems to be one of the brain regions mostly affected by low TH levels, as shown in thyroidectomized Wistar rats, which resulted affected by hippocampal hypothyroidism. Thyroidectomized rats were characterized by increased serum TSH, decreased T4 and T3 serum levels, and a reduced hippocampal expression of MCT8, thyroid hormone receptor alfa, deiodinase type 2, ectonucleotide pyrophosphatase/phosphodiesterase 2 and brain-derived neurotrophic factor (BDNF) genes (da Conceição et al., 2016). These data altogether support direct associations between levels of serum T4 and levels of TH-dependent signalling in the brain (i.e., brain TH levels).

The ability of the developing brain to compensate for TH insufficiency is not well known. Limited data is available that demonstrates that the developing brain can compensate for chemical-induced alterations in TH concentrations (e.g., Calvo et al., 1990; Morse et al., 1996; Sharlin et al., 2010). There are likely different quantitative relationships between these two KEs depending on the compensatory ability based on both developmental stage and specific brain region (Sharlin et al., 2010). For these reasons, the empirical support for this linkage is rated as moderate. In addition, future work on transport mechanisms and deiodinase activity may add additional KEs and KERs to this AOP.

While it is well established that decreased in serum TH levels result in decreased brain TH concentrations, particularly fetal brain concentrations, a major gap is the lack of empirical data that allow direct quantification of this relationship.

There may also be different quantitative relationships between these two KEs depending on the compensatory ability of different developing brain regions (Sharlin et al., 2010).

- Fisher et al., (2013) recently published a quantitative biologically-based dose-response model (BBDR) for iodine deficiency in the rat. In particular, HPT axis adaptations to dietary iodide intake in euthyroid (4.1-39 µg iodide/day) and iodide-deficient (0.31 and 1.2 µg iodide/day) conditions were evaluated. In rat pups that were iodide deficient during gestation and lactation, decreases in serum T4 levels were associated with declines in TH levels in the fetal brain and a suppression of synaptic responses in the hippocampal region of the brain of the adult offspring (Gilbert et al., 2013).

- Anık et al., 2014 Four males with AHDS from two unrelated Turkish families (age from 1.5 to 11 years) presented high plasma T3, low plasma T4 and rT3, and normal/mildly elevated TSH levels. Functional analysis of a novel missense MCT8 mutation (p.G495A) revealed lowered TH transport to organs (including the brain) (from 20 to 85%) depending on the cell/tissue context.

- López-Espíndola et al., 2014 This study analyzed brain sections from a 30^th gestational week male fetus and an 11-year-old boy in comparison with  brain tissue from a 30^th and 28^th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy, as controls. The MCT8-deficient fetal cerebral cortex showed 50% reduction of TH (i.e., T4, T3, and rT3), while T3 and T4 levels were normal in the liver. This TH deficiency in the brain produced an expected increase in type 2 deiodinase and decrease in type 3 deiodinase mRNA expression.

- Stohn et al., 2016 In this study, mice characterized by single MCT8 deficiency showed low serum T4 (~ 47% decrease vs control mice, at age P21), elevated serum TSH (~ 125% increase vs control mice, at age P21)  and T3 (~ 33% increase vs control mice, at age P21), and decreased T3-dependent gene expression in both the hypothalamus (~ 57% decrease vs control mice, at age P21) and the cortex (~ 40% decrease vs control mice, at age P21), as indicated by the lower expression of the rat hairless (hr) gene, which is highly up-regulated by TH in the developing CNS.

- Müller et al., 2014 This study reported that MCT8 knock-out mice were characterized by high serum T3 (~ 100% increase at P21, and ~ 300% increase at 2.5 months of age, compared to control mice), low serum T4 (~ 62% decrease at P21, and ~ 50% decrease at 2.5 months of age, compared to control mice), and decreased TH content in the forebrain (~ 43% decrease for both T3 and T4, at 2.5 months of age, compared to control mice), while TH concentrations in the liver, kidneys, and thyroid gland resulted increased.

- da Conceição et al., 2016 Thyroidectomized Wistar rats resulted affected by hippocampal hypothyroidism. Thyroidectomized rats were characterized by increased serum TSH (~ 750% increase vs control rats), decreased T4 (~ 80% decrease vs control rats) and T3 (~ 45% decrease vs control rats) serum levels, and a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), thyroid hormone receptor alfa (Trα1, ~ 77% decrease vs control rats), deiodinase type 2 (Dio2, ~ 90% decrease vs control rats), ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2, ~ 80% decrease vs control rats) and brain-derived neurotrophic factor (BDNF, ~ 75% decrease vs control rats) mRNAs.

References

Anık A, Kersseboom S, Demir K, Catlı G, Yiş U, Böber E, van Mullem A, van Herebeek RE, Hız S, Abacı A, Visser TJ. (2014). Psychomotor retardation caused by a defective thyroid hormone transporter: report of two families with different MCT8 mutations. Horm Res Paediatr. 82(4):261-71.

Alshehri B, D'Souza DG, Lee JY, Petratos S, Richardson SJ.  The diversity of mechanisms influenced by transthyretin in neurobiology: development, disease and endocrine disruption. J Neuroendocrinol. 2015 May;27(5):303-23.

Bastian TW, Anderson JA, Fretham SJ, Prohaska JR, Georgieff MK, Anderson GW (2012), Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex. Endocrinology 153:5668-5680.

Bastian TW, Prohaska JR, Georgieff MK, Anderson GW (2014) Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 55:1157-1167.

Braverman LE. (2012). Various aspects of thyroid physiology and pathology contributed by international experts. Curr Opin Endocrinol Diabetes Obes. Oct;19(5):381.

Broedel, O., Eravci, M., Fuxius, S., Smolarz, T., Jeitner, A., Grau, H., Stoltenburg-Didinger, G., Plueckhan, H., Meinhold, H., and Baumgartner, A. (2003). Effects of hyper and hypothyroidism on thyroid hormone concentrations in regions of the rat brain. Am. J. Physiol. Endocrinol. Metab. 285:E470–480.

Calvo, R., Obregon, M.J., Escobar del Rey, F., and Morreale de Escobar, G. (1992). The rat placenta and the transfer of thyroid hormones from the mother to the fetus. Effects of maternal thyroid status. Endocrinology 131:357–365.

Calvo R, Obregon MJ, Ruiz de Ona C, Escobar del Rey F, Morreale de Escobar G 1990 Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3’-triiodothyronine in the protection of the fetal brain. J Clin Invest 86:889–899.

da Conceição RR, Laureano-Melo R, Oliveira KC, de Carvalho Melo MC, Kasamatsu TS, de Barros Maciel RM, de Souza JS, Giannocco G. (2016). Antidepressant behavior in thyroidectomized Wistar rats is induced by hippocampal hypothyroidism. Physiol Behav. Apr 1;157:158-64.

Denver, RJ. (1998). The molecular basis of thyroid hormone-dependent central nervous system remodeling during amphibian metamorphosis. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 119:219-228.

Dratman MB, Crutchfield FL, Schoenhoff MB. Transport of iodothyronines from bloodstream to brain: contributions by blood:brain and choroid plexus:cerebrospinal fluid barriers.  Brain Res. 1991 Jul 19;554(1-2):229-36.

Escobar del Rey F, Ruiz de Oña C, Bernal J, Obregón MJ, Morreale de Escobar G. Generalized deficiency of 3,5,3'-triiodo-L-thyronine (T3) in tissues from rats on a low iodine intake, despite normal circulating T3 levels. Acta Endocrinol (Copenh). 1989 Apr;120(4):490-8.

Escobar-Morreale HF, Obregón MJ, Escobar del Rey F, Morreale de Escobar G. Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats. J Clin Invest. 1995 Dec;96(6):2828-38.

Escobar-Morreale HF1, Obregón MJ, Hernandez A, Escobar del Rey F, Morreale de Escobar G. Regulation of iodothyronine deiodinase activity as studied in thyroidectomized rats infused with thyroxine or triiodothyronine. Endocrinology. 1997 Jun;138(6):2559-68.

Fekete C, Lechan RM.  Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions.  Endocr Rev. 2014 Apr;35(2):159-94

Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME. (2013). Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 132(1):75-86.

Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW (2013) An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195.

Lavado-Autric R, Calvo RM, de Mena RM, de Escobar GM, Obregon MJ. Deiodinase activities in thyroids and tissues of iodine-deficient female rats. Endocrinology. 2013 Jan;154(1):529-36.

López-Espíndola D, Morales-Bastos C, Grijota-Martínez C, Liao XH, Lev D, Sugo E, Verge CF, Refetoff S, Bernal J, Guadaño-Ferraz A. (2014). Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. Dec;99(12):E2799-804.

McAninch EA, Bianco AC. (2014). Thyroid hormone signaling in energy homeostasis and energy metabolism. Ann N Y Acad Sci. Apr;1311:77-87.

McLean TR, Rank MM, Smooker PM, Richardson SJ.  Evolution of thyroid hormone distributor proteins. Mol Cell Endocrinol. 2017 Feb 27. pii: S0303-7207(17)30151-X. doi: 10.1016/j.mce.2017.02.038. [Epub ahead of print]

Morreale de Escobar, G., Obregon, M.J., and Escobar del Ray, F. (1987). Fetal and maternal thyroid hormones. Hormone Res. 26:12–27.

Morreale de Escobar, G., Calvo, R., Obregon, M.J., and Escobar del Rey, F. (1990). Contribution of maternal thyroxine to fetal thyroxine pools in normal rats near term. Endocrinology 126:2765–2767.

Müller J, Mayerl S, Visser TJ, Darras VM, Boelen A, Frappart L, Mariotta L, Verrey F, Heuer H. (2014). Tissue-specific alterations in thyroid hormone homeostasis in combined Mct10 and Mct8 deficiency. Endocrinology. Jan;155(1):315-25.

Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254).Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79

Obregon MJ, Ruiz de Ona C, Calvo R, Escobar del Rey F, Morreale de Escobar G 1991 Outer ring iodothyronine deiodinases and thyroid hormone economy: Responses to iodine deficiency in the rat fetus and neonate. Endocrinology 129:2663–2673.

Oppenheimer, J.H. (1983). The nuclear Receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action. In: Molecular Basis of Thyroid Hormone Action, eds. J.H. Oppenheimer and H.H. Samuels, pp. 1–35. New York: Academic Press.

Pedraza PE, Obregon MJ, Escobar-Morreale HF, del Rey FE, de Escobar GM (2006) Mechanisms of adaptation to iodine deficiency in rats: thyroid status is tissue specific. Its relevance for man. Endocrinology 147:2098-2108.

Porterfield, S.P. and Hendrich, C.E. (1992). Tissue iodothyronine levels in fetuses of control and hypothyroid rats at 13 and 16 days gestation. Endocrinology 131:195–200.

Porterfield, S.P. and Hendrich, C.E. (1993). The role of thyroid hormones in prenatal neonatal neurological development-current perspectives. Endocrine Rev. 14:94–106.

Porterfield SP. (1994). Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system. Environ Health Perspect. Jun;102 Suppl 2:125-30.

Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. (2001). Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. Dec;130(4):447-59.

Roberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, Grindstaff KK, Mengesha W, Raman C, Zerangue N.  Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology. 2008 Dec;149(12):6251-61.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Sharlin DS, Gilbert ME, Taylor MA, Ferguson DC, Zoeller RT. (2010).The nature of the compensatory response to low thyroid hormone in the developing brain. J Neuroendocrinol.  Mar;22(3):153-65.

St. Germain, D.L. and Galton, V.A. (1997). The deiodinase family of selenoproteins. Thyroid 7:655–668.

Stohn JP, Martinez ME, Matoin K, Morte B, Bernal J, Galton VA, St Germain D, Hernandez A. (2016). MCT8 Deficiency in Male Mice Mitigates the Phenotypic Abnormalities Associated With the Absence of a Functional Type 3 Deiodinase. Endocrinology. Aug;157(8):3266-77.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC (2008) Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Utiger RD. (2006). Iodine nutrition--more is better. N Engl J Med. Jun 29;354(26):2819-21.

Van Herck SL, Geysens S, Delbaere J, Darras VM. (2013). Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol.190:96-104.

Visser WE, Friesema EC, Visser TJ.  Minireview: thyroid hormone transporters: the knowns and the unknowns.  Mol Endocrinol. 2011 Jan;25(1):1-14.

Zoeller RT, Tan SW, Tyl RW. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology. Jan-Feb;37:11-53.

T4 in neuronal tissue, Decreased leads to BDNF, Reduced

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The connection between TH levels and BDNF expression has been studied only in rodent models up to date (see above studies).

How Does This Key Event Relationship Work

It is widely accepted that the thyroid hormones (TH) have a prominent role in the development and function of the Central Nervous System (CNS) and their action has been closely linked to the cognitive function because of their importance in the neocortical development (Gilbert et al., 2012). During the early cortical network development TH has been shown to influence the number of cholinergic neurons and the degree of innervation of hippocampal CA3 and CA1 regions (Oh et al., 1991; Thompson and Potter 2000), and to regulate the morphology and function of GABAergic neurons (Westerholz et al., 2010).

One of the mediators of this regulation has been suggested to be the brain derived neurotrophic factor (BDNF), whose role in brain development and function has been very well-documented (Binder and Scharfman, 2004) and which function has been associated with TH levels in the brain (Gilbert and Lasley, 2013). Several studies have shown that TH can regulate BDNF expression in the brain (Koibuchi et al., 1999; Koibuchi and Chin, 2000; Sui and Li, 2010), with the subsequent neurodevelopmental consequences.

In view of the above evidence, it has been shown that the thyroid insufficiency (lower TH levels) results in reduction of BDNF levels (mRNA or protein) in the developmental brain.

Weight of Evidence

The importance of thyroid hormones (TH) in brain development has been recognised and investigated for many decades (Bernal, 2011). Several human studies have shown that low levels of circulating maternal TH, even in the modest degree, can lead to neurophysiological deficits in the offspring, including learning and memory deficits, or even cretinism in most severe cases (Zoeller and Rovet, 2004; Henrichs et al., 2010). The levels of serum TH at birth are not always informative, as most of the neurological deficits are present despite the normal thyroid status of the newborn. That means that the cause of these impairments is rooted in the early stages of the neuronal development during the gestational period. The nature and the temporal occurrence of these defects suggest that TH may exert their effects through the neurotrophins, as they are the main regulators of neuronal system development (Lu and Figurov, 1997). Among them, BDNF represents the prime candidate because of its critical role in CNS development and its ability to regulate synaptic transmission, dendritic structure and synaptic plasticity in adulthood (Binder and Scharfman, 2004). Additionally, hippocampus and neocortex are two of the regions characterized by the highest BDNF expression (Kawamoto et al., 1996), and are also key brain areas for learning and memory functions.

The empirical support for this direct KER is weak. There are a limited number of studies investigating TH concentrations in the brain and BDNF levels. This is due to the fact that TH is difficult to measure in the brain and TH-induced changes in BDNF expression (mRNA or protein levels) can be subtle.

For the current AOP the temporal nature of this KER is described in the context of different developmental stages (Seed et al., 2005). The impact of brain TH concentrations on regulation of TH receptor (TR) regulated genes is age-dependent for a number of genes critical for normal hippocampal development. It is widely accepted that different genes, including BDNF, are downregulated under conditions of TH insufficiency (Pathak et al, 2011). TH supplementation has been shown to reverse some of the effects on gene expression (Liu et al., 2010; Pathak et al., 2011), including also BDNF expression (Wang et al., 2012).

Many in vivo studies have focused on the determination of the relationship between TH-mediated effects and BDNF expression in the brain. The majority of the work has been performed by evaluating the effects of TH insufficiency on BDNF developmental expression profile. The results, despite some differences, are showing a trend toward BDNF down-regulation.

Reductions in BDNF mRNA and protein were observed in hypothyroid rat models, created by exposing the animals to the TPO inhibitors methimazole (MMI) (Sinha et al., 2009) or propylthiouracil (PTU) (Neveu and Arenas, 1996; Lasley and Gilbert, 2011), and perchlorate (NIS inhibitor) (Koibuchi et al., 1999; 2001). These studies supported direct associations between lower level of TH and lower BDNF expression in the developmental cerebellum, hippocampus and cortex. The dose-response relationship could not be evaluated in these studies, as they were conducted in conditions of severe maternal hypothyroidism, namely after exposure to very high doses of the chemicals.

Additionally, in more complex models of maternal hypothyroidism in rats a reduction of hippocampal and cortex BDNF expression was observed (Wang et al., 2012; Liu et al., 2010). In these latter cases, hypothyroidism was developed via thyroidectomies, and T4 supplementation was performed at specific stages during gestation.

Indirect evidences supporting this KER:

- Koibuchi et al., 2001 Here newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Since TH levels in the brain were not measured, the same study was also included in the indirect KER "TH synthesis decrease leads to BDFN reduction".

- Morte et al., 2010 induced maternal and fetal hypothyroidism by maternal thyroidectomy (Tx) followed by the antithyroid drug MMI (TPO inhibitor) treatment. Tx dams treated with MMI showed increased TSH (~10 fold increase) and decreased serum T4 (~95%) and serum T3 (~90%). Whilst fetuses from Tx rats had normal serum TSH and cerebral cortex T4 and T3, pups born from dams treated with MMI showed increase of TSH (~8 fold), decreased cortex T4 (~75%) and cortex T3 (~95%). Additionally, analysis of gene expression in the fetal cerebral cortex showed a reduction of Camk4 (Ca(2+) and calmodulin-activated kinase) gene expression (~70%) induced by maternal and fetal hypothyroidism. As Camk4 during development is known to induce BDNF expression (Shieh et al., 1998), a reduction of cortical TH levels (leading to Camk4 mRNA downregulation) may lead to reduction of BDNF expression and/or signaling; however, BDNF expression was not measured in Morte et al. 2010.

- da Conceição et al., 2016 Thyroidectomized (i.e,. hypothyroid) adult Wistar rats showed significant increase of serum TSH (~ 750% increase vs control rats), decrease of T4 (~ 80% decrease vs control) and T3 serum levels (~ 45% decrease vs control), together with a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), TH receptor alfa (TRα1) (~ 77 % decrease vs control), deiodinase type 2 (DIO2) (~ 90% decrease vs control), and BDNF mRNA expression in hippocampus (~ 75% decrease vs control). The reduced gene expression of TRα1 (expressed in nearly all neurons in the brain (Schwartz et al., 1992)), MCT8 and DIO2 (both essential for the regulation of glia-neuron cell interaction in TH metabolism in the brain (Remaud et al., 2014)), indicate the presence of low TH levels in the hippocampus (hippocampal hypothyroidism) in thyroidectomized rats. However, direct measures of TH (T3 or T4) brain levels were not assessed in this study.

 - Pathak et al, 2011 Here the effect of maternal TH deficiency on neocortical development was investigated. Rat dams were exposed to MMI (TPO inhibitor) from GD6 until sacrifice. Decreased number and length of radial glia, loss of neuronal bipolarity, and impaired neuronal migration were recovered with early TH replacement (at E13-15). BDNF mRNA resulted downregulated (80% decrease at E14) while trkB expression was increased (2-fold) in hypothyroid fetuses at E14 stage. However, TH levels in the brain were not measured in this study.

Hypothyroidism (i.e., induced by chemicals known to inhibit TPO or NIS, or by thyroidectomy, leading to low TH serum levels) is generally associated with lower levels of BDNF in brain tissues. As described in Conceição et al., 2016, thyroidectomized adult rats, apart from showing reduced TH levels, also presented reduced hippocampal gene expression of MCT8, TRα1, DIO2 and BDNF, which support a link between hippocampal hypothyroidism and reduced BDNF levels.

However, despite the fact that many in vivo studies have shown a correlation between hypothyroidism and BDNF expression in the brain, there are no studies simultaneously measuring the levels of both TH and BDNF in the brain. Therefore, no clear consensus can be reached by the overall evaluation of the existing data. There are numerous conflicting studies showing no significant decrease in BDNF mRNA or protein levels under hypothyroid conditions (Alvarez-Dolado et al., 1994; Bastian et al., 2010; 2012; Royland et al., 2008; Lasley and Gilbert, 2011). However, the results of these studies cannot exclude the possibility of temporal- or region-specific decreased BDNF effects as a consequence of foetal hypothyroidism. A transient TH-dependent BDNF reduction in early postnatal life can be followed by a period of normal BDNF levels or, on the contrary, normal BDNF expression in the early developmental stages is not predictive of equally normal BDNF expression throughout development. Moreover, significant differences in study design, the assessed brain regions, the age and the method of assessment in the existing studies, further complicate result interpretation.

While PTU (TPO inhibitor) has been shown to decrease serum TH levels and brain BDNF protein levels and mRNA expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in Cortés et al., 2012 study, treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB, the receptor for BDNF, resulted reduced at the postsynaptic density (PSD) of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD in hypothyroid animals. While these data indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain, downregulation of TrkB receptors still leads to decrease signalling pathways regulated by BDNF.

There are no consistent quantitative data linking TH levels in the brain and the level of BDNF expression (mRNA or protein), due to differences in study designs, dose regimes and the methods used to assess the endpoints.

As discussed in the Empirical support section, there are some quantitative data in support of the indirect KER “Decreased of TH synthesis leads to reduced BDNF release”, but not to this direct KER.

References

Alvarez-Dolado M, Iglesias T, Rodrıguez-Pena A, Bernal J, Munoz A. (1994). Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain. Brain Res Mol Brain Res. 27:249–257.

Bastian TW, Anderson JA, Fretham SJ, Prohaska JR, Georgieff MK, Anderson GW. (2012). Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex. Endocrinology 153: 5668–5680.

Bastian TW, Prohaska JR, Georgieff MK, Anderson GW. (2010). Perinatal iron and copper deficiencies alter neonatal rat circulating and brain thyroid hormone concentrations. Endocrinology 151:4055–4065.

Bernal J. (2011). Thyroid hormone transport in developing brain. Curr Opin Endocrinol Diab Obes 18:295–299.

Binder DK, Scharfman HE. (2004). Brain-derived neurotrophic factor. Growth Factors. 22(3):123–131

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Cortés C, Eugenin E, Aliaga E, Carreño LJ, Bueno SM, Gonzalez PA, Gayol S, Naranjo D, Noches V, Marassi MP, Rosenthal D, Jadue C, Ibarra P, Keitel C, Wohllk N, Court F, Kalergis AM, Riedel CA. (2012). Hypothyroidism in the adult rat causes incremental changes in brain-derived neurotrophic factor, neuronal and astrocyte apoptosis, gliosis, and deterioration of postsynaptic density. Thyroid. Sep;22(9):951-63.

da Conceição RR, Laureano-Melo R, Oliveira KC, de Carvalho Melo MC, Kasamatsu TS, de Barros Maciel RM, de Souza JS, Giannocco G. (2016). Antidepressant behavior in thyroidectomized Wistar rats is induced by hippocampal hypothyroidism. Physiol Behav. Apr 1;157:158-64.

Gilbert ME, Lasley SM. (2013). Developmental thyroid hormone insufficiency and brain development: a role for brain-derived neurotrophic factor (BDNF)? Neurosci 239: 253-270.

Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012). Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology 33(4):842-852.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology, Feb;157(2):774-87

Henrichs J, Bongers-Schokking JJ, Schenk JJ, Ghassabian A, Schmidt HG, Visser TJ, Hooijkaas H, de Muinck Keizer-Schrama SM, Hofman A, Jaddoe VV, Visser W, Steegers EA, Verhulst FC, de Rijke YB, Tiemeier H. (2010). Maternal thyroid function during early pregnancy and cognitive functioning in early childhood: the generation R study. J Clin Endocrinol Metab 95:4227–4234.

Kawamoto Y, Nakamura S, Nakano S, Oka N, Akiguchi I, Kimura J. (1996). Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain. Neurosci 74(4):1209-1226.

Koibuchi N, Chin WW. (2000). Thyroid hormone action and brain development. Trends Endocrinol Metab. 11(4):123-128.

Koibuchi N, Yamaoka S, Chin WW. (2001). Effect of altered thyroid status on neurotrophin gene expression during postnatal development of the mouse cerebellum. Thyroid 11:205–210.

Koibuchi N, Fukuda H, Chin WW. (1999). Promoter-specific regulation of the brain-derived neurotrophic factor gene by thyroid hormone in the developing rat cerebellum. Endocrinol 140: 3955–3961.

Lasley SM, Gilbert ME. (2011). Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates. Neurotoxicol Teratol 33:464–472.

Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H. (2010). The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring. Thyroid 20:909–915.

Lu B, Figurov A. (1997). Role of neurotrophins in synapse development and plasticity. Rev Neurosci 8:1–12.

Morte B, Diez D, Auso E, Belinchon MM, Gil-Ibanez P, Grijota-Martinez C, Navarro D, de Escobar GM, Berbel P, Bernal J (2010a) Thyroid hormone regulation of gene expression in the developing rat fetal cerebral cortex: prominent role of the Ca2+/calmodulin-dependent protein kinase IV pathway. Endocrinology 151:810-820.

Neveu I, Arenas E. (1996.) Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. J Cell Biol 133:631–646.

Oh JD, Butcher LL, Woolf NJ (1991). Thyroid hormone modulates the development of cholingergic terminal fields in the rat forebrain: relation to nerve growth factor receptor. Devl Brain Res  59:133–142.

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM. 2011. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cerebral cortex. Jan;21:11-21.

Remaud S, Gothié JD, Morvan-Dubois G, Demeneix BA (2014). Thyroid hrmone signaling and adult neurogenesis in mammals. Front. Endocrinol., 5, p. 40

Royland JE, Parker JS, Gilbert ME. (2008). A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol 20:1319–1338.

Schwartz HL, Strait KA, Ling NC, Oppenheimer JH (1992). Quantitation of rat-tissue thyroid-hormone binding-receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding-capacity. J. Biol. Chem., 267 (17), pp. 11794–11799.

Shafiee SM, Vafaei AA, Rashidy-Pour A. (2016). Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise. Neuroscience. Aug 4;329:151-61.

Shieh PB, Hu SC, Bobb K, Timmusk T, Ghosh A (1998). Identification of a signaling pathway involved in calcium regulation of BDNF expression. Neuron  20:727–740

Sinha RA, Pathak A, Kumar A, Tiwari M, Shrivastava A, Godbole MM. (2009). Enhanced neuronal loss under perinatal hypothyroidism involves impaired neurotrophic signaling and increased proteolysis of p75(NTR). Mol Cell Neurosci 40:354–364.

Sui L, Li BM. (2010). Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: role of DNA methylation and histone acetylation. Steroids 75:988–997.

Thompson CC, Potter GB. (2000). Thyroid hormone action in neural development. Cereb Cortex. Oct;10(10):939-45.

Wang S, Teng W, Gao Y, Fan C, Zhang H, Shan Z. (2012). Early levothyroxine treatment on maternal subclinical hypothyroidism improves spatial learning of offspring in rats. J Neuroendocrinol 24:841–848.

Westerholz S, deLima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neurosci 168:573–589.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Zoeller RT, Rovet J. (2004). Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J Neuroendocrinol 16:809–818.

BDNF, Reduced leads to GABAergic interneurons, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from work with laboratory rodents (rats and mice). No data are available for other species.

How Does This Key Event Relationship Work

GABAergic interneurons are remarkably diverse and complex in nature and they are believed to play a key role in numerous neurodevelopmental processes (Southwell et al., 2014). Among them, those that express parvalbumin (PV) (marker of GABAergic interneurons) as their calcium-binding protein are the ones subjected to regulations by neurotrophins and BDNF specifically (Woo and Lu, 2006). These neurons do not express the BDNF protein but its functional receptor, Trk-B (Cellerino et al., 1996; Marty et al., 1996; Gorba and Wahle, 1999). BDNF is released by the BDNF-producing neurons of the CNS and binds to Trk-B of the GABA PV-interneurons, an interaction necessary for the subsequent developmental effects mediated by BDNF (Polleux et al., 2002; Jin et al., 2003; Rico et al., 2002; Aguado et al., 2003). BDNF promotes the morphological and neurochemical maturation of hippocampal and neocortical interneurons and promotes GABAergic synaptogenesis (Danglot et al., 2006 and Hu and Russek, 2008). BDNF also regulates the expression of the GABA-specific K(+)/Cl(-) co-transporter, KCC2, which is responsible for switching of GABA action from excitatory to inhibitory, and consequently determines the nature of GABA-induced development of glutamatergic (excitatory) synapses (Wang and Kriegstein, 2009; Blaesse et al., 2009).

Weight of Evidence

Proper function of the Central Nervous System (CNS) results from the closely regulated development and function of the different neuronal subtypes and is driven by the overall balance between excitation and inhibition. In the cerebral cortex the synaptic inhibition is mediated by the GABAergic interneurons, which regulate also the neuronal developmental excitability and thereby the function and maturation of the neuronal networks (Voigt et al., 2001; Cherubini et al., 2011).

Many trophic factors are implicated in the regulation of these processes but among them BDNF stands out as the prime candidate due to do its effects on interneuron development (Palizvan et al., 2004; Patz et al., 2004; Woo and Lu, 2006; Huang et al., 2007; Huang, 2009). Exogenous application of BDNF in developing neocortical and hippocampal GABAergic interneurons has demonstrated an enhanced dendritic elongation and branching in cultures (Jin et al., 2003; Vicario-Abejon et al., 1998). Interneuron differentiation was also affected by endogenous BDNF, as the length and branching of GABAergic interneurons (GFP-positive (i.e., BDNF^+/+)), was promoted only when they were innervated by BDNF-releasing interneurons (Kohara et al., 2003). Due to these dendritic effects of BDNF on GABAergic interneurons, this neurotrophin was suggested to promote also the formation of inhibitory synapses, which was further supported by several in vitro studies. Exogenous application of BDNF significantly increased the number of functional synapses in culture (Vicario-Abejon et al., 1998; Marty et al., 2000), while blocking BDNF with antibodies greatly reduced the formation of inhibitory synapses (Seil and Drake-Baumann, 2000). Similar results were observed in vivo in transgenic mice with deleted Trk-B gene in cerebellar precursors, in which Trk-B receptor was found to be the prerequisite for inhibitory synapses formation (Rico et al., 2002). Additionally, BDNF was reported to elicit presynaptic changes in GABAergic interneurons, as several presynaptic proteins were up-regulated after BDNF application (Yamada et al., 2002; Berghuis et al., 2004). A significant increase of GABAA receptor density was observed in cultured hippocampus-derived neurons after treatment with BDNF (Yamada et al., 2002).

BDNF is also a potent regulator of spontaneous neuronal activity (Aguado et al., 2003; Carmona et al., 2006), a major milestone of the developing hippocampus and an important feature of the CNS. Further supporting studies have shown that it has the ability to depolarize cortical neurons in culture (Kafitz et al., 1999), an effect which has been linked to the developmentally regulated spontaneous network activity (Feller, 1999; O'Donovan, 1999).

The spontaneous neuronal activity early in development is also closely related to Cl-homeostasis, which is developmentally regulated by KCC2, the main K+ Cl- co-transporter in the brain (Rivera et al., 1999). Because neuronal expression of KCC2 is low during early development, the intracellular [Cl-] cannot be extruded leading to the depolarizing effect of GABA during this period (Ben-Ari et al., 2004). Taking these under consideration, it was demonstrated that the effects of BDNF on neuronal activity was mediated by the KCC2 regulation, as observed in several in vitro and in vivo studies (Ludwig et al., 2011a and 2011b; Yeo et al., 2009; Aguado et al., 2003; Carmona et al., 2006).

In support to this KER, recent studies have demonstrated that injured hippocampal neurons can survive and be regenerated through the same mechanism (Shulga et al., 2013). Indeed, after mature nerve injury, KCC2 is down-regulated and the GABA responses switch to depolarization, in a way similar to the early developmental stages. The rescue and re-generation of these neurons requires the switch of GABA from depolarization to hyperpolarization, a process driven by BDNF and the subsequent KCC2 up-regulation in hippocampal neurons (Shulga et al., 2009) during brain development.

It is widely accepted that BDNF expression is regulated by TH (Koibuchi et al., 1999; 2001; Chakraborty et al., 2012). Deregulation of BDNF signaling has been shown to decrease cortical GABA interneuron markers (Kelsom and Lu, 2013; Fiumelli et al., 2000; Arenas et al., 1996; Jones et al., 1994).

- Westerholz et al., 2013 In recent in vitro studies in rat T3-deficient cultures of cortical PV+ interneurons, it was shown that the number of synaptic boutons was reduced, an effect that was abolished after exogenous BDNF application. Additionally, inhibition of BDNF by K252a (a TrK antagonist) in cultures containing T3 resulted also in decreased number of synaptic boutons, as in the T3-deprived cultures. These results suggest that BDNF signaling promotes the formation of synaptic boutons and that this function is mediated by TH (T3 and T4).

- Chen et al., 2016 BDNF-Val66Met knock-in mice (BDNF^Met/Met) are known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. This study showed that GABAergic innervations of pyramidal neurons of BDNF^Met/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, compared to wild type mice.

- Kong et al., 2014 This study showed that chronic seizure rats 6 months after treatment with cyclothiazide (CTZ, a seizure inducer), underwent decrease of both GAD (from 75.2 ± 13.0 in CA1, 79.7 ± 9.7 in CA3, and 251.5 ± 4.3 in DG, respectively, to 3.0 ± 0.5 in CA1, 3.6 ± 0.9 in CA3, and 5.3 ± 1.8 in DG) and GAT-1 (from 60.7 ± 3.0 in CA1, 55.7 ± 9.1 in CA3, and 212.3 ± 11.3 in DG, respectively, to 20.7 ± 8.6 in CA1, 24.3 ± 3.4 in CA3, and 24.7 ± 13.3 in DG) across CA1, CA3, and dentate gyrus area of the hippocampus. Also, hippocampal decrease of both BDNF⁺ cells (from 70.7 ± 9.0 in CA1, 72.2 ± 3,7 in CA3, and 138.3 ± 15.9 in DG, respectively, to 4.1 ± 1.0 in CA1, 2.9 ± 0.1 in CA3, and 21.2 ± 16.2 in DG) and TrkB⁺ (BDNF receptor) cells (from 126.7 ± 7.2 in CA1, 275.7 ± 56.3 in CA3, and 399.2 ± 22.4 in DG, respectively, to 64.7 ± 16.2 in CA1, 158.3 ± 41.7 in CA3, and 250.3 ± 46.8 in DG) was observed.

- Aguado et al., 2003 BDNF overexpression in transgenic embryos raised the spontaneous activity of E18 hippocampal neurons, as shown by increased number of synapses (63% more synapses in the hippocampus of BDNF transgenic embryos than in controls), and increased spontaneous neuronal activity (2.3 times more active neurons than wild type embryos, and 36.3% greater rates of activation). Moreover, BDNF transgenic embryos had higher number of GABAergic interneuron synapses, as shown by higher GAD67 mRNA (by 3-fold) and K(+)/Cl(-) KCC2 mRNA expression (by 4.3-fold) (responsible for the conversion of GABA responses from depolarizing to inhibitory), without altering the expression of GABA and glutamate ionotropic receptors. These data indicate that BDNF controls both GABAergic pre- and postsynaptic sites.

The role of BDNF on differentiation and maturation of GABAergic interneurons is supported by the studies described in Weight of Evidence section. However, in a recent publication (Puskarjov et al., 2015) BDNF-/- mice were utilized to show that in the absence of BDNF the seizure-induced up regulation of KCC2 was eliminated, but interestingly no change in early (P5-6) or later (P13-14) postnatal KCC2 expression was observed compared to the wild type littermates, but neither the functionality of KCC2 protein was investigated, nor the ability of the neurons to extrude Cl^- in the absence of BDNF.

Additionally, other studies have shown that the up-regulation of KCC2 via the transcription factor Egr4 is also regulated by a different neurotrophic factor, neurturin (Ludwig et al., 2011b). These results reveal that the same transcriptional pathways, such as KCC2, can be activated by different neurotrophic factors and might lead to the same outcome under different conditions. This hypothesis should be further investigated, as it could explain the compensation mechanisms that are activated in the total absence of BDNF, and which might be different from those that are triggered by a decrease of BDNF levels.

There is a lack of quantitative studies linking brain BDNF levels (gene and/or protein) and the amount of GABAergic interneurons, resulting in changes of their morphology and function, therefore no robust quantitative information can be provided.

References

Aguado F, Carmona MA, Pozas E, Aguiló A, Martínez-Guijarro FJ, Alcantara S, Borrell V, Yuste R, Ibañez CF, SorianoE. (2003). BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl–co-transporter KCC2. Development 130:1267-1280.

Arenas E, Akerud P, Wong V, Boylan C, Persson H, Lindsay RM, Altar CA. (1996). Effects of BDNF and NT-4/5 on striatonigral neuropeptides or nigral GABA neurons in vivo. Eur J Neurosci. 8(8):1707–1717.

Ben-Ari Y, Khalilov I, Represa A, Gozlan H. (2004). Interneurons set the tune of developing networks. Trends Neurosci 27: 422–427.

Berghuis P, Dobszay MB, Sousa KM, Schulte G, Mager PP, Hartig W, Gorcs TJ, Zilberter Y, Ernfors P, Harkany T. (2004). Brain derived neurotrophic factor controls functional differentiation and microcircuit formation of selectively isolated fast-spiking GABAergic interneurons. Eur J Neurosci 20:1290–1306.

Blaesse P, Airaksinen MS, Rivera C, Kaila K. (2009). Cation chloride co-transporters and neuronal function. Neuron 61:820–838

Carmona MA, Pozas E, Martínez A, Espinosa-Parrilla JF, Soriano E, Aguado F. (2006). Age-dependent spontaneous hyperexcitability and impairment of GABAergic function in the hippocampus of mice lacking trkB. Cereb Cortex 16:47– 63.

Cellerino A, Maffei L, Domenici L. (1996). The distribution of brain-derived neurotrophic factor and its receptor trkB in parvalbumin-containing neurons of the rat visual cortex. Eur J Neurosci 8:1190–1197.

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Chen YW, Surgent O, Rana BS, Lee F, Aoki C. (2016). Variant BDNF-Val66Met Polymorphism is Associated with Layer-Specific Alterations in GABAergic Innervation of Pyramidal Neurons, Elevated Anxiety and Reduced Vulnerability of Adolescent Male Mice to Activity-Based Anorexia. Cereb Cortex. Aug 30.

Cherubini E, Griguoli M, Safiulina V, Lagostena L. (2011). The depolarizing action of GABA controls early network activity in the developing hippocampus. Mol Neurobiol. 43:97-106.

Danglot L, Triller A, Marty S. (2006). The development of hippocampal interneurons in rodents. Hippocampus. 16:1032-1060.

Feller MB. (1999). Spontaneous correlated activity in developing neural circuits. Neuron 22: 653-656.

Fiumelli H, Kiraly M, Ambrus A, Magistretti PJ, Martin JL. (2000). Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons. J Neurochem. 74(5):1870–1877.

Gorba T, Wahle P. (1999). Expression of TrkB and TrkC but not BDNF mRNA in neurochemically identified interneurons in rat visual cortex in vivo and in organotypic cultures. Eur J Neurosci 11:1179–90.

Hu Y, Russek SJ. (2008). BDNF and the diseased nervous system: a delicate balance between adaptive and pathological processes of gene regulation. J Neurochem. 105:1-17.

Huang ZJ. (2009). Activity-dependent development of inhibitory synapses and innervation pattern: roleof GABA signalling and beyond. J.Physiol. 587: 1881–1888.

Huang ZJ, DiCristo G, Ango F. (2007). Development of GABA innervation in the cerebral and cerebellar cortices. Nat.Rev.Neurosci. 8: 673–686.

Jin X, Hu H, Mathers PH, Agmon A. (2003). Brain-derived neurotrophic factor mediates activity-dependent dendritic growth in nonpyramidal neocortical interneurons in developing organotypic cultures. J Neurosci 23:5662–5673.

Jones KR, Farinas I, Backus C, Reichardt LF. (1994). Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cell. 76(6):989–999.

Kafitz KW, Rose CR, Thoenen H, Konnerth A. (1999). Neurotrophin-evoked rapid excitation through trkB receptors. Nature 401:918–921.

Kelsom C, Lu W. (2013). Development and specification of GABAergic cortical interneurons. Cell Biosci. Apr 23;3(1):19.

Kohara K, Kitamura A, Adachi N, Nishida M, Itami C, Nakamura S, et al. (2003). Inhibitory but not excitatory cortical neurons require presynaptic brain-derived neurotrophic factor for dendritic development, as revealed by chimera cell culture. J Neurosci 23: 6123–6131.

Koibuchi N, Yamaoka S, Chin WW. (2001). Effect of altered thyroid status on neurotrophin gene expression during postnatal development of the mouse cerebellum. Thyroid 11:205–210.

Koibuchi N, Fukuda H, Chin WW. (1999). Promoter-specific regulation of the brain-derived neurotrophic factor gene by thyroid hormone in the developing rat cerebellum. Endocrinology 140: 3955–3961.

Kong S, Cheng Z, Liu J, Wang Y. (2014). Downregulated GABA and BDNF-TrkB pathway in chronic cyclothiazide seizure model. Neural Plast. 2014:310146.

Ludwig A, Uvarov P, Soni S, Thomas-Crusells J, Airaksinen MS, Rivera C. (2011a). Early growth response 4 mediates BDNF induction of potassium chloride co-transporter 2 transcription. J Neurosci 31:644-649.

Ludwig A, Uvarov P, Pellegrino C, Thomas-Crusells J, Schuchmann S, Saarma M, Airaksinen MS, Rivera C. (2011b). Neurturin evokes MAPK dependent up-regulation of Egr4 and KCC2 in developing neurons. Neural Plast 1-8.

Marty S, Berninger B, Carroll P, Thoenen H. (1996). GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor. Neuron 16:565–570.

Marty S, Wehrle R, Sotelo C. (2000). Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus. J Neurosci 20: 8087–8095.

O’Donovan MJ. (1999). The origin of spontaneous activity in developing networks of the vertebrate nervous system. Curr Opin Neurobiol 9:94–104.

Palizvan MR, Sohya K, Kohara K, Maruyama A, Yasuda H, Kimura F, et al. (2004). Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex. Neurosci 126: 955–966.

Patz S, Grabert J, Gorba T, Wirth MJ, Wahle P. (2004). Parvalbumin expression in visual cortical interneurons depends on neuronal activity and TrkB ligands during an early period of postnatal development. Cereb Cortex 14:342–51.

Polleux F, Whitford KL, Dijkhuizen PA, Vitalis T, Ghosh A. (2002). Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling. Development 129:3147–60.

Puskarjov M, Ahmad F, Khirug S, Sivakumaran S, Kaila K. (2015). BDNF is required for seizure-induced but not developmental up-regulation of KCC2 in the neonatal hippocampus. Neuropharmacology. Jan;88:103-9.

Rico B, Xu B, Reichardt LF. (2002). TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum. Nat Neurosci 5:225–233.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K. (1999). The K-/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255.

Seil FJ, Drake-Baumann R. (2000). TrkB receptor ligands promote activity-dependent inhibitory synaptogenesis. J Neurosci 20: 5367–73.

Shulga A, Blaesse A, Kysenius K, Huttunen HJ, Tanhuanpää K, Saarma M, Rivera C. (2009). Thyroxin regulates BDNF expression to promote survival of injured neurons. Mol Cell Neurosci. 42:408-418.

Shulga A, Rivera C. (2013). Interplay between thyroxin, BDNF and GABA in injured neurons. Neurosci. 239: 241-252.

Southwell DG, Nicholas CR, Basbaum AI, Stryker MP, Kriegstein AR, Rubenstein JL, Alvarez-Buylla A. (2014). Interneurons from embryonic development to cell-based therapy. Science. 44:1240622.

Vicario-Abejon C, Collin C, McKay RD, Segal M. (1998). Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons. J Neurosci 18:7256–71.

Voigt T, Opitz T, De Lima AD. (2001). Synchronous oscillatory activity in immature cortical network is driven by GABAergic preplate neurons. J Neurosci 21: 8895–8905.

Wang DD, Kriegstein AR. (2009). Defining the role of GABA in cortical development. J Physiol. 587:1873-1879.

Wake, H., Watanabe, M., Moorhouse, A.J., Kanematsu, T., Horibe, S., Matsukawa, N., Asai, K., Ojika, K., Hirata, M. & Nabekura, J. (2007). Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation. J. Neurosci., 27, 1642–1650.

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Woo NH, Lu B. (2006). Regulation of Cortical Interneurons by neurotrophins: from development to cognitive disorders. Neuroscientist. 12: 43-56.

Yamada MK, Nakanishi K, Ohba S, Nakamura T, Ikegaya Y, Nishiyama N, et al. (2002). Brain-derived neurotrophic factor promotes the maturation of GABAergic mechanisms in cultured hippocampal neurons. J Neurosci 22:7580–5.

Yeo M, Berglund K, Augustine G, Liedtke W. (2009). Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride. J Neurosci 29:14652–14662.

GABAergic interneurons, Decreased leads to Synaptogenesis, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Most of the available studies have been performed in rodent models and human cortical neurons, referenced in the "Biological plausibility" section.

The relationship between KCC2 function and GABA signalling has been also demonstrated in the retinotectal circuit of Xenopus (Akerman and Cline, 2006).

How Does This Key Event Relationship Work

Early in cortical development, the GABAergic interneurons have been found to contribute to key aspects of the brain development. A precise balance between excitatory and inhibitory synapses in cortical neurons is crucial for the formation and maturation of the neuronal connections and eventually the proper neural circuitry function. In the cerebral cortex, the young neurons first receive GABAergic depolarizing inputs before forming any synapses (Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002), and thus the GABAergic system is believed to be the initial regulator of synaptogenesis.  Indeed, initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006). Nascent GABAergic synapses contain both presynaptic and postsynaptic elements, and produce synaptic transmission (Ahmari and Smith, 2002). GABAA receptors form clusters before presynaptic terminals emerge (Scotti and Reuter, 2001), and this clustering occur in the absence of scaffolding proteins and GABA release (Scotti and Reuter, 2001; Christie et al., 2002). Also, during maturation, GABAA receptors become selectively clustered across from terminals that release the neurotransmitter GABA (Craig et al., 1994; Swanwick et al., 2006).

Weight of Evidence

Early in the development of the neocortex, GABAergic interneurons play a role in the formation of spontaneous synchronized activity, which has a fundamental role in the activation of glutamatergic synapses, the synchronization of synaptogenesis and the establishment of long–range cortico-cortical connections (Voigt et al., 2001; 2005). Increasing evidence suggests that GABAergic signaling is the main regulator of this early neuronal activity, as it is established before the glutamatergic one in the neocortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006). Despite the fact that GABA is the main inhibitory neurotransmitter in the adult CNS, it exerts depolarizing actions in the immature brain (Ben-Ari et al., 2007), caused by the low levels of Cl^- concentration in the post-synaptic cells (Rivera et al., 1999; Ehrlich et al., 1999). K-Cl co-transporter 2 (KCC2) is the main Cl^- efflux mechanism with a developmentally-regulated expression profile in the brain and it is therefore thought to be the regulator of GABA signalling during early neuronal development. The effects of KCC2 on the levels of [Cl-]I in immature neurons and the subsequent effects on the shift of the GABA signaling has been extensively studied during the last decades:

• Existing data indicate that KCC2 expressed by GABA neurons is sufficient to shift from the depolarizing and excitatory period of GABA during cortical neuron development (Lee et al., 2005; Chudotvorova et al., 2005) and to effectively decrease the [Cl-]I in immature rat neurons (Chudotvorova et al., 2005).

• Transcriptional repression of KCC2 in rat cortical neurons delayed the GABA switch corresponding to significant changes of [Cl-]I in the same neurons (Yeo et al., 2009).

Several studies focused on the effects of GABA signaling on synaptogenesis and they all had convergent results leading to a strong biological plausibility of this KER.

• Too early shift of GABA-induced excitation-to-inhibition not only affects synaptic integration, but it also results in deficient circuitry development (Wang and Kriegstein, 2008). This has been demonstrated in rodents and mammals cortical neurons in culture.

• Premature GABA switch has also morphological effects in cortical neurons, as it has been shown to drive in fewer and shorter dendrites with defective effects in synaptic formation (Cancedda et al., 2007).

• In the dentate gyrus of the adult hippocampus, newborn granule cells are tonically activated by ambient GABA before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic Cl^- ion content (Ge et al., 2006).

• An early hyperpolarizing shift in Cl⁻ reversal potential, by premature expression of KCC2, has been shown to increase the ratio of inhibitory-to-excitatory inputs both in Xenopus tectal neurons and rat cortical neurons in vitro (Chudotvorova et al., 2005; Akerman and Cline, 2006).

The mechanistic details of this relationship are not entirely known, but the most possible mechanism entails a functional relationship between GABA and NMDA receptor activation (Wang and Kriegstein, 2008; Cserép et al., 2012). Cortical neurons begin to express functional NMDA receptors when they migrate to the cortical plate, but these initial glutamatergic synapses are “silent” because of the Mg²⁺ block of NMDA receptors at the resting membrane potential (LoTurco et al., 1991; Akerman and Cline, 2006). GABAergic depolarization can facilitate relief of this voltage-dependent Mg²⁺ block and allow Ca²⁺ entry to initiate intracellular signalling cascades (Leinekugel et al., 1997). This mechanism suggests that the initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009). 

The correlation between the GABA function and synaptogenesis has been mainly studied through the developmental modifications of intracellular Cl^- gradient and the subsequent GABA switch. In all available cases, this is performed by disturbing KCC2 or NKCC1 expression with genetic or mechanical manipulations of the neuronal models.

The temporal concordance of GABA shift and synaptogenesis is extensively reviewed by Ben-Ari et al., 2007 and 2012. It is widely accepted that the first spontaneous synaptic activity in the cortex is driven by the GABA-mediated depolarization and it is necessary for the subsequent synapse formation in the brain. Furthermore, the absence of T3 in cultures of cortical GABAergic interneurons can delay the typical developmental KCC2 up-regulation and subsequently the GABA shift, with a profound decrease in the number of synapses (Westerholz et al., 2010; 2013).

- Westerholz et al., 2013 This study showed that in rat T3-deficient cultures of cortical GABAergic PV⁺ interneurons, the number of synaptic boutons (presynaptic terminals containing the presynaptic marker synaptophysin) was reduced, an effect that was abolished after exogenous BDNF application.

- López-Espíndola et al., 2014: Human brain sections from MCT8-deficient subjects (30^th gestational week male fetus and an 11-year-old boy) were studied in comparison with relevant healthy control brain tissues. The MCT8-deficient fetal cerebral cortex showed 50% reduction of TH (i.e., T4, T3, and rT3), while T3 and T4 levels were normal in the liver. This TH deficiency in the brain produced an expected increase in type 2 deiodinase and decrease in type 3 deiodinase mRNA expression. Also, MCT8-deficient fetus showed a delay in cortical and cerebellar development and myelination, loss of parvalbumin (marker of GABAergic interneurons) expression, abnormal calbindin-D28k content, impaired axonal maturation (impaired synaptogenesis), and diminished biochemical differentiation of Purkinje cells. The 11-year-old boy displayed altered cerebellar structure, deficient myelination, deficient synaptophysin (presynaptic marker of synapse) and parvalbumin expression and abnormal calbindin-D28k expression.

Possible indirect KERs (not created due to limited evidence)

- Fisher et al., (2013), recently published a quantitative biologically-based dose-response model (BBDR) for iodine deficiency in the rat. In particular, HPT axis adaptations to dietary iodide intake in euthyroid (4.1-39 µg iodide/day) and iodide-deficient (0.31 and 1.2 µg iodide/day) conditions were evaluated. In rat pups that were iodide deficient during gestation and lactation, decreases in serum T4 levels were associated with declines in TH levels in the fetal brain. A 15% reduction in cortical T4 in the fetal brain was sufficient to induce permanent reductions in synaptic function in adults, confirming that even modest developmental TH disruption can cause synaptic dysfunctions also in hippocampal region (critical for learning and memory) of the adult brain (Gilbert et al., 2013). These data support indirect KER between decrease TH level in the brain and decreased synaptogenesis.

In regards to toxicological studies, bisphenol A (BPA), an environmental toxicant known to inhibit NIS-mediated iodide uptake (Wu Y et al., 2016), has also been found to delay and decrease both KCC2 expression and the developmental Cl^- shift (Yeo et al., 2013):

- Yeo et al., 2013 In primary rat cortical neurons and primary human cortical neurons (obtained from human fetal brain tissue specimens), 100 nM BPA caused decrease of KCC2 mRNA expression (≥ 25% decrease in rat cells at 4-5 DIV, ~ 70% decrease in human cells at 10 DIV) and attenuated [Cl⁻]_i shift in migrating cortical inhibitory precursor neurons. These data support indirect KER between NIS inhibition and GABAergic interneuron alteration.

These findings also concur with studies in other brain areas, such as the auditory brainstem and the hippocampus (Friauf et al., 2008; Hadjab-Lallemend et al., 2010), supporting correlation between KCC2 expression and GABA presence in the brain, and their implication in synaptogenesis.

In vivo evidence for the role of GABA in synaptogenesis is controversial. Ji et al., 1999 have shown that in GAD65/67-deficient mice, in which the production of GABA was reduced to less than 5%, the development of brain morphology until birth was normal. These mice die at birth and therefore synaptogenesis and circuit development could not be controlled, however no morphological defects were detected in the neocortex, cerebellum and hippocampus of these animals by the time of their death. The authors of this study suggested that GABA may not be crucial for development. However, functional changes were not assessed in this study. One hypothesis is that glutamate, glycine and taurine could compensate for the lack of GABA (LoTurco et al., 1995; Flint et al., 1998).

In KCC2 knock out mice, apart from lung atelectasis, no other obvious histological changes in the brain were observed in neonatal mice (Hubner et al., 2001). Moreover, these mice died at birth, before the GABA switch takes place, and neuronal electrical activity or synaptogenesis were not evaluated.

Additionally, after premature expression of KCC2 transporter an increase of the excitatory synapses was observed, but the glutamatergic synapses were not affected (Chudotvorova et al., 2005), as in the case of NKCC1 knock out mice (Wang and Kriegstein, 2008). These contradictory results reveal the complexity of the developmental brain and suggest that many different mechanisms are involved in the regulation of the temporal profile of the two main neuronal co-transporters, namely the KNCC1 and KCC2. However, in all cases the importance of Cl^- homeostasis in the developmental cortex and its correlation with the proper synapse formation is demonstrated.

There is a lack of studies directly linking alteration of GABAergic interneurons (morphology and function) with quantitative analyses of synaptogenesis modifications, and therefore no robust quantitative information can be provided.

References

Ahmari SE, Smith SJ. (2002). Knowing a nascent synapse when you see it. Neuron. 34:333–336.

Akerman CJ, Cline HT. (2006). Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci 26: 5117–5130.

Ben-Ari Y. (2006). Basic developmental rules and their implications for epilepsy in the immature brain. Epileptic Disord. Jun; 8(2):91-102.

Ben Ari Y, Gaiarsa JL, Tyzio R, Khazipov R. (2007). GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev 87:1215–84.

Ben-Ari Y, Khalilov I, Kahle KT, Cherubini E. (2012). The GABA excitatory/inhibitory shift in brain maturation and neurological disorders. Neuroscientist. 18(5):467-486.

Cancedda L, Fiumelli H, Chen K, Poo MM. (2007). Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo. J Neurosci 27: 5224–5235.

Chudotvorova I, Ivanov A, Rama S, Hubner CA, Pellegrino C, Ben-Ari Y, Medina I. (2005). Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses. J Physiol 566: 671–679.

Christie SB, Miralles CP, De Blas AL. GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons. J Neurosci. 2002;22:684–697.

Craig AM, Blackstone CD, Huganir RL, Banker G. Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc Natl Acad Sci U S A. 1994;91:12373–12377.

Cserép C, Szabadits E, Szőnyi A, Watanabe M, Freund TF, Nyiri G. (2012). NMDA receptors in GABAergic synapses during postnatal development. PLoS One. 7(5):e37753.

Ehrlich I, Lohrke S, Friauf E. (1999). Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation. J Physiol. 1:121-137.

Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME. (2013). Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 132(1):75-86.

Flint AC, Liu X, Kriegstein AR. (1998). Nonsynaptic glycine receptor activation during early neocortical development. Neuron 20: 43–53.

Friauf E, Wenz M, Oberhofer M, Nothwang HG, Balakrishnan V, Knipper M, Löhrke S. (2008). Hypothyroidism impairs chloride homeostasis and onset οφ inhibitory neurotransmission in developing auditory brainstem and hippocampal neurons. Eur J Neurosci 28:2371-2380.

Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H. (2006). GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 43: 589–593.

Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW (2013) An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195.

Hadjab-Lallemend S, Wallis K, van Hogerlinden M, Dudazy S, Nordström K, Vennström B, Fisahn A. (2010). A mutant thyroid hormone receptor alpha1 alters hippocampal circuitry and reduces seizure susceptibility in mice. Neuropharmacol 58(7):1130-9.

Hennou S, Khalilov I, Diabira D, Ben-Ari Y, Gozlan H. (2002). Early sequential formation of functional GABAA and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus. Eur J Neurosci 16: 197–208.

Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ. (2001). Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron. 30:515-524.

Ji F, Kanbara N, Obata K. (1999). GABA and histogenesis in fetal and neonatal mouse brain lacking both the isoforms of glutamic acid decarboxylase. Neurosci Res 33: 187–194.

Lee H, Chen CX, Liu YJ, Aizenman E, Kandler K. (2005). KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses. Eur J Neurosci. 21: 2593-2599.

Leinekugel X, Medina I, Khalilov I, Ben-Ari Y, Khazipov R. (1997). Ca2+ oscillations mediated by the synergistic excitatory actions of GABAA and NMDA receptors in the neonatal hippocampus. Neuron 18: 243–255.

López-Espíndola D, Morales-Bastos C, Grijota-Martínez C, Liao XH, Lev D, Sugo E, Verge CF, Refetoff S, Bernal J, Guadaño-Ferraz A. (2014). Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. Dec;99(12):E2799-804.

LoTurco JJ, Blanton MG. Kriegstein AR (1991). Initial expression and endogenous activation of NMDA channels in early neocortical development. J Neurosci 11: 792–799.

LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR. (1995). GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15: 1287–1298.

Owens DF, Liu X, Kriegstein AR. (1999). Changing properties of GABAA receptor-mediated signalling during early neocortical development. J Neurophysiol 82: 570–583.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K. (1999). The K/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251–255.

Scotti AL, Reuter H. Synaptic and extrasynaptic gamma -aminobutyric acid type A receptor clusters in rat hippocampal cultures during development. Proc Natl Acad Sci U S A. 2001;98:3489–3494.

Swanwick CC, Murthy NR, Mtchedlishvili Z, Sieghart W, Kapur J. Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons. J Comp Neurol. 2006 Apr 10;495(5):497-510

Tyzio R, Represa A, Jorquera I, Ben-Ari Y, Gozlan H, Aniksztejn L. (1999). The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite. J Neurosci 19: 10372–10382.

Voigt T, Opitz T, De Lima AD. (2001). Synchronous oscillatory activity in immature cortical network is driven by GABAergic preplate neurons. J Neurosci 21: 8895–8905.

Voigt T,Opitz T, deLima AD. (2005). Activation of early silent synapses by spontaneous synchronous network activity limits the range of neocortical connections. J. Neurosci. 25: 4605–4615.

Wang DD, Kriegstein AR. (2008). GABA regulates excitatory synapse formation in the neocortex via NMDA receptor activation. J Neurosci 28: 5547–5558.

Wang DD, Kriegstein AR. (2009). Defining the role of GABA in cortical development. J Physiol. 587:1873-1879.

Westerholz S, de Lima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience 168: 573–589.

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Yeo M, Berglund K, Augustine G, Liedtke W. (2009). Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride. J Neurosci 29:14652–14662.

Yeo M, Berglund K, Hanna M, Guo JU, Kittur J, Torres MD, Abramowitz J, Busciglio J, Gao Y, Birnbaumer L, Liedtke WB. (2013). Bisphenol A delays the perinatal chloride shift in cortical neurons by epigenetic effects on the Kcc2 promoter. Proc Natl Acad Sci U S A. 110(11):4315-20.

Synaptogenesis, Decreased leads to Neuronal network function, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The main proof of evidence comes from in vivo studies in rodents. However, Colón-Ramos (2009) has recently reviewed the early developmental events that take place during the process of synaptogenesis in invertebrates, pointing out the importance of this process in neural network formation and function. The experimental findings reviewed in this paper derive from knowledge acquired in the field of neuroscience using C. elegans and Drosophila; at the same time, emerging findings derived from vertebrates are also discussed (Colón-Ramos, 2009).

How Does This Key Event Relationship Work

The ability of a neuron to communicate is based on neural network formation that relies on functional synapse establishment (Colón-Ramos, 2009). The main roles of synapses are the regulation of intercellular communication in the nervous system, and the information flow within neural networks. The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation during the process of synaptogenesis is critical and leads to decrease of neural network formation and function in the adult brain.

Synaptic transmission and plasticity require the integrity of the anatomical substrate. The connectivity of axons emanating from one set of cells to post-synaptic side of synapse on the dendrites of the receiving cells must be intact for effective communication between neurons. Changes in the placement of cells within the network due to delays in neuronal migration, the absence of a full formation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will individually and cumulatively impair synaptic function. These anatomical alterations are responsible for many structural anomalies reported in various regions of the brain following severe developmental hypothyroidism. Although the primary evidence of synaptic transmission impairments in hypothyroid models have come from studying the hippocampus, it is assumed that the role thyroid hormones play in these processes is likely similar across different brain regions. Altered hippocampal structure induced by decreased TH levels impacts neurogenesis in the developing hippocampus or cortex, contributing to deficits in synaptic function.

Weight of Evidence

The weight of evidence supporting the relationship between decreased synaptogenesis induced by TH insufficiency and altered neuronal network and synaptic function is moderate. Functional change as exemplified by alterations in synaptic transmission may be more easily detected than structural abnormalities. The exact alignment between the neuroanatomical effects (such as decreased synaptogenesis and alteration of GABAergic interneurons) that have been associated with developmental hypothyroidism (e.g., elicited by exposing rat dams to TPO inhibitors) and the neurophysiological impairments has not been entirely elucidated.

Neuronal network formation and function are established via the process of synaptogenesis. The developmental period of synaptogenesis is critical for the formation of the basic circuitry of the nervous system, although neurons are able to form new synapses throughout life (Rodier, 1995). The brain electrical activity dependence on synapse formation is critical for proper neuronal communication.

Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD-95 arrests the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).

The biological plausibility of the known effects of TH insufficiency on brain structure having an impact on synaptic function and plasticity in brain is strong. Reductions in myelination of axons, cell number, dendritic arborization, and synaptogenesis have been described in models of severe hormone deprivation, as comprehensively summarized by Thompson and Potter, 2000. Because synaptic transmission relies on the integrity of synaptic contacts and the electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference with process of synapse formation (morphological unit of neuronal network) will very much impact the neural network function.

Most of the information on developmental hypothyroidism and altered synaptic function has been derived from studies of the hippocampus. It is presumed that structural changes of synaptic connectivity at certain level may lead to functional deficits in synaptic transmission and plasticity impairments (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005), but the precise structural aberration is not known. Within the hippocampus, area CA1 has been investigated primarily with in vitro techniques, using slices of hippocampus from animals exposed to TPO inhibitors (MMI or PTU) and measuring synaptic function across CA1-pyramidal cell synapses (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Taylor et al., 2008). Pyramidal neurons of hypothyroid animals have fewer synapses and an impoverished dendritic arbor (Rami et al., 1986a, Madeira et al., 1992), with reductions ranging between 14.2 and 22.5% as observed in 30 and 180-day-old hypothyroid rats (as described by Madeira et al., 1992).

The other major region in hippocampus investigated in hypothyroid models is the perforant path-dentate gyrus synapse (Gilbert, 2011). Granule cells are the principal cell type of the dentate gyrus region of the hippocampal formation and receive input from cortical neurons in the entorhinal cortex. TPO inhibitors like PTU and MMI decrease the volume of the granule cell layer, the density of cells within the layer, and estimates of total granule cell number (Madeira et al., 1991). Migration of granule cells from the proliferative zone to the granule cell layer is retarded by thyroid deficiency as is dendritic arborization and synaptogenesis assessed by immunohistochemistry for the synaptic protein, synaptophysin (Rami et al., 1986b, Rami and Rabie, 1990, Dong et al., 2005). Impairments in synaptic function from both rodent and human studies are summarized below.

Excitatory and inhibitory synaptic transmission is reduced in CA region of hippocampus in animals with TH insufficiencies in early life (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005). Similarly, excitatory and inhibitory synaptic transmission is reduced in the CA1 and dentate gyrus regions of the hippocampus (Gilbert and Paczkowski, 2003, Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2013) under decreased TH levels. Parvalbumin (PV) is a calcium binding protein expressed exclusively in GABA inhibitory neurons of the hippocampus. Impairments in inhibitory synaptic transmission are associated with reductions in the number of PV⁺ cells (Gilbert et al., 2007).

- Vara et al., 2002 This study assessed effects of TH on short-term synaptic plasticity (associated with short-term memory) in control vs hypothyroid rats. Specifically, dams were treated with 0.02% methylmercaptoimidazole (MMI, TPO inhibitor) continually from GD9. A group of pups were also treated with T3 (i.p. daily injections of T3 (20 μg /100 g body weight)) starting 72 h before killing. Data showed that hypothyroid rats presented increase in the Ca(2+)-dependent neurotransmitter release, indicative of altered neuronal network function (i.e., decreased paired-pulse facilitation), and these alterations were reverted by T3 administration. These synaptic changes were determined by an increase of synapsin I and synaptotagmin I levels in the hypothyroid rats, suggesting that TH modulate neurotransmitter release. These results are in contrast with those of Di Liegro et al. (1995), who showed that in primary cultures T₃ induces the expression of synapsin I. This difference could be the result of the multiple differences between the two models: different brain regions (hippocampus vs. cortex), the age of the neurons (neonatal vs. embryonic), the preparations (slice vs. culture).

- Sui and Gilbert, 2003 Here developing rats were exposed in utero and postnatally to 0, 3, or 10 ppm propylthiouracil (PTU, TPO inhibitor), administered in the drinking water of dams from GD6 until PND30. Excitatory postsynaptic potentials and population spikes (indicative of neuronal network function) were recorded in area CA1 of hippocampal slices from offspring between PND21 and PND30. PTU caused at PND30 a decrease of maternal total T₄ (43.9% with 3-ppm, and 65.0% with 10-ppm) compared to controls, with no change in total T_3.  Maternal TSH was increased above control levels in a dose-dependent manner. In pups, total T₄ was depressed (by 75%) in both treated groups relative to the controls pups, and total T₃ was depressed (35.8% in the 3-ppm group and 66.5% in the 10-ppm group) relative to the controls. TH insufficiency was dose-dependently associated with a reduction of paired-pulse facilitation and long-term potentiation of the excitatory postsynaptic potential and elimination of paired-pulse depression of the population spike. Excitatory synaptic transmission was increased by developmental exposure to PTU. This suggests that TH insufficiency compromises synaptic communication in area CA1 of developing rat hippocampus (involved in learning and memory).

- Gilbert, 2004 Here developing rats were transiently exposed to PTU (0 or 15 ppm), through the drinking water of pregnant dams beginning on GD18 until PND21. This regimen markedly reduced circulating levels of TH in pups (T3: ∼50% lower than control at PND21; T4: T4 ∼40% below control levels). Analysis of field potentials in area CA1 of hippocampal slices derived from adult male offspring exposed to PTU showed a reduction of somatic population spike amplitudes, with no differences in excitatory postsynaptic potentials (EPSP).  Short-term plasticity of the EPSP (as indexed by paired pulse facilitation) was markedly decreased by PTU exposure. These data confirm associations between decrease of synaptic function in the hippocampus and decrease of neuronal network function as a consequence of TH insufficiency.

- Dong et al., 2005 Through gestation and lactation, iodine-deficient or hypothyroid dam rats were administered with either iodine-deficient diet or MMI (TPO inhibitor) added to drinking water. Exposure was terminated on PND30. Both treated groups showed lower concentrations of serum FT3 (~60% decrease on PND30) and FT4 (~80% decrease on PND30), smaller population spike amplitude (~50% decrease) and field-excitatory postsynaptic potential (f-EPSP, 50-60% decrease) slope induced by high-frequency stimulation (HFS). Also, TH insufficiency decreased the levels of c-fos (by ~60% at PND30 in MMI group) and c-jun proteins (by ~20% at PND30 in MMI group) in the hippocampus. C-fos and c-jun expression is regulated by synaptic activity, both play an important role in the neuroplastic mechanisms (synaptogenesis) critical to memory consolidation, and play an essential role in neuronal differentiation (Herdegen et al., 1997).

KEs proceeding the AO (learning and memory deficits), such as "Decreased synaptogenesis" (KEup) and "Decreased Neural Network Function" (KEdown) are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER (Decreased synaptogenesis leads to decreased neuronal network function in developing brain) described in the present AOP.

- Otto and Reiter, 1984: At low Pb²⁺ levels (less than 30 µg/dl), slow surface-positive cortical potentials have been observed in children under five years old, which may reflect axodendritic inhibitory processes, whilst negative slow potentials are observed in children over five years. This is due to the fact that during maturation of the cortex, the locus of inhibitory activity shifts deeper to axosomatic connections. However, age-related polarity reversal has been observed in children with higher Pb²⁺ levels.

- Kumar and Desiraju, 1992: In experiments carried out in Wistar rats that have been fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings show statistically significant reduction in the delta, theta, alpha and beta band of EEG spectral power in motor cortex and hippocampus with the exception of the delta and beta bands power of motor cortex in wakeful state.

- McCarren and Eccles, 1983: Male Sprague-Dawley rats have been exposed to Pb²⁺ from parturition to weaning though their dams' milk (dams received drinking water containing 1.0, 2.5, or 5.0 mg/ml lead acetate). Starting from 15 weeks of age, the characteristics of the electrically elicited hippocampal after discharge (AD) and its alteration by phenytoin (PHT) showed significant increase in primary AD duration only in the animals exposed to the higher dose of Pb²⁺, whereas all groups responded to PHT with increases in primary AD duration.

The exact mechanism by which a change in cell number, reduced dendritric arborization and synaptogenesis may lead to decreased neuronal network function has not been fully elucidated. Dose-dependent reductions in synaptic function in hippocampus have been demonstrated in models of moderate degrees of TH reduction, but studies of the anatomical integrity of the specific cell populations examined electrophysiologically have largely been evaluated in models of severe hypothyroidism and often in brain regions distinct from the hippocampus.

The exact mechanism by which a change in cell number, reduced dendritric arborization and synaptogenesis may lead to decreased neuronal network function has not been fully elucidated. Dose-dependent reductions in synaptic function in hippocampus have been demonstrated in models of moderate degrees of TH reduction, but studies of the anatomical integrity of the specific cell populations examined electrophysiologically have largely been evaluated in models of severe hypothyroidism and often in brain regions distinct from the hippocampus.

There are no data on the quantitative linkages between altered (hippocampal or cortical) synaptogenesis and impaired neuronal network function. Developmental window of exposure and duration of exposure can modulate response-response relationships.

References

Cline H, Haas K. (2008). The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: A review of the synaptotrophic hypothesis. J Physiol 586: 1509-1517.

Colón-Ramos DA. (2009). Synapse formation in developing neural circuits. Curr Top Dev Biol. 87: 53-79.

Di Liegro I, Savettieri G, Coppolino M, Scaturro M, Monte M, Nastasi T, Salemi G, Castiglia D, Cesterlli A (1995). Expression of synapsin I gene in primary cultures of differentiating rat cortical neurons. Neurochem. Res., 20, pp. 239–243

Dong J, Yin H, Liu W, Wang P, Jiang Y, Chen J. (2005). Congenital iodine deficiency and hypothyroidism impair LTP and decrease C-fos and C-jun expression in rat hippocampus. Neurotoxicology 26:417-426.

Ehrlich I, Klein M, Rumpel S, Malinow R. (2007). PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A. 104: 4176-4181.

Gilbert ME. (2004). Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev Brain Res 148:11-18.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW. (2013). An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195.

Gilbert ME, Paczkowski C. (2003). Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus. Brain Res Dev Brain Res 145:19-29.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Gilbert ME, Sui L. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH. (2007). Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology 148:92-102.

Herdegen T, Skene P, Bahr M (1997). The c-Jun transcription factor-bipotential mediator of neuronal death, survival and regeneration. Trends Neurosci, 20, pp. 227–231

Kumar MV, Desiraju T. (1992). EEG spectral power reduction and learning disability in rats exposed to lead through postnatal developing age. Indian J Physiol Pharmacol. 36: 15-20.

Madeira MD, Cadete-Leite A, Andrade JP, Paula-Barbosa MM. (1991). Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study. J Comp Neurol 314:171-186.

Madeira MD, Sousa N, Lima-Andrade MT, Calheiros F, Cadete-Leite A, Paula-Barbosa MM. (1992). Selective vulnerability of the hippocampal pyramidal neurons to hypothyroidism in male and female rats. J Comp Neurol 322:501-518.

McCarren M, Eccles CU. (1983). Neonatal lead exposure in rats: II. Effects on the hippocampal afterdischarge. Neurobehav Toxicol Teratol. 5: 533-540.

Otto D, Reiter L. (1984). Developmental changes in slow cortical potentials of young children with elevated body lead burden. Neurophysiological considerations. Ann N Y Acad Sci. 425: 377-383.

Rami A, Patel AJ, Rabie A. (1986a). Thyroid hormone and development of the rat hippocampus: morphological alterations in granule and pyramidal cells. Neuroscience 19:1217-1226.

Rami A, Rabie A, Patel AJ. (1986b). Thyroid hormone and development of the rat hippocampus: cell acquisition in the dentate gyrus. Neuroscience 19:1207-1216.

Rami A, Rabie A. (1990). Delayed synaptogenesis in the dentate gyrus of the thyroid-deficient developing rat. Dev Neurosci 12:398-405.

Rodier PM. (1995). Developing brain as a target of toxicity. Environ. Health Perspect. 103: 73-76.

Sui L, Anderson WL, Gilbert ME. (2005). Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampal area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci 85:647-656.

Sui L, Gilbert ME. (2003). Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology 144:4195-4203.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. (2008). Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Thompson CC, Potter GB. (2000). Thyroid hormone action in neural development. Cereb Cortex. Oct;10(10):939-45.

Vara H, Martinez B, Santos A, Colino A. (2002). Thyroid hormone regulates neurotransmitter release in neonatal rat hippocampus. Neuroscience 110:19-28.

Neuronal network function, Decreased leads to Impairment, Learning and memory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Synaptic transmission and plasticity are achieved via mechanisms common across taxonomies. LTP has been recorded in aplysia, lizards, turtles, birds, mice, guinea pigs, rabbits and rats. Deficiencies in hippocampally based learning and memory following developmental hypothyroidism have been documented mainly in rodents and humans.

How Does This Key Event Relationship Work

Learning and memory is one of the outcomes of the functional expression of neurons and neural networks from mammalian to invertebrates. Damage or destruction of neurons by chemical compounds during development when they are in the process of synapses formation, integration and formation of neural networks, will derange the organization and function of these networks, thereby setting the stage for subsequent impairment of learning and memory. Exposure to the potential developmental toxicants during neuronal differentiation and synaptogenesis will increase risk of functional neuronal network damage leading to learning and memory impairment.

Impairments in learning and memory are measured using behavioral techniques. It is well accepted that these alterations in behavior are the result of structural or functional changes in neurocircuitry. Functional impairments are often measured using field potentials of critical synaptic circuits in hippocampus and cortex. A number of studies have been performed in rodent models that reveal deficits in both excitatory and inhibitory synaptic transmission in the hippocampus as a result of developmental thyroid insufficiency (Wang et al., 2012; Oerbeck et al., 2003; Wheeler et al., 2011; Wheeler et al., 2015; Willoughby et al., 2014; Davenport and Dorcey, 1972; Tamasy et al., 1986; Akaike, 1991; Axelstad et al., 2008; Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011; Gilbert et al., 2016). A well-established functional readout of memory at the synaptic level is known as long-term potentiation (LTP) (i.e., a persistent strengthening of synapses based on recent patterns of activity). Deficiencies in LTP are generally regarded as potential substrates of learning and memory impairments. In rodent models where synaptic function is impaired by TH deficiencies, deficits in hippocampus-mediated memory are also prevalent (Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011; Gilbert et al., 2016).

Weight of Evidence

A number of studies have consistently reported alterations in synaptic transmission resulting from developmental TH disruption, and leading to decreased cognition.

Long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy (not always and not always high frequency stimulation leads to LTP), and its discovery suggested that changes in synaptic strength could provide the substrate for learning and memory (reviewed in Lynch, 2004). Moreover, LTP is intimately related to the theta rhythm, an oscillation long associated with learning. Learning-induced enhancement in neuronal excitability, a measurement of neural network function, has also been shown in hippocampal neurons following classical conditioning in several experimental approaches (reviewed in Saar and Barkai, 2003).

On the other hand, memory requires the increase in magnitude of EPSCs to be developed quickly and to be persistent for few weeks at least without disturbing already potentiated contacts. Once again, a substantial body of evidence has demonstrated that tight connection between LTP and diverse instances of memory exist (reviewed in Lynch, 2004).

A review on Morris water maze (MWM) as a tool to investigate spatial learning and memory in laboratory rats also pointed out that the disconnection between neuronal networks rather than the brain damage of certain regions is responsible for the impairment of MWM performance. Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and MWM performance (D'Hooge and De Deyn, 2001).

Moreover, it is well accepted that alterations in synaptic transmission and plasticity contribute to deficits in cognitive function. There are a number of studies that have linked exposure to TPO inhibitors (e.g., PTU, MMI), as well as iodine deficient diets, to changes in serum TH levels, which result in alterations in both synaptic function and cognitive behaviors (Akaike et al., 1991; Vara et al., 2002; Gilbert and Sui, 2006; Axelstad et al., 2008; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016), described in the indirect KER "Decrease of TH synthesis leads to learning and memory deficits".

Developmental hypothyroidism reduces the functional integrity in brain regions critical for learning and memory. Neurophysiological indices of synaptic transmission of excitatory and inhibitory circuitry are impaired in the hippocampus of hypothyroid animals. Both hippocampal regions (area CA1 and dentate gyrus) exhibit alterations in excitatory and inhibitory synaptic transmission following reductions in serum TH in the pre and early postnatal period (Vara et al., 2002; Sui and Gilbert, 2003; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). These alterations persist into adulthood despite a recovery to euthyroid conditions in blood. The latter observation indicates that these alterations represent permanent changes in brain function caused by transient hormones insufficiencies induced during critical window of development.  

Because the adult hippocampus is involved in learning and memory, it is a brain region of remarkable plasticity. Use-dependent synaptic plasticity is critical during brain development for synaptogenesis and fine tuning of synaptic connectivity. In the adult brain, similar plasticity mechanisms underlie use-dependency that underlies learning and memory, as exhibited in LTP model of synaptic memory. Hypothyroidism during development reduces the capacity for synaptic plasticity in juvenile and adult offspring (Vara et al., 2002; Sui and Gilbert, 2003; Dong et al., 2005; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). Decrease of neuronal network function and plasticity are observed coincident with deficits in learning tasks that require the hippocampus.

- Wang et al., 2012: This study showed that maternal subclinical hypothyroidism impairs spatial learning in the offspring, as well as the efficacy and optimal time of T4 treatment in pregnancy. Female adult Wistar rats were randomly divided into six groups: control, hypothyroid (H), subclinical hypothyroid (SCH) and SCH treated with T4, starting from GD10, GD13 and GD17, respectively, to restore normal TH levels. Results indicate that progenies of SCH and H groups demonstrated significantly longer mean latency in the water maze test (on the 2^nd training day, latency was ~83% higher in H group, and ~50% higher in SCH), and a lower amplification percentage of the amplitude (~15% lower in H group, and 12% lower in SCH), and slope of the field excitatory postsynaptic potential (fEPSP) recording (~20% lower in H group, and 17% lower in SCH), compared to control group. T4 treatment at GD10 and GD13 significantly shortened mean latency and increased the amplification percentage of the amplitude and slope of the fEPSPs of the progeny of rats with subclinical hypothyroidism. However, T4 treatment at GD17 showed only minimal effects on spatial learning in the offspring. Altogether these data indicate direct correlation between decrease of neural network function and learning and memory deficits.

- Liu et al., 2010 This study assessed the effects of hypothyroidism in 60 female rats who were divided into three groups: (i) maternal subclinical hypothyroidism (total thyroidectomy with T4 infusion), (ii) maternal hypothyroidism (total thyroidectomy without T4 infusion), and (iii) control (sham operated). The Morris water maze tests revealed that pups from the subclinical hypothyroidism group showed long-term memory deficits, and a trend toward short-term memory deficits.

- Gilbert and Sui, 2006 Administration of 3 or 10 ppm PTU to pregnant and lactating dams via the drinking water from GD6 until PND30 caused a 47% and 65% reduction in serum T4, in the dams of the low and high-dose groups, respectively. Baseline synaptic transmission was impaired in PTU-exposed animals: mean EPSP slope (by ~60% with 10 ppm PTU) and population spike amplitudes (by ~70% with 10 ppm PTU) in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams. High-dose animals (10 ppm) demonstrated very little evidence of learning despite 16 consecutive days of training (~5-fold higher mean latency to find the hidden platform, used as an index of learning).

- Gilbert et al., 2016 Exposure to PTU during development produced dose-dependent reductions in mRNA expression of nerve growth factor (Ngf) in whole hippocampus of neonates. These changes in basal expression persisted to adulthood despite the return to euthyroid conditions in blood. Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2^nd trial resulted ~60% higher in rats treated with 10 ppm PTU).

- Gilbert, 2011 Trace fear conditioning deficits to context and to cue reported in animals treated with PTU and who also displayed synaptic transmission and LTP deficits in hippocampus. Baseline synaptic transmission was impaired in PTU-exposed animals (by ~50% in animal treated with 3 ppm PTU). EPSP slope amplitudes in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams.

BPA, an environmental toxicant known to inhibit NIS-mediated iodide uptake (Wu Y et al., 2016) has been found to cause learning and memory deficits in rodents as described below:

- Jang et al., 2012 In this study, pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. Exposure of F0 mice to BPA (10 mg/kg) decreased hippocampal neurogenesis (~ 30% decrease of hippocampal BrdU⁺ cells vs control) in F2 female mice. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (NIS inhibitor) in pregnant mothers could decrease hippocampal neurogenesis (decreased number of neurons) and cognitive function in future generations.

In humans, the data linking these two specific KE are much more limited, but certainly clear reductions in IQ, with specific impairments in hippocampus-mediated functions have been observed.

- Wheeler et al., 2015 This study assessed hippocampal functioning in adolescents with congenital hypothyroidism (CH), using functional magnetic resonance imaging (fMRI). 14 adolescents with CH and 14 typically developing controls (TDC) were studied. Hippocampal activation was greater for pairs than items in both groups, but this difference was only significant in TDC. When the groups were directly compared, the right anterior hippocampus was the primary region in which the TDC and CH groups differed for this pair memory effect. Results signify that adolescents with CH show abnormal hippocampal functioning during verbal memory processing, in order to compensate for the effects induced by TH deficit in the brain.

- Wheeler et al., 2012 In this study hippocampal neuronal network function was measured based on synaptic performance using fMRI and was altered while subjects engaged in a memory task. Data showed paired word recognition deficits in adolescents with congenital hypothyroidism (N = 14; age range, 11.5-14.7 years) compared with controls (N = 15; age range, 11.2-15.5 years), with no impairment on simple word lists. Analysis of functional magnetic resonance imaging showed that adolescents with congenital hypothyroidism had both increased magnitude of hippocampal activation relative to controls and bilateral hippocampal activation when only the left was observed in controls. Furthermore, the increased activation in the congenital hypothyroidism group was correlated with the severity of the hypothyroidism experienced early in life.

- Willoughby et al., 2013 Analogously, in this study, fMRI revealed increased hippocampus activation with word pair recognition task in CH and children born to women with hypothyroxinemia during midgestation. These differences in functional activation were not seen with single word recognition, but were revealed when retention of word pair associations was probed. The latter is a task requiring engagement of the hippocampus.

A series of important findings suggest that the biochemical changes that happen after induction of LTP also occur during memory acquisition, showing temporality between the two KEs (reviewed in Lynch, 2004).

- Morris et al., 1986 This study found that blocking the NMDA receptor of the neuronal network with AP5 inhibits spatial learning in rats. Most importantly, in the same study they measured brain electrical activity and recorded that this agent also inhibits LTP, however, they have not proven that spatial learning and LTP inhibition are causally related.

Since then a number of NMDA receptor antagonists have been studied towards their ability to induce impairment of learning and memory. It is worth mentioning that similar findings have been found in human subjects:

- Grunwald et al., 1999 By combining behavioural and electrophysiological data from patients with temporal lobe epilepsy exposed to ketamine, involvement of NMDA receptors in human memory processes was demonstrated.

The last KE preceding the AO (learning and memory deficits), i.e. "Decreased Neural Network Function", is also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER described in the present AOP.

Pb2+: Exposure to low levels of Pb2+, during early development, has been implicated in long-lasting behavioural abnormalities and cognitive deficits in children (Needleman et al., 1975; Needleman and Gatsonis, 1990; Bellinger et al., 1991; 1992; Baghurst et al., 1992; Leviton et al., 1993; Needleman et al., 1996; Finkelstein et al., 1998; Lanphear et al., 2000; 2005; Canfield et al., 2003; Bellinger 2004; Lanphear et al., 2005; Surkan et al., 2007; Jusko et al., 2008; Neal and Guilarte, 2010) and experimental animals (Brockel and Cory-Slechta, 1998; Murphy and Regan, 1999; Moreira et al., 2001). Multiple lines of evidence suggest that Pb2+ can impair hippocampus-mediated learning in animal models (reviewed in Toscano and Guilarte, 2005).

- Jett et al., 1997 Female rats exposed to Pb²⁺ through gestation and lactation have shown more severe impairment of memory than male rats with similar Pb²⁺ exposures.

- De Souza Lisboa et al., 2005 This study reported that exposure to Pb²⁺ during both pregnancy and lactation caused depressive-like behaviour (detected in the forced swimming test) in female but not male rats.

- Anderson et al., 2012 This study investigated the neurobehavioral outcomes in Pb²⁺-exposed rats (250, 750 and 1500 ppm Pb²⁺ acetate in food) during gestation and through weaning and demonstrated that these outcomes are very much influenced by sex and rearing environment. In females, Pb²⁺ exposure lessened some of the benefits of enriched environment on learning, whereas, in males, enrichment does help to overcome detrimental effects of Pb²⁺ on learning. Regarding reference memory, environmental enrichment has not been beneficial in females when exposure to Pb²⁺ occurs, in contrast to males.

- Jaako-Movits et al., 2005 Wistar rat pups were exposed to 0.2% Pb²⁺ via their dams' drinking water from PND 1 to PND 21 and directly via drinking water from weaning until PND 30. At PND 60 and 80, the neurobehavioural assessment has revealed that developmental Pb²⁺ exposure induces persistent increase in the level of anxiety and inhibition of contextual fear conditioning. The same behavioural syndrome in rats has been described in Salinas and Huff, 2002.

- Finkelstein et al., 1998 These observations are in agreement with observations on humans, as children exposed to low levels of Pb²⁺ displayed attention deficit, increased emotional reactivity and impaired memory and learning.

- Kumar and Desiraju, 1992 In Wistar rats fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings showed statistically significant reduction in the delta, theta, alpha and beta band EEG spectral power in motor cortex and hippocampus, but not in delta and beta bands power of motor cortex in wakeful state. After 40 days of recovery, animals were assessed for their neurobehaviour, and revealed that Pb²⁺ treated animals showed more time and sessions in attaining criterion of learning than controls.

Further data obtained using animal behavioral techniques demonstrate that NMDA mediated synaptic transmission is decreased by Pb²⁺ exposure (Cory-Slechta, 1995; Cohn and Cory-Slechta, 1993 and 1994).

- Xiao et al., 2014 Rat pups from parents exposed to 2 mM PbCl₂ three weeks before mating until their weaning (pre-weaning Pb²⁺) and weaned pups exposed to 2 mM PbCl₂ for nine weeks (post-weaning Pb²⁺) were assessed for their spatial learning and memory by MWM on PND 85-90. The study revealed that both rat pups in pre-weaning Pb²⁺ and post-weaning Pb²⁺ groups performed significantly worse than those in the control group. The number of synapses in pre-weaning Pb²⁺ group increased significantly, but it was still less than that of control group. The number of synapses in post-weaning Pb²⁺ group was also less than that of control group, although the number of synapses had no differences between post-weaning Pb²⁺ and control groups before MWM. In both pre-weaning Pb²⁺ and post-weaning Pb²⁺ groups, synaptic structural parameters such as thickness of postsynaptic density (PSD), length of synaptic active zone and synaptic curvature increased, whereas width of synaptic cleft decreased compared to controls.

One of the most difficult issues for neuroscientists is to link neuronal network function to cognition, including learning and memory. It is still unclear what modifications of neuronal circuits need to happen in order to alter motor behaviour as it is recorded in a learning and memory test (Mayford et al., 2012), meaning that there is no clear understanding about the how these two KEs are connected.

Several epidemiological studies where Pb2+ exposure levels have been studied in relation to neurobehavioural alterations in children have been reviewed in Koller et al. 2004. This review has concluded that in some occasions there is negative correlation between Pb2+ dose and cognitive deficits of the subjects due to high influence of social and parenting factors in cognitive ability like learning and memory (Koller et al. 2004), meaning that not always Pb2+ exposure is positively associated with learning and memory impairment in children.

The direct relationship of alterations in neural network function and specific cognitive deficits is difficult to ascertain given the many forms that learning and memory can take and the complexity of synaptic interactions in even the simplest brain circuit. Linking of neurophysiological assessments to learning and memory processes have, by necessity, been made across simple monosynaptic connections and largely focused on the hippocampus. Alterations in synaptic function have been found in the absence of behavioral impairments. This may result from measuring only one component in the complex brain circuitry that underlies 'cognition', behavioral tests that are not sufficiently sensitive for the detection of subtle cognitive impairments, and behavioral plasticity whereby tasks are solved by the animal via different strategies developed as a consequence of developmental insult.

Finally, in order to provide empirical support for this KER, data on the effects of lead (Pb) exposure are reported. However, Pb exposure is not always associated with learning and memory impairment in children. In this regard, Koller's review has commented that in some occasions, low-level Pb dose and cognitive deficits of the subjects are negatively correlated, and this may be due to the high influence of social and parenting factors in cognitive ability, like learning and memory (Koller et al., 2004).

There is not enough quantitative information how much change decrease of neuronal network functions leads to learning and memory deficits. However, qualitatively is well documented that decrease of LTP is directly linked to learning and memory deficits.

There is very limited information on the degree of quantitative change in neural network function required to alter cognitive behaviors. This is a result of the diversity of methods for measuring both neuronal network function and learning and memory deficits, which hamper cross-study analyses. This highlights the need to develop empirical data based models of this KER. It is well known that the altered balance between excitatory and inhibitory synapses affects learning and memory, although no quantitative data are available.

References

Akaike M, Kato, N., Ohno, H., Kobayashi, T. (1991). Hyperactivity and spatial maze learning impairment of adult rats with temporary neonatal hypothyroidism. Neurotoxicol Teratol 13:317-322.

Anderson DW, Pothakos K, Schneider JS. (2012). Sex and rearing condition modify the effects of perinatal lead exposure on learning and memory. Neurotoxicology 33: 985-995.

Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, U H. (2008). Developmental neurotoxicity of Propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol 232:1-13.

Baghurst PA, Tong S, Sawyer MG, Burns J, McMichael AJ. (1992). Environmental exposure to lead and children’s intelligence at the age of seven years. The Port Pirie Cohort Study. N Engl J Med. 327: 1279-1284.

Bellinger D, Sloman J, Leviton A, Rabinowitz M, Needleman HL, Waternaux C. (1991). Low-level lead exposure and children's cognitive function in the preschool years. Pediatrics. 87: 219-227.

Bellinger DC, Stiles KM, Needleman HL. (1992). Low-level lead exposure, intelligence and academic achievement: a long-term follow-up study. Pediatrics. 90: 855-861.

Bellinger DC. (2004). Lead. Pediatrics 113: 1016-1022.

Brockel BJ, Cory-Slechta DA. (1998). Lead, attention, and impulsive behavior: changes in a fixed-ratio waiting-for-reward paradigm. Pharmacol Biochem Behav. 60: 545-552.

Canfield RL, Henderson CR Jr, Cory-Slechta D, Cox C, Jusko TA, Lanphear BP. (2003). Intellectual impairment in children with blood lead concentrations below 10 μg per deciliter. N Engl J Med. 348: 1517-1526.

Cao X, Huang S, Ruan D. (2008). Enriched environment restores impaired hippocampal long-term potentiation and water maze performance induced by developmental lead exposure in rats. Dev Psychobiol. 50: 307-313.

Cohn J, Cory-Slechta DA. (1993). Subsensitivity of lead-exposed rats to the accuracy-impairing and rate-altering effects of MK-801 on a multiple schedule of repeated learning and performance. Brain Res. 600: 208-218.

Cohn J, Cory-Slechta DA. (1994). Lead exposure potentiates the effects of N-methyl-D-asparate on repeated learning. Neurotoxicol Teratol. 16: 455-465.

Cory-Slechta DA. (1995). MK-801 subsensitivity following postweaning lead exposure. Neurotoxicology 16: 83-95.

de Souza Lisboa S F, Gonzalves G, Komatsu F, Salci Queiroz CA, Aparecido Almeida A, Gastaldello Moreira EN. (2005). Developmental lead exposure induces depressive-like behavior in female rats. Drug Chem Toxicol. 28: 67-77.

D'Hooge R, De Deyn PP. (2001). Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev. 36: 60-90.

Dong J, Yin H, Liu W, Wang P, Jiang Y, Chen J. (2005). Congenital iodine deficiency and hypothyroidism impair LTP and decrease C-fos and C-jun expression in rat hippocampus. Neurotoxicology 26:417-426.

Finkelstein Y, Markowitz ME, Rosen JF. (1998). Low-level lead-induced neurotoxicity in children: an update on central nervous system effects. Brain Res Rev. 27: 168-176.

Gilbert ME, Kelly ME, Samsam TE, Goodman JH. (2005). Chronic developmental lead exposure reduces neurogenesis in adult rat hippocampus but does not impair spatial learning. Toxicol Sci. 86: 365-374.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Gilbert ME, Sui L. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Grunwald T, Beck H, Lehnertz K, Blümcke I, Pezer N, Kurthen M, Fernández G, Van Roost D, Heinze HJ, Kutas M, Elger CE. (1999). Evidence relating human verbal memory to hippocampal N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A. 96: 12085-12089.

Jaako-Movits K, Zharkovsky T, Romantchik O, Jurgenson M, Merisalu E, Heidmets LT, Zharkovsky A. (2005). Developmental lead exposure impairs contextual fear conditioning and reduces adult hippocampal neurogenesis in the rat brain. Int J Dev Neurosci. 23: 627-635.

Jang YJ, Park HR, Kim TH, Yang WJ, Lee JJ, Choi SY, Oh SB, Lee E, Park JH, Kim HP, Kim HS, Lee J. (2012). High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations. Toxicology. Jun 14;296(1-3):73-82.

Jett DA, Kuhlmann AC, Farmer SJ, Guilarte TR. (1997). Age-dependent effects of developmental lead exposure on performance in the Morris water maze. Pharmacol Biochem Behav. 57: 271-279.

Jusko TA, Henderson CR, Lanphear BP, Cory-Slechta DA, Parsons PJ, Canfield RL. (2008). Blood lead concentrations < 10 microg/dL and child intelligence at 6 years of age. Environ Health Perspect 116:243-248.

Koller K, Brown T, Spurgeon A, Levy L. (2004). Recent developments in low-level lead exposure and intellectual impairment in children. Environ Health Perspect. 112: 987-994.

Kumar MV, Desiraju T. (1992). EEG spectral power reduction and learning disability in rats exposed to lead through postnatal developing age. Indian J Physiol Pharmacol. 36: 15-20.

Lanphear BP, Dietrich K, Auinger P, Cox C. (2000). Cognitive deficits associated with blood lead concentrations <10 microg/dL in US children and adolescents. Public Health Rep. 115: 521-529.

Lanphear BP, Hornung R, Khoury J, Yolton K, Baghurst P, Bellinger DC, Canfield RL, Dietrich KN, Bornschein R, Greene T, Rothenberg SJ, Needleman HL, Schnaas L, Wasserman G, Graziano J, Roberts R. (2005). Low-level environmental lead exposure and children’s intellectual function: an international pooled analysis. Environ Health Perspect. 113: 894-899.

Leviton A, Bellinger D, Allred EH, Rabinowitz M, Needleman H, Schoenbaum S. (1993). Pre- and postnatal low-level lead exposure and children's dysfunction in school. Environ Res 60:30-43.

Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H. (2010). The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring. Thyroid 20:909–915.

Lynch MA. (2004). Long-term potentiation and memory. Physiol Rev. 84: 87-136.

Mayford M, Siegelbaum SA, Kandel ER. (2012). Synapses and memory storage. Cold Spring Harb Perspect Biol. 4. pii: a005751.

Moreira EG, Vassilieff I, Vassilieff VS. (2001). Developmental lead exposure: behavioral alterations in the short and long term. Neurotoxicol Teratol. 23: 489-495.

Morris RG, Anderson E, Lynch GS, Baudry M. (1986). Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319: 774-776.

Murphy KJ, Regan CM. (1999). Low level lead exposure in the early postnatal period results in persisting neuroplastic deficits associated with memory consolidation. J Neurochem. 72: 2099-2104.

Neal AP, Guilarte TR. (2010). Molecular Neurobiology of Lead (Pb2+): Effects on Synaptic Function. Mol Neurobiol. 42: 151-160.

Needleman HL, Epstein S, Carnow B, Scanlon J, Parkinson D, Samuels S, Mazzochi A, David O. (1975). Letter: Blood-lead levels, behaviour and intelligence. Lancet 1: 751-752.

Needleman HL, Gatsonis CA. (1990). Low-level lead exposure and the IQ of children. A meta-analysis of modern studies. Jama 263: 673-678.

Needleman HL, Riess JA, Tobin MJ, Biesecker GE, Greenhouse JB. (1996). Bone Lead Levels and Delinquent Behavior. Jama 275: 363-369.

Saar D, Barkai E. (2003). Long-term modifications in intrinsic neuronal properties and rule learning in rats. Eur J Neurosci. 17: 2727-2734.

Salinas JA, Huff NC. (2002). Lead and conditioned fear to contextual and discrete cues. Neurotoxicol Teratol. 24: 541-550.

Surkan PJ, Zhang A, Trachtenberg F, Daniel DB, McKinlay S, Bellinger DC. (2007). Neuropsychological function in children with blood lead levels <10 microg/dL. Neurotoxicology. 28: 1170-1177.

Sui L, Anderson WL, Gilbert ME. (2005). Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampal area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci 85:647-656.

Sui L, Gilbert ME. (2003). Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology 144:4195-4203.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. (2008). Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Toscano CD, Guilarte TR. (2005). Lead neurotoxicity: From exposure to molecular effects. Brain Res Rev. 49: 529-554.

Vara H, Martinez B, Santos A, Colino A. (2002). Thyroid hormone regulates neurotransmitter release in neonatal rat hippocampus. Neuroscience 110:19-28.

Wang S, Teng W, Gao Y, Fan C, Zhang H, Shan Z. (2012). Early levothyroxine treatment on maternal subclinical hypothyroidism improves spatial learning of offspring in rats. J Neuroendocrinol 24:841–848.

Wheeler SM, McAndrews MP, Sheard ED, Rovet J. (2012). Visuospatial associative memory and hippocampal functioning in congenital hypothyroidism. J Int Neuropsychol Soc 18:49-56.

Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF. (2015). Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163.

Willoughby KA, McAndrews MP, Rovet J. (2013). Effects of early thyroid hormone deficiency on children's autobiographical memory performance. J Int Neuropsychol Soc 19:419-429.

Willoughby KA, McAndrews MP, Rovet JF. (2014). Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Xiao Y, Fu H, Han X, Hu X, Gu H, Chen Y, Wei Q, Hu Q. (2014). Role of synaptic structural plasticity in impairments of spatial learning and memory induced by developmental lead exposure in Wistar rats. PLoS One. 23;9(12):e115556.

Xu J, Yan HC, Yang B, Tong LS, Zou YX, Tian Y. (2009). Effects of lead exposure on hippocampal metabotropic glutamate receptor subtype 3 and 7 in developmental rats. J Negat Results Biomed. 8: 5.

Inhibition, Na+/I- symporter (NIS) leads to Impairment, Learning and memory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

As described in the Empirical Support section, the association between NIS inhibition and learning and memory impairment has been studied only in rodent models and in humans.

How Does This Key Event Relationship Work

NIS is a membrane protein responsible for iodide transport into the follicular cells of the thyroid, which is the first and most critical step leading to T4 biosynthesis (Dohan et al., 2000). TH synthesis is dramatically suppressed in case of NIS dysfunction or inhibition (Spitzweg and Morris, 2010; Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006), resulting in the decreased TH levels in the serum and consequently in the brain. Hypothyroid brain development results in sever functional impairments including ataxia, spasticity, sever mental retardation, including impairment of learning and memory. 

NIS inhibition occurring as a consequence of exposure to certain pollutants has been associated with learning and memory deficits in rodents and humans (Wang et al, 2016; Jang et al, 2012; Taylor et al., 2014; Chen et al., 2014; Roze et al., 2009; van Wijk et al., 2008; Wu Y et al., 2016).

Weight of Evidence

The weight of evidence supporting an indirect linkage between the MIE, NIS inhibition, and the adverse outcome Impairment of learning and Memory is moderate.

Some epidemiological and in vivo studies have indicated associations between NIS inhibition (e.g., as a consequence of exposure to perchlorate or other pollutants, such as BPA and BDE-47, NIS inhibitors) (Wu Y et al., 2016) and decreased cognition. For instance:

- Wang et al., 2016 Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on PND 21 with the object recognition task. BPA-exposed male offspring underwent memory and cognitive impairments: they not only spent more time (~ 43% more, at 1.5 hr after training) in exploring the familiar object at the highest dose than the control, but also displayed a significant decrease in the object recognition index (at 50 mg/kg BW/day, ~ 54% lower short term memory measured 1.5 hr after training).

- Jang et al., 2012 In this study pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. These results suggest that BPA exposure (NIS inhibition) in pregnant mothers could decrease hippocampal neurogenesis and cognitive function in future generations.

- Taylor et al., 2014 In this historical cohort study of 21,846 women in Cardiff, United Kingdom, and Turin, Italy, who were pregnant from 2002 to 2006, levels of urinary perchlorate (a NIS inhibitor) in the highest 10% were associated with a higher risk for having children with IQ scores in the lowest decile at age three, as described in 487 mother–child pairs in mothers who were hypothyroid/hypothyroxinemic during pregnancy.

- Chen et al., 2014 In this prospective birth cohort, maternal serum concentrations of BDE-47 and other PBDE congeners were measured in 309 women at 16 weeks of gestation, and associated with neurodevelopment in children. Importantly, BDE-47 and other chemicals, such as triclosan, triclocarban and BPA, have been reported to disturb TH homeostasis by inhibiting NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis in rat thyroid follicular FRTL-5 cells (Wu Y et al., 2016). A 10-fold increase in prenatal BDE-47 exposure was associated with a 4.5-point decrease in Full-Scale IQ and a 3.3-point increase in the hyperactivity score at age 5 years in children.

- Roze et al., 2009 Similarly, this study assessed the level of several compounds, including BDE-47 (i.e., 2,2'-bis-(4 chlorophenyl)-1,1'-dichloroethene, pentachlorophenol (PCP), PCB-153, 4OH-CB-107, 4OH-CB-146, 4OH-CB-187, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, and hexabromocyclododecane), in 62 mothers during the 35^th week of pregnancy, and possible associations with the neuropsychological level in their children at 5-6 years of age. THs were determined in umbilical cord blood. Brominated flame retardants correlated with worse fine manipulative abilities, worse attention, better coordination, better visual perception, and better behavior. Chlorinated OHCs correlated with less choreiform dyskinesia. Hydroxylated polychlorinated biphenyls correlated with worse fine manipulative abilities, better attention, and better visual perception. The wood protective agent (PCP) correlated with worse coordination, less sensory integrity, worse attention, and worse visuomotor integration.

- van Wijk et al., 2008 This in vivo study assessed the behavioural effects of perinatal and chronic hypothyroidism during development in both male and female offspring of hypothyroid rats. To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate (NIS inhibitor). Treatment was started in dams 2 weeks prior to mating, and in pups either until the day of killing (i.e., chronic hypothyroidism) or only until weaning (i.e., perinatal hypothyroidism) to test for reversibility of the effects observed. Early neuromotor competence, as assessed in the grip test and balance beam test, was impaired by both chronic and perinatal hypothyroidism. The open field test, assessing locomotor activity, revealed hyperactive locomotor behavioural patterns in chronic hypothyroid animals only. The Morris water maze test, used to assess cognitive performance, showed that chronic hypothyroidism affected spatial memory in a negative manner. Perinatal hypothyroidism was found to impair spatial memory in female rats only. In general, the effects of chronic hypothyroidism on development were more pronounced than the effects of perinatal hypothyroidism. This suggests that the early effects of hypothyroidism on functional alterations of the developing brain may be partly reversible.

Single NIS mutations, causing decreased thyroidal iodide uptake, may not necessarily lead to cognitive disorders. In this regard, Nicola and coworkers (Nicola et al., 2015) recently identified a new NIS mutation (V270E) in a patient (full-term girl born to healthy, non-consanguineous Jamaican parents), who resulted to be heterozygous for this NIS mutation (R124H/V270E). The presence of the mutation V270E markedly reduces iodide uptake (5.4% 24 hours after the oral administration of 100 μCi ¹²³I− (normal range, 10–40%)) via a pronounced (but not total) impairment of the protein's plasma membrane targeting. However, the retaining of a minimal iodide uptake was enough to enable sufficient TH biosynthesis and prevent cognitive impairment.

Temporal concordance of insufficient iodide uptake (as a consequence of NIS inhibition occurring upon exposure to perchlorate or other pollutants, such as BPA and BDE-47) and decreased cognitive functions has been documented by some epidemiological studies (Taylor et al., 2014; Chen et al., 2014; Roze et al. 2009) and have demonstrated that there are critical exposure windows during gestation/development where permanent changes are triggered.

For example, different degrees of TH insufficiency have been produced in dams and pups by administering varying doses of NIS inhibitor (e.g., sodium perchlorate) (van Wijk et al., 2008) or TPO inhibitors and assessing the dose-dependency of the observed effects on a variety of measurements. In these low dose model studies, the absence of overt signs of maternal or neonatal toxicity is taken as evidence of the temporal concordance of hormone insufficiency and neurodevelopmental impairment and the specificity of the observed effects on brain development to be mediated by TH insufficiency (van Wijk  et al., 2008; Gilbert and Sui, 2006; Sharlin et al., 2007; Axelstad et al., 2008; Sharlin et al., 2008; Babu et al., 2011; Gilbert, 2011; Powell et al., 2012; Gilbert et al., 2013; Bastian et al., 2014; Gilbert et al., 2014; Gilbert et al., 2016).

References

Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, U H. (2008). Developmental neurotoxicity of Propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol 232:1-13.

Babu S, Sinha RA, Mohan V, Rao G, Pal A, Pathak A, Singh M, Godbole MM. (2011). Effect of hypothyroxinemia on thyroid hormone responsiveness and action during rat postnatal neocortical development. Exp Neurol 228:91-98.

Bastian TW, Prohaska JR, Georgieff MK, Anderson GW. (2014). Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 155:1157-1167.

Chen A, Yolton K, Rauch SA, Webster GM, Hornung R, Sjödin A, Dietrich KN, Lanphear BP. (2014). Prenatal polybrominated diphenyl ether exposures and neurodevelopment in U.S. children through 5 years of age: the HOME study. Environ Health Perspect. Aug;122(8):856-62.

De Groef B, Decallonne BR, Van der Geyten S, Darras VM, Bouillon R. (2006). Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Europ J Endocr. 155:17-25.

Dohan O, De la Vieja A, Carrasco N. (2000) Molecular study of the sodium-iodide symporter (NIS): a new field in thyroidology. Trends Endocrinol Metab. Apr;11(3):99-105.

Gilbert ME, Sui L. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW. (2013). An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195.

Gilbert ME, Ramos RL, McCloskey DP, Goodman JH. (2014). Subcortical band heterotopia in rat offspring following maternal hypothyroxinaemia: structural and functional characteristics. J Neuroendocrinol 26:528-541.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Jang YJ, Park HR, Kim TH, Yang WJ, Lee JJ, Choi SY, Oh SB, Lee E, Park JH, Kim HP, Kim HS, Lee J. (2012). High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations. Toxicology. Jun 14;296(1-3):73-82.

Jones PA, Pendlington RU, Earl LK, Sharma RK, Barrat MD. (1996). In vitro investigations of the direct effects of complex anions on thyroidal iodide uptake: identification of novel inhibitors. Toxicol. In Vitro. 10: 149-160.

Nicola JP, Reyna-Neyra A, Saenger P, Rodriguez-Buritica DF, Gamez Godoy JD, Muzumdar R, Amzel LM, Carrasco N. (2015). Sodium/Iodide Symporter Mutant V270E Causes Stunted Growth but No Cognitive Deficiency. J Clin Endocrinol Metab. Oct;100(10):E1353-61.

Powell MH, Nguyen HV, Gilbert M, Parekh M, Colon-Perez LM, Mareci TH, Montie E. (2012). Magnetic resonance imaging and volumetric analysis: novel tools to study the effects of thyroid hormone disruption on white matter development. Neurotoxicology 33:1322-1329.

Roze E, Meijer L, Bakker A, Van Braeckel KN, Sauer PJ, Bos AF. (2009). Prenatal exposure to organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance at school age. Environ Health Perspect. Dec;117(12):1953-8.

Sharlin DS TD, Bansal R, Gilbert ME, and Zoeller RT. (2007). The Thyroid Hormone Transporter, MCT8, Selectively Responds to Thyroid Hormone Insufficiency in the Developing Rat Brain. In: Endocrinology.

Sharlin DS, Tighe D, Gilbert ME, Zoeller RT. (2008). The balance between oligodendrocyte and astrocyte production in major white matter tracts is linearly related to serum total thyroxine. Endocrinology 149:2527-2536.

Spitzweg C, Morris JC. (2010). Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Mol Cell Endocrinol. Jun 30;322(1-2):56-63.

Taylor PN, Okosieme OE, Murphy R, Hales C, Chiusano E, Maina A, Joomun M, Bestwick JP, Smyth P, Paradice R, Channon S, Braverman LE, Dayan CM, Lazarus JH, Pearce EN. (2014). Maternal perchlorate levels in women with borderline thyroid function during pregnancy and the cognitive development of their offspring: data from the Controlled Antenatal Thyroid Study.J Clin Endocrinol Metab. Nov; 99(11):4291-8.

Tonacchera M, Agretti P, de Marco G, Elisei R, Perri A, Ambrogini E, De Servi M, Ceccarelli C, Viacava P, Refetoff S, Panunzi C, Bitti ML, Vitti P, Chiovato L, Pinchera A. (2003). Congenital hypothyroidism due to a new deletion in the sodium/iodide symporter protein. Clin Endocrinol. 59: 500–506.

van Wijk N1, Rijntjes E, van de Heijning BJ. (2008). Perinatal and chronic hypothyroidism impair behavioural development in male and female rats. Exp Physiol. Nov;93(11):1199-209.

Wang C, Li Z, Han H, Luo G, Zhou B, Wang S, Wang J. (2016). Impairment of object recognition memory by maternal bisphenol A exposure is associated with inhibition of Akt and ERK/CREB/BDNF pathway in the male offspring hippocampus. Toxicology. Feb 3;341-343:56-64.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

TH synthesis, Decreased leads to Impairment, Learning and memory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Deficiencies in learning and memory following developmental hypothyroidism (TH synthesis inhibition) have been documented mainly in rodents and humans.

How Does This Key Event Relationship Work

It is widely accepted that the thyroid hormones (TH) play a prominent role in the development and function of the CNS, including hippocampus and neocortex, two critical brain structure closely linked to the cognitive function (Gilbert et al., 2012). Brain concentrations of T4 are dependent on transfer of T4 from serum, through the vascular endothelia, into astrocytes.  In astrocytes, T4 is converted to T3 by deiodinase and subsequently transferred to neurons cellular membrane transporters. In the brain T3 controls transcription and translation of genes responsible for normal hippocampal structural and functional development. Normal hippocampal structure and physiology are critical for the development of cognitive function. Thus, there is an indisputable indirect link between TH synthesis, controlling the levels of T4 in serum, and cognitive function, including learning and memory processes.

Weight of Evidence

The weight of evidence supporting the relationship between decreased TH synthesis and learning and memory impairments (occurring as a consequence of altered neuronal network and synaptic function) is strong (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005). This is consistent with the well understood and documented relationship between TH synthesis that is responsible for TH concentrations in serum, and consequently in brain. TH controls brain development and function, including learning and memory processes, in humans and animals.

The importance of thyroid hormones (TH) in brain development has been recognised and investigated for many decades (Bernal, 2011; Williams 2008). Several human studies have shown that low levels of circulating maternal TH (as a consequence of a decrease of TH synthesis) can lead to neurophysiological deficits in the offspring, including learning and memory deficits, or even cretinism in most severe cases (Zoeller and Rovet, 2004; Henrichs et al., 2010).

A number of studies have consistently reported alterations in synaptic transmission resulting from developmental TH disruption, and leading to decreased cognition. Developmental hypothyroidism reduces the functional integrity in brain regions critical for learning and memory. For example, pyramidal neurons of hypothyroid animals have fewer synapses and an impoverished dendritic arbor (Rami et al., 1986a, Madeira et al., 1992) that would lead to cognitive impairments.

Both hippocampal regions (area CA1 and dentate gyrus) exhibit alterations in excitatory and inhibitory synaptic transmission following reductions in serum TH in the pre and early postnatal period (Vara et al., 2002; Sui and Gilbert, 2003; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). These deficits persist into adulthood long after recovery to euthyroid status.

Hypothyroidism, induced by different approaches, including inhibition of TH synthesis, during development reduces the capacity for synaptic plasticity in juvenile and adult offspring (Vara et al., 2002; Sui and Gilbert, 2003; Dong et al., 2005; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). Impairments in synaptic function and plasticity are observed coincident with deficits in learning tasks that require the hippocampus.

Several studies have reported associations between decrease of TH synthesis (induced by TPO inhibitors) and learning and memory impairments:

- Davenport and Dorcey, 1972; Tamasy et al., 1986: Deficits in passive avoidance learning have been reported, but these early observations are often limited to animals suffering fairly severe hormonal deprivation.

- Akaike, 1991 Temporary hypothyroidism was induced in neonatal rats by 0.02% PTU administration to lactating dams during days 0-19 after delivery.  The radial arm maze test started at 13 weeks revealed that the PTU animals required more trials until they showed the first well-performed trial (with 3-fold more errors on day 3). The total number of choices was also larger, with less correct choices. These results suggest an involvement of temporary neonatal hypothyroidism in learning and memory impairment.

- Axelstad et al., 2008 They showed radial arm maze deficits in adult offspring of rat dams treated with high doses of the TPO inhibitor, propylthiouracil (PTU), throughout gestation and lactation. Data showed a ~66% increase in the total number of errors made in the radial arm maze during 3 weeks of testing, in adult male rats perinatally exposed to 1.6 mg/kg/day of PTU from GD7 to PND17.

- Shafiee et al., 2016 This study investigated the effects of PTU (TPO inhibitor, 100 mg/L) in pregnant rats to evaluate the effects elicited by maternal hypothyroidism in the offspring. PTU was added to the drinking water from gestation day 6 to PND 21. Analysis of hippocampal BDNF levels, and learning and memory tests were performed on PNDs 45-52 on pups. These results indicated that hypothyroidism during the fetal period and the early postnatal period was associated with: (i) ~ 70% reduction of total serum T4, (ii) ~ 5-fold increase of total serum TSH levels (on PND 21; no significant differences could be found at the end of the behavioral testing, on PND 52), (iii) reduction of hippocampal BDNF protein levels (~ 8% decrease vs control), (iv) impairment of spatial learning and memory, in both male and female rat offspring.

- Gilbert et al., 2016 Exposure to PTU during development produced dose-dependent reductions in mRNA expression of nerve growth factor (Ngf) in whole hippocampus of neonates. These changes in basal expression persisted to adulthood despite the return to euthyroid conditions in blood. Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2^nd trial resulted ~60% higher in rats treated with 10 ppm PTU).

- Gilbert and Sui, 2006 Administration of 3 or 10 ppm PTU to pregnant and lactating dams via the drinking water from GD6 until PND30 caused a 47% and 65% reduction in serum T4, in the dams of the low and high-dose groups, respectively. Baseline synaptic transmission was impaired in PTU-exposed animals: mean EPSP slope (by ~60% with 10 ppm PTU) and population spike amplitudes (by ~70% with 10 ppm PTU) in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams. High-dose animals (10 ppm) demonstrated very little evidence of learning despite 16 consecutive days of training (~5-fold higher mean latency to find the hidden platform, used as an index of learning).

- Gilbert, 2011 Trace fear conditioning deficits to context and to cue reported in animals treated with PTU and who also displayed synaptic transmission and LTP deficits in hippocampus. Baseline synaptic transmission was impaired in PTU-exposed animals (by ~50% in animal treated with 3 ppm PTU). EPSP slope amplitudes in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams.

In addition, studies in humans have shown associations between hypothyroidism and decreased memory and learning:

- Oerbeck et al., 2003 This study reported visual and verbal memory deficits in congenitally hypothyroid (CH) children. The CH group attained significantly lower scores than control subjects on intellectual, motor, and school-associated tests (total IQ: 102.4 vs 111.4). All verbal memory tasks were impaired; visual memory domain gave less consistent findings.

- Wheeler et al., 2011 Functional magnetic resonance imaging (fMRI) data from humans support the relationship between decreased serum concentrations of TH (occurring as a consequence of a decrease of TH synthesis) and memory deficits. CH subjects scored significantly below controls on indices of verbal but not visual memory as well as aspects of everyday memory functioning.

- Willoughby et al., 2013 The present study analysed 26 children with early-treated congenital hypothyroidism (CH), 23 children born to women with inadequately treated hypothyroidism during pregnancy (HYPO), and 30 typically developing controls. Results showed that relative to controls, CH and HYPO groups both exhibited weaknesses in episodic autobiographical memory, but not semantic autobiographical memory. In particular, CH and HYPO groups showed difficulty in recalling event details (i.e., the main happenings) and visual details from past experiences, confirming memory deficits.

- Willoughby et al., 2014 Congenitally hypothyroid children and children born to women with high TSH during pregnancy (biomarker of decreased TH synthesis) were weaker in recalling event and perceptual details from past naturally-occurring autobiographical events than age matched controls. HYPO cases scored significantly below controls on one objective and several subjective memory indices, and these were correlated with lower hippocampal volumes (brain region critical for learning and memory).

Numerous studies reported that iodine deficiency in critical periods of brain development and growth causes severe and permanent growth and cognitive impairment (cretinism) (Pesce and Kopp, 2014; de Escobar et al., 2007; de Escobar et al., 2008; Zimmermann, 2007; Melse-Boonstra and Jaiswal, 2010; Horn and Heuer, 2010; Zimmermann, 2012). However, direct quantitative correlation between decreased TH synthesis (as a consequence of TPO inhibition) and decreased cognition, in support to this KER, were not assessed in these reports.

Moreover, Wheeler et al., 2012 used fMRI visuospatial memory task to assess hippocampal activation in adolescents with CH (N = 14; age range, 11.5-14.7 years) compared with controls (N = 15; age range, 11.2-15.5 years). Despite, adolescents with congenital hypothyroidism showed both increased magnitude of hippocampal activation relative to controls and bilateral hippocampal activation when only the left was observed in controls, no group differences were recorded in task performance.

References

Akaike M, Kato, N., Ohno, H., Kobayashi, T. (1991). Hyperactivity and spatial maze learning impairment of adult rats with temporary neonatal hypothyroidism. Neurotoxicol Teratol 13:317-322.

Auso E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G, Berbel P. (2004). A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology 145:4037-4047.

Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, U H. (2008). Developmental neurotoxicity of Propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol 232:1-13.

Berbel P, Navarro D, Auso E, Varea E, Rodriguez AE, Ballesta JJ, Salinas M, Flores E, Faura CC, de Escobar GM. (2010). Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity. Cereb Cortex 20:1462-1475.

Bernal J. (2011). Thyroid hormone transport in developing brain. Curr Opin Endocrinol Diab Obes 18:295–299.

Davenport JW, Dorcey TP. (1972). Hypothyroidism: learning deficit induced in rats by early exposure to thiouracil. Horm Behav 3:97-112.

de Escobar GM, Obregon MJ, del Rey FE. (2007). Iodine deficiency and brain development in the first half of pregnancy. Public Health Nutr.10(12A):1554–1570.

de Escobar GM, Ares S, Berbel P, Obregon MJ, del Rey FE. (2008). The changing role of maternal thyroid hormone in fetal brain development. Semin Perinatol. 32(6):380–386.

Dong J, Yin H, Liu W, Wang P, Jiang Y, Chen J. (2005). Congenital iodine deficiency and hypothyroidism impair LTP and decrease C-fos and C-jun expression in rat hippocampus. Neurotoxicology 26:417-426.

Gilbert ME. (2004). Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev Brain Res 148:11-18.

Gilbert ME, Sui L. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012). Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology 33(4):842-852.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Goodman JH, Gilbert ME. (2007). Modest thyroid hormone insufficiency during development induces a cellular malformation in the corpus callosum: a model of cortical dysplasia. Endocrinology. 2007 Jun;148(6):2593-7.

Henrichs J, Bongers-Schokking JJ, Schenk JJ, Ghassabian A, Schmidt HG, Visser TJ, Hooijkaas H, de Muinck Keizer-Schrama SM, Hofman A, Jaddoe VV, Visser W, Steegers EA, Verhulst FC, de Rijke YB, Tiemeier H. (2010). Maternal thyroid function during early pregnancy and cognitive functioning in early childhood: the generation R study. J Clin Endocrinol Metab 95:4227–4234.

Horn S, Heuer H. (2010). Thyroid hormone action during brain development: more questions than answers. Mol Cell Endocrinol. 315(1–2):19–26.

Koibuchi N, Chin WW. (2000). Thyroid hormone action and brain development. Trends Endocrinol Metab 11:123-128.

Lavado-Autric R, Auso E, Garcia-Velasco JV, Arufe Mdel C, Escobar del Rey F, Berbel P, Morreale de Escobar G. (2003). Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. J Clin Invest 111:1073-1082.

Madeira MD, Sousa N, Lima-Andrade MT, Calheiros F, Cadete-Leite A, Paula-Barbosa MM. (1992). Selective vulnerability of the hippocampal pyramidal neurons to hypothyroidism in male and female rats. J Comp Neurol 322:501-518.

Melse-Boonstra A, Jaiswal N. (2010). Iodine deficiency in pregnancy, infancy and childhood and its consequences for brain development. Best Pract Res Clin Endocrinol Metab. 24(1):29–38.

Oerbeck B, Sundet K, Kase BF, Heyerdahl S. (2003). Congenital hypothyroidism: influence of disease severity and L-thyroxine treatment on intellectual, motor, and school-associated outcomes in young adults. Pediatrics 112:923-930.

Pesce L, Kopp P. (2014). Iodide transport: implications for health and disease. Int J Pediatr Endocrinol. 2014(1):8.

Rami A, Patel AJ, Rabie A. (1986a). Thyroid hormone and development of the rat hippocampus: morphological alterations in granule and pyramidal cells. Neuroscience 19:1217-1226.

Shafiee SM, Vafaei AA, Rashidy-Pour A. (2016). Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise. Neuroscience. Aug 4;329:151-61.

Sui L, Anderson WL, Gilbert ME. (2005). Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampal area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci 85:647-656.

Sui L, Gilbert ME. (2003). Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology 144:4195-4203.

Tamasy V, Meisami E, Vallerga A, Timiras PS. (1986). Rehabilitation from neonatal hypothyroidism: spontaneous motor activity, exploratory behavior, avoidance learning and responses of pituitary--thyroid axis to stress in male rats. Psychoneuroendocrinology 11:91-103.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. (2008). Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Vara H, Martinez B, Santos A, Colino A. (2002). Thyroid hormone regulates neurotransmitter release in neonatal rat hippocampus. Neuroscience 110:19-28.

Wheeler SM, McAndrews MP, Sheard ED, Rovet J. (2012). Visuospatial associative memory and hippocampal functioning in congenital hypothyroidism. J Int Neuropsychol Soc 18:49-56.

Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF. (2015). Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163.

Wheeler SM, Willoughby KA, McAndrews MP, Rovet JF. (2011). Hippocampal size and memory functioning in children and adolescents with congenital hypothyroidism. J Clin Endocrinol Metab 96:E1427-1434.

Williams GR. (2008). Neurodevelopmental and Neurophysiological Actions of Thyroid Hormone. Journal of Neuroendocrinology ,  20,  784–794.

Willoughby KA, McAndrews MP, Rovet J. (2013). Effects of early thyroid hormone deficiency on children's autobiographical memory performance. J Int Neuropsychol Soc 19:419-429.

Willoughby KA, McAndrews MP, Rovet JF. (2014). Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584.

Zimmermann MB. (2007). The adverse effects of mild-to-moderate iodine deficiency during pregnancy and childhood: a review. Thyroid. 17(9):829–835.

Zimmermann MB. (2012). The effects of iodine deficiency in pregnancy and infancy. Paediatr Perinat Epidemiol. 26(Suppl 1):108–117.

Zoeller RT, Rovet J. (2004). Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J Neuroendocrinol 16:809–818.

TH synthesis, Decreased leads to BDNF, Reduced

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The connection between synthesis of TH and BDNF expression has been studied only in rodent models up to date.

How Does This Key Event Relationship Work

Several studies have shown that THs regulate BDNF expression in the brain (Koibuchi et al., 1999; Koibuchi and Chin, 2000; Sui and Li, 2010), with the subsequent neurodevelopmental consequences, as described in the direct KER. For example, during the early cortical network development TH has been shown to regulate the morphology and function of the GABAergic neurons (Westerholz et al., 2010) and BDNF is one of the mediators of this regulation (Binder and Scharfman, 2004; Gilbert and Lasley, 2013).

In view of the above evidence, it has been suggested that the thyroid insufficiency triggered by inhibition of TPO or NIS functions, resulting in decreased TH synthesis and subsequent lowered TH levels in serum and brain, may lead to reduction of the levels of BDNF mRNA or protein in the developmental brain.

Weight of Evidence

The importance of TH in brain development has been recognised and investigated for many decades (Bernal, 2011; Williams 2008). Several human studies have shown that low levels of circulating maternal TH, even in the modest degree, can lead to neurophysiological deficits in the offspring, including learning and memory deficits, or even cretinism in most severe cases (Zoeller and Rovet, 2004; Henrichs et al., 2010). The levels of serum TH at birth are not always informative, as most of the neurological deficits are present despite the normal thyroid status of the newborn. That means that the cause of these impairments is rooted in the early stages of the neuronal development during the gestational period. The nature and the temporal occurrence of these defects suggest that TH may exert their effects through the neurotrophins, as they are the main regulators of neuronal system development (Lu and Figurov, 1997). Among them, BDNF represents the prime candidate because of its critical role in CNS development and its ability to regulate synaptic transmission, dendritic structure and synaptic plasticity in adulthood (Binder and Scharfman, 2004). Additionally, hippocampus and neocortex are two of the regions characterized by the highest BDNF expression (Kawamoto et al., 1996), and are also key brain areas for learning and memory functions.

Many in vivo studies have focused on the determination of the relationship between TH-mediated effects and BDNF expression in the brain. The majority of the work has been performed by evaluating the effects of TH insufficiency on BDNF developmental expression profile. The results, despite some differences, are showing a trend toward BDNF down-regulation triggered by decrease of TH synthesis.

Reductions in BDNF mRNA and protein were observed in hypothyroid rat models exposed to the TPO inhibitors methimazole (MMI) or propylthiouracil (PTU), and perchlorate (NIS inhibitor) (Koibuchi et al., 1999; 2001; Sinha et al., 2009; Neveu and Arenas, 1996; Lasley and Gilbert, 2011). These studies supported direct associations between decreased levels of TH and reduced BDNF expression in the developmental cerebellum, hippocampus and cortex. The dose-response relationship could not be evaluated in these studies, as they were conducted in conditions of severe maternal hypothyroidism, namely after exposure to very high doses of the chemicals.

- Koibuchi et al., 2001 Here newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (ROR-alpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats, suggesting impairment in neuronal differentiation.

- Chakraborty et al., 2012 PTU (TPO inhibitor) exposure in rat dams (4 ppm in drinking water) significantly decreased the levels of free T4 (~ 33% decrease vs control, at PND 7) and total T4 (~ 38% decrease vs control, at PND 7) in the offspring, and hippocampal BDNF protein levels in the offspring at 3 and 7 PNDs (~ 25% decrease of hippocampal BDNF, at PND 7, in female pups vs untreated female control). No significant BDNF reductions were observed in either the cerebellum or brain stem.

- Blanco et al., 2013 A significant dose-dependent down-regulation of hippocampal BDNF mRNA (~ 32% decrease vs control) in combination with the dose-dependent reduction of plasma TH (T4: ~ 25% decrease vs control; T3: ~ 14% decrease vs control), was also shown in Sprague Dawley rats after exposure to BDE-99 (2 mg/kg/day, through gavage, from gestation day 6 to PND 21).

- Shafiee et al., 2016 This study investigated the effects of PTU (TPO inhibitor, 100 mg/L) in pregnant rats to evaluate the effects elicited by maternal hypothyroidism in the offspring. PTU was added to the drinking water from gestation day 6 to PND 21. Analysis of hippocampal BDNF levels, and learning and memory tests were performed on PNDs 45-52 on pups. These results indicated that hypothyroidism during the fetal period and the early postnatal period was associated with: (i) ~ 70% reduction of total serum T4, (ii) ~ 5-fold increase of total serum TSH levels (on PND 21; no significant differences could be found at the end of the behavioral testing, on PND 52), (iii) reduction of hippocampal BDNF protein levels (~ 8% decrease vs control), (iv) impairment of spatial learning and memory, in both male and female rat offspring.

- Gilbert et al., 2016 Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus (e.g., at PND14, rats treated with 3 ppm PTU, underwent reduction of Bdnft by ~25%, Bdnfiv by ~10%, Ngf by ~25%, and Pval by ~50%) and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2^nd trial resulted ~60% higher in rats treated with 10 ppm PTU).

- Abedelhaffez and Hassan, 2013 This study in rats reported that methimazole (MMI, a TPO inhibitor)-induced hypothyroidism reduced plasma free T3, free T4 and significantly increased TSH in the pups, showing also reduced hippocampal and cerebellar BDNF levels.

- da Conceição et al., 2016 Thyroidectomized (i.e,. hypothyroid) adult Wistar rats showed significant increase of serum TSH (~ 750% increase vs control rats), decrease of T4 (~ 80% decrease vs control) and T3 serum levels (~ 45% decrease vs control), together with a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), TH receptor alfa (TRα1) (~ 77 % decrease vs control), deiodinase type 2 (DIO2) (~ 90% decrease vs control), and BDNF mRNA expression in hippocampus (~ 75% decrease vs control).

- Cortés et al., 2012 Adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU, a TPO inhibitor) (0.05% in drinking water) for 20 days to induce hypothyroidism. PTU-treated rats showed decrease serum fT4 (~ 70% decrease vs control) and tT3 (~ 45% decrease vs control) levels, and increased TSH levels (~ 9.5-fold increase over control). The hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. The glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained lower postsynaptic density (PSD) than control rats (~ 25% lower PSD than control). This observation was in agreement with a reduced content of NMDAR subunits (NR1 and NR2A/B subunits, both subunits: ~ 25% decrease vs control) at the PSD in hypothyroid animals. Additionally, the hippocampal amount of BDNF mRNA (assessed by in situ hybridization) was higher (~ 4.8-fold increase over control) of hypothyroid rats, while the content of TrkB protein (BDNF receptor) was reduced (~ 30% decrease vs control) at the PSD of the CA3 region of hypothyroid rats, compared with controls. Even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.

- Jang et al., 2012 In this study pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (NIS inhibitor) in pregnant mothers could decrease hippocampal neurogenesis and cognitive function in future generations.

- Pathak et al, 2011 Here the effect of maternal TH deficiency on neocortical development was investigated. Rat dams were maintained on MMI (TPO inhibitor) from GD6 until sacrifice. Decreased number and length of radial glia, loss of neuronal bipolarity, and impaired neuronal migration were recovered with early TH replacement (at E13-15). BDNF mRNA resulted downregulated (80% decrease at E14) while trkB expression was increased (2-fold) in hypothyroid fetuses at E14 stage. TH levels in the brain were not measured in this study.

- Neveu and Arenas, 1996 This study found that early hypothyroidism (by PTU administration to rat dams) decreased the expression of neurotrophin 3 (NT-3) and BDNF mRNA (70% reduction in BDNF level at P30). Grafting of P3 hypothyroid rats with cell lines expressing high levels of NT-3 or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15.

Despite the fact that many in vivo studies have shown a correlation between hypothyroidism and decreased BDNF expression in the brain, no clear consensus can be reached by the overall evaluation of the existing data. There are numerous conflicting studies showing no significant alterations in BDNF mRNA or protein levels (Alvarez-Dolado et al., 1994; Bastian et al., 2010; 2012; Royland et al., 2008; Lasley and Gilbert, 2011). However, the results of these studies cannot exclude the possibility of temporal- or region-specific BDNF effects as a consequence of foetal hypothyroidism. A transient TH-dependent BDNF reduction in early postnatal life can be followed by a period of normal BDNF levels or, on the contrary, normal BDNF expression in the early developmental stages is not predictive of equally normal BDNF expression throughout development. Moreover, significant differences in study design, the assessed brain regions, the age and the method of assessment in the existing studies, further complicate result interpretation.

While PTU (TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in Cortés et al., 2012 study, treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB, the receptor for BDNF, resulted reduced at the postsynaptic density (PSD) of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD in hypothyroid animals. These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain. However, even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.

As discussed in the Empirical support section, there are some quantitative data in support of this indirect KER (Decreased of TH synthesis leads to Reduced BDNF release).

References

Abedelhaffez AS, Hassan A. (2013). Brain derived neurotrophic factor and oxidative stress index in pups with developmental hypothyroidism: neuroprotective effects of selenium. Acta Physiol Hung. Jun;100(2):197-210.

Alvarez-Dolado M, Iglesias T, Rodrıguez-Pena A, Bernal J, Munoz A. (1994). Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain. Brain Res Mol Brain Res. 27:249–257.

Bastian TW, Prohaska JR, Georgieff MK, Anderson GW. (2010). Perinatal iron and copper deficiencies alter neonatal rat circulating and brain thyroid hormone concentrations. Endocrinology 151:4055–4065.

Bernal J. (2011). Thyroid hormone transport in developing brain. Curr Opin Endocrinol Diab Obes 18:295–299.

Binder DK, Scharfman HE. (2004). Brain-derived neurotrophic factor. Growth Factors. 22(3):123–131

Blanco J, Mulero M, Heredia L, Pujol A, Domingo JL, Sanchez Dc. (2013). Perinatal exposure to BDE-99 causes learning disorders and decreases serum thyroid hormone levels and BDNF gene expression in hippocampus in rat offspring. Toxicol 308:122-128.

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Cortés C, Eugenin E, Aliaga E, Carreño LJ, Bueno SM, Gonzalez PA, Gayol S, Naranjo D, Noches V, Marassi MP, Rosenthal D, Jadue C, Ibarra P, Keitel C, Wohllk N, Court F, Kalergis AM, Riedel CA. (2012). Hypothyroidism in the adult rat causes incremental changes in brain-derived neurotrophic factor, neuronal and astrocyte apoptosis, gliosis, and deterioration of postsynaptic density. Thyroid. Sep;22(9):951-63.

da Conceição RR, Laureano-Melo R, Oliveira KC, de Carvalho Melo MC, Kasamatsu TS, de Barros Maciel RM, de Souza JS, Giannocco G. (2016). Antidepressant behavior in thyroidectomized Wistar rats is induced by hippocampal hypothyroidism. Physiol Behav. Apr 1;157:158-64.

Gilbert ME, Lasley SM. (2013). Developmental thyroid hormone insufficiency and brain development: a role for brain-derived neurotrophic factor (BDNF)? Neurosci 239: 253-270.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology, Feb;157(2):774-87

Henrichs J, Bongers-Schokking JJ, Schenk JJ, Ghassabian A, Schmidt HG, Visser TJ, Hooijkaas H, de Muinck Keizer-Schrama SM, Hofman A, Jaddoe VV, Visser W, Steegers EA, Verhulst FC, de Rijke YB, Tiemeier H. (2010). Maternal thyroid function during early pregnancy and cognitive functioning in early childhood: the generation R study. J Clin Endocrinol Metab 95:4227–4234.

Jang YJ, Park HR, Kim TH, Yang WJ, Lee JJ, Choi SY, Oh SB, Lee E, Park JH, Kim HP, Kim HS, Lee J. (2012). High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations. Toxicology. Jun 14;296(1-3):73-82.

Kawamoto Y, Nakamura S, Nakano S, Oka N, Akiguchi I, Kimura J. (1996). Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain. Neurosci 74(4):1209-1226.

Koibuchi N, Chin WW. (2000). Thyroid hormone action and brain development. Trends Endocrinol Metab. 11(4):123-128.

Koibuchi N, Yamaoka S, Chin WW. (2001). Effect of altered thyroid status on neurotrophin gene expression during postnatal development of the mouse cerebellum. Thyroid 11:205–210.

Koibuchi N, Fukuda H, Chin WW. (1999). Promoter-specific regulation of the brain-derived neurotrophic factor gene by thyroid hormone in the developing rat cerebellum. Endocrinol 140: 3955–3961.

Lasley SM, Gilbert ME. (2011). Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates. Neurotoxicol Teratol 33:464–472.

Lu B, Figurov A. (1997). Role of neurotrophins in synapse development and plasticity. Rev Neurosci 8:1–12.

Neveu I, Arenas E. (1996.) Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. J Cell Biol 133:631–646.

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM. 2011. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cerebral cortex. Jan;21:11-21.

Royland JE, Parker JS, Gilbert ME. (2008). A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol 20:1319–1338.

Shafiee SM, Vafaei AA, Rashidy-Pour A. (2016). Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise. Neuroscience. Aug 4;329:151-61.

Sinha RA, Pathak A, Kumar A, Tiwari M, Shrivastava A, Godbole MM. (2009). Enhanced neuronal loss under perinatal hypothyroidism involves impaired neurotrophic signaling and increased proteolysis of p75(NTR). Mol Cell Neurosci 40:354–364.

Sui L, Li BM. (2010). Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: role of DNA methylation and histone acetylation. Steroids 75:988–997.

Westerholz S, deLima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neurosci 168:573–589.

Williams G.R. (2008). Neurodevelopmental and Neurophysiological Actions of Thyroid Hormone. Journal of Neuroendocrinology ,  20,  784–794.

Zoeller RT, Rovet J. (2004). Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J Neuroendocrinol 16:809–818.

TH synthesis, Decreased leads to GABAergic interneurons, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from work with laboratory rodents (rats and mice).

How Does This Key Event Relationship Work

Thyroid hormone synthesis is responsible for physiological TH serum levels that subsequently correlate with TH brain concentrations. It has been shown that TH regulates function of different neuronal subtypes, including GABAergic neurons. TH increases glutamic acid decarboxylase (GAD) activity (responsible for GABA-synthesis) in neonatal brain, and GABA transaminase (responsible for GABA degradation) activity (Shulga and Rivera, 2013). GABAergic interneurons are remarkably diverse and complex in nature and they are believed to play a key role in numerous neurodevelopmental processes (Southwell et al., 2014). During the early cortical network development TH has been shown to regulate the morphology and function of the GABAergic neurons (Westerholz et al., 2010). It is well documented that decreased TH synthesis triggered by TPO and NIS inhibitors affects survival of GABAergic interneurons, as well as their morphology and function.

Weight of Evidence

TH levels influence the development of cortical GABAergic circuits (Friauf et al., 2008; Westerholz et al., 2010). In hypothyroid rats the expression of parvalbumin, the marker of a subpopulation of GABAergic neurons, is reduced (Gilbert et al., 2007). TH increase glutamic acid decarboxylase (GAD, GABA-synthesizing enzyme) activity in neonatal brain. Also GABA transaminase (GABA-T, GABA-degrading enzyme) activity appears to be increased by TH. Therefore, both GABA synthesis and degradation are increased by TH. This might reflect either the specific regulation of GABA levels, or general regulation of gene expression maintenance by TH, as commented by Shulga and Rivera, 2013. This strongly supports the link between the two KEs described in this indirect KER (decrease of TH synthesis leads to GABAergic interneuron decrease). It was also shown that low concentrations of T3 increase by non-genomic mechanism the depolarization-dependent release of GABA. GABA appears to provide negative feedback to thyroid endocrine axis, as TSH release is inhibited by GABA (Wiens and Trudeau, 2006).

TPO inhibitors like PTU and MMI decrease the volume of the granule cell layer of the dentate gyrus, the density of cells within the layer, and estimates of total granule cell number (Madeira et al., 1991). Migration of granule cells from the proliferative zone to the granule cell layer (with different neuronal subtypes, including GABAergic neurons) is retarded by thyroid deficiency as is dendritic arborization and synaptogenesis assessed by immunohistochemistry for the synaptic protein, synaptophysin (Rami et al., 1986a, 1986b, Rami and Rabie, 1990).

- Sawano et al., 2013 This study investigated the effects methimazole (MMI, a TPO inhibitor) on the developing rat hippocampus, one of the brain regions most sensitive to TH status. MMI was administered at the concentration of 0.025% in drinking water to pregnant dams from gestational day 15 until 4 weeks postpartum. Looking at the pre- and post-synaptic components of the GABAergic system, the level of glutamic acid decarboxylase 65 (GAD65) protein was reduced to less than 50% of control in the hippocampus of hypothyroid rats, and recovered to control levels by daily thyroxine-replacement after birth. Reduction in GAD65 protein was correlated immunohistochemically with a 37% reduction in the number of GAD65⁺ cells, as well as a reduction in GAD65⁺ processes. In contrast, GAD67 was not affected by MMI treatment. A subpopulation of GABAergic neurons containing PV was also confirmed to be highly dependent on TH status (with a 33% reduction in total PV⁺ neurons compared with the control). Moreover, the physiologically occurring transient rise of KCC2 expression observed at PND 10 (followed by a large increase in KCC2 protein at PND 15) in the euthyroid hippocampus, was completely suppressed by MMI (~ 80% reduction in KCC2 protein at PND 15 vs control).

- Shiraki et al., 2012 this study compared the differential effects of MMI (0, 50, 200 ppm in the drinking water) comparing the developmental and adult-stage, in particular comparing pregnant rats treated from gestation day 10 to PND 21 (i.e., developmental hypothyroidism) and adult male rats treated from PND 46 through to PND 77 (i.e., adult-stage hypothyroidism). With regard to precursor granule cells, a sustained reduction of Pax6⁺ stem or early progenitor cells and a transient reduction of doublecortin⁺ late-stage progenitor cells were observed after developmental hypothyroidism with MMI at 50 and 200 ppm. These cells were unchanged by adult-stage hypothyroidism. The number of PV⁺ cells (a GABAergic interneuron subpopulation in the dentate hilus) was decreased (~ 60% reduction at PND 21, with 200 ppm MMI) and the number of calretinin⁺ cells was increased (~ 85% increase at PND 21, with 200 ppm MMI) after both developmental and adult-stage hypothyroidism.

- Gilbert et al., 2007 In this study pregnant rat dams were exposed to propylthiouracil (PTU, a TPO inhibitor, administered at 0, 3, 10 ppm in the drinking water, from gestational day 6 until PND 30). PTU decreased maternal serum T4 by ~ 50-75% and increased TSH. At weaning, T4 was reduced by approximately 70% in offspring in the low-dose group and fell below detectable levels in high-dose animals. PV⁺ cells were diminished in the hippocampus and neocortex of offspring sacrificed on PND 21 (~ 45% reduction in the cortex, and ~ 55% reduction in the dentate gyrus, with 3 ppm treatment), and altered staining persisted to adulthood despite the return of TH to control levels.

- Westerholz et al., 2010; 2013: In the developing cortex, spontaneous activity is characterized by synchronous bursts of action potentials in populations of glutamatergic and GABAergic neurons which propagate throughout developing neural networks. In cortical neurons in culture, synthesis of TH, and T3, in particular, augments the density and growth of GABAergic neurons and accelerates the maturation of neural networks.

While some studies (Sawano et al., 2013; Shiraki et al., 2012) have shown a decrease of GABAergic cell populations upon induction of hypothyroidism, Saegusa and co-workers (Saegusa et al., 2010) reported about an increase of GABAergic interneurons. In Saegusa's study, rat dams were treated with either PTU or MMI in the drinking water, and male offspring were immunohistochemically examined on PND 20 and at the adult stage (i.e., 11-week-old). MMI and PTU caused in the offspring growth retardation, lasting into the adult stage. All exposure groups showed a sustained increase of GAD67⁺ cells in the adult stage, indicating an increase in GABAergic interneurons.

It should be noticed that in Saegusa et al., 2010 study, increase of GAD67⁺ cells was mainly observed in the adult stage (11-week-old rats) and analysis of GABAergic interneurons.  PV⁺ cells, which appear to be the GABAergic population most affected by TH dysregulation, was not evaluated. On the opposite, Sawano's and Shiraki's studies reported a decrease of GABAergic PV⁺ neurons at earlier stages, respectively on PND 15 and 21 induced by hypothyroidism (Sawano et al., 2013; Shiraki et al., 2012). Discrepancies in results are due to the fact that THs have effects on multiple components of the GABA system. For instance, in the developing brain, hypothyroidism generally decreases enzyme activities and GABA levels, whereas in adult brain, hypothyroidism generally increases enzyme activities and GABA levels.

There are also conflicting results on effects of long term changes in TH levels on GABA reuptake. Therefore, results variability from study to study is due to different experimental study designs, accounting for differences in brain development stages (PND vs adult), times of exposures to chemicals, and regional brain differences (Wiens and Trudeau, 2006).

There is a lack of quantitative studies defining a threshold that would link inhibition of TH synthesis and decrease of GABAergic interneuron populations. Therefore no quantitative information can be provided.

References

Aguado F, Carmona MA, Pozas E, Aguiló A, Martínez-Guijarro FJ, Alcantara S, Borrell V, Yuste R, Ibañez CF, SorianoE. (2003). BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl–co-transporter KCC2. Development 130:1267-1280.

Blaesse P, Airaksinen MS, Rivera C, Kaila K. (2009). Cation chloride co-transporters and neuronal function. Neuron 61:820–838

Friauf E, Wenz M, Oberhofer M, Nothwang HG, Balakrishnan V, Knipper M, Lohrke S. (2008). Hypothyroidism impairs chloride homeostasis and onset of inhibitory neurotransmission in developing auditory brainstem and hippocampal neurons. Eur J Neurosci, 28, pp. 2371–2380

Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH. (2007). Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology. Jan;148(1):92-102.

Madeira MD, Cadete-Leite A, Andrade JP, Paula-Barbosa MM. (1991). Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study. J Comp Neurol 314:171-186.

Rami A, Patel AJ, Rabie A. (1986a). Thyroid hormone and development of the rat hippocampus: morphological alterations in granule and pyramidal cells. Neuroscience 19:1217-1226.

Rami A, Rabie A, Patel AJ. (1986b). Thyroid hormone and development of the rat hippocampus: cell acquisition in the dentate gyrus. Neuroscience 19:1207-1216.

Rami A, Rabie A. (1990). Delayed synaptogenesis in the dentate gyrus of the thyroid-deficient developing rat. Dev Neurosci 12:398-405.

Rivera, C., Li, H., Thomas-Crusells, J., Lahtinen, H., Vilkman, V., Nanobashvili, A., Kokaia, Z., Airaksinen, M.S., Voipio, J., Kaila, K. & Saarma, M. (2002). BDNF-induced TrkB activation down-regulates the K+–Cl− cotransporter KCC2 and impairs neuronal Cl− extrusion. J. Cell Biol., 159, 747–752.

Rivera, C., Voipio, J., Thomas-Crusells, J., Li, H., Emri, Z., Sipilä, S., Payne, J.A., Minichiello, L., Saarma, M. & Kaila, K. (2004). Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J. Neurosci., 24, 4683–4691.

Sawano E, Takahashi M, Negishi T, Tashiro T. (2013). Thyroid hormone-dependent development of the GABAergic pre- and post-synaptic components in the rat hippocampus. Int J Dev Neurosci. Dec;31(8):751-61.

Shiraki A, Akane H, Ohishi T, Wang L, Morita R, Suzuki K, Mitsumori K, Shibutani M. (2012). Similar distribution changes of GABAergic interneuron subpopulations in contrast to the different impact on neurogenesis between developmental and adult-stage hypothyroidism in the hippocampal dentate gyrus in rats. Arch Toxicol. Oct;86(10):1559-69.

Shulga A, Rivera C. (2013). Interplay between thyroxin, BDNF and GABA in injured neurons. Neuroscience. Jun 3;239:241-52.

Southwell DG, Nicholas CR, Basbaum AI, Stryker MP, Kriegstein AR, Rubenstein JL, Alvarez-Buylla A. (2014). Interneurons from embryonic development to cell-based therapy. Science. 44:1240622.

Wake, H., Watanabe, M., Moorhouse, A.J., Kanematsu, T., Horibe, S., Matsukawa, N., Asai, K., Ojika, K., Hirata, M. & Nabekura, J. (2007). Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation. J. Neurosci., 27, 1642–1650.

Westerholz S, de Lima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience 168:573-589.

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Wiens SC, Trudeau VL. (2006). Thyroid hormone and gamma-aminobutyric acid (GABA) interactions in neuroendocrine systems. Comp Biochem Physiol A Mol Integr Physiol. Jul;144(3):332-44. Epub 2006 Mar 9.

BDNF, Reduced leads to Synaptogenesis, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from work with laboratory rodent-derived cells and brain slices, and rodent in vivo studies.

How Does This Key Event Relationship Work

Disruption of BDNF signaling during development was shown to interfere with synaptogenesis in the hippocampus (Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012). In the adult brain, BDNF is involved in synaptic plasticity (Lu et al., 2013; Leal et al., 2014), which is a fundamental process linked with learning and memory. Synaptic dysfunction is a key pathophysiological hallmark in neurodegenerative disorders, including Alzheimer's disease, and synaptic repair therapies based on the use of trophic factors, such as BDNF, are currently under consideration (Lu et al., 2013).

BDNF is released by the BDNF-producing neurons of the CNS and binds to Trk-B of the PV-interneurons, an interaction necessary for the subsequent developmental effects of this neurotrophin (Polleux et al., 2002; Jin et al., 2003; Rico et al., 2002; Aguado et al., 2003). BDNF promotes the morphological and neurochemical maturation of hippocampal and neocortical interneurons and promotes GABAergic synaptogenesis (Danglot et al., 2006; Hu and Russek, 2008).

BDNF plays an important role in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development (Chapleau et al., 2009). Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, as well as in mouse models of these disorders (Chapleau et al., 2009).

Weight of Evidence

BDNF, in addition to its pro-survival effects, has powerful synaptic effects, promoting synaptic transmission, synaptic plasticity and synaptogenesis (Lu et al., 2013; Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012; Danglot et al., 2006; Hu and Russek, 2008). NMDAR activity has been linked to the signaling of the trans-synaptic neurotorophin BDNF (Neal et al., 2010).

Use of selective agonist or antagonist of BDNF receptor TrkB demonstrates the contribution of BDNF in synaptogenesis in adult-generated neurons in the rat dentate gyrus (Ambrogini et al., 2013). In this regard, exogenous application of BDNF significantly increased the number of functional synapses in culture (Vicario-Abejon et al., 1998; Marty et al., 2000), while blocking of BDNF with antibodies greatly reduced the formation of inhibitory synapses (Seil and Drake-Baumann, 2000). Similar results were described also in an in vivo study on mutant mice characterized by deletion of the trkB gene in cerebellar precursors (obtained by Wnt1-driven Cre--mediated recombination). TrkB mutant mice showed reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations, whilst granule and Purkinje cell dendrites appeared normal and the former presented typical numbers of excitatory synapses. This study demonstrated that TrkB is essential to the development of GABAergic neurons and the regulation of synapse formation (Rico et al., 2002). BDNF is also a potent regulator of spontaneous neuronal activity in GABAergic neurons and interneurons, as shown in in embryonic (E18) hippocampal slices (Aguado et al., 2003), and plays a critical role in controlling the emergence, complexity and networking properties of spontaneous networks.

- Westerholz et al., 2013 In recent in vitro studies with rat T3-deficient cultures of cortical GABAergic PV⁺ interneurons, which are subject to BDNF regulation, it was shown that the number of synaptic boutons (i.e., presynaptic terminals containing the presynaptic marker synaptophysin) was reduced, an effect that was abolished after exogenous BDNF application. Additionally, inhibition of BDNF TrkB receptors by K252a in cultures containing T3 resulted also in decreased number of synaptic boutons, as in the T3-deprived cultures. These results indicate that BDNF signaling promotes the formation of synaptic boutons and that this function is mediated by THs (T3 and T4). Additionally, T3-related increase of spontaneous network activity was remarkably reduced after addition of K252a, and also upon inhibition of mTOR pathway (with rapamycin), a pathway known to control synaptogenesis (Buckmaster et al., 2009).

- Sato et al., 2007 This study on rat cultured hippocampal slices showed that beta-estradiol (E2) induced synaptogenesis between mossy fibers (one of the major inputs to cerebellum) and hippocampal CA3 neurons by enhancing BDNF release from dentate gyrus (DG) granule cells, by increasing the expression of PSD95, a postsynaptic marker. E2 effects on in hippocampal slice cultures and subregional neuron cultures were completely inhibited by blocking the BDNF receptor (TrkB) with K252a (200 nM) or by using a function-blocking antibody to BDNF (10 μg/ml), which inhibited the expression of PSD95 induced by E2. Both K252a and the antibody anti-BDNF elicited ~ 60-70% decrease of spine density and ~ 55% decrease of presynaptic sites in dentate gyrus granule cells (measured as number of puncta/neuron).

- Schjetnan and Escobar, 2012 In this study, intrahippocampal microinfusion of BDNF (3 µg/3 µl; 0.2 µl/min,) in adult rats modified the ability of the hippocampal mossy fiber pathway to present long-term potentiation (LTP, i.e., a persistent strengthening of synapses based on recent patterns of activity) by high frequency stimulation (HFS). This indicates that BDNF initiates the metaplastic mechanisms that allow the modifications of the ability of the mossy fiber pathway to present LTP induced by subsequent HFS. On the contrary, microinfusion of K252a (administered in combination with BDNF: 3 μg of BDNF/3 μl of K252a 20 μM; 0.2 μl/min) blocked the functional and morphological effects produced by BDNF (shown by densitometric analysis on synaptic reorganization: ~ 30% reduction of the relative area of the dorsal hippocampus in the contralateral side of HFS, and ~ 70% reduction in the ipsilateral side of HFS, compared to BDNF administered alone), supporting the role of BDNF in the regulation of synaptic plasticity.

- Schildt et al., 2013 Using field potential recordings in CA3 of adult heterozygous BDNF knockout (BDNF+/-) mice, an impairment of NMDAR-independent mossy fiber (MF)-LTP (~ 50% decrease) was observed. Additionally, inhibition of TrkB/BDNF with K252a (slices preincubated for 3 hr with 100 nM), or with the selective BDNF scavenger TrkB-Fc (slices preincubated for 3 hr with 5 μg/ml), both inhibited MF-LTP to the same extent as observed in BDNF+/- mice (K252a: ~ 60% decrease vs control slices; TrkB-Fc: ~ 50% decrease vs control slices).

- Cortés et al., 2012 Adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU, a TPO inhibitor) (0.05% in drinking water) for 20 days to induce hypothyroidism. PTU-treated rats showed decrease serum fT4 (~ 70% decrease vs control) and tT3 (~ 45% decrease vs control) levels, and increased TSH levels (~ 9.5-fold increase over control). The hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. The glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained lower postsynaptic density (PSD) than control rats (~ 25% lower PSD than control). This observation was in agreement with a reduced content of NMDAR subunits (NR1 and NR2A/B subunits, both subunits: ~ 25% decrease vs control) at the PSD in hypothyroid animals. Additionally, the hippocampal amount of BDNF mRNA (assessed by in situ hybridization) was higher (~ 4.8-fold increase over control) of hypothyroid rats, while the content of TrkB protein (BDNF receptor) was reduced (~ 30% decrease vs control) at the PSD of the CA3 region of hypothyroid rats, compared with controls. Even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.

- Koibuchi et al., 2001 Here newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (ROR-alpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased in hypothyroid rats. Dendritic arborization is used as readout for synapse formation, as post-synaptic side (synaptogenesis) is mainly located on dendrites.

- Aguado et al., 2003 BDNF overexpression in transgenic embryos raised the spontaneous activity of E18 hippocampal neurons, as shown by increased number of synapses (63% more synapses in the hippocampus of BDNF transgenic embryos than in controls), and increased spontaneous neuronal activity (2.3 times more active neurons than wild type embryos, and 36.3% greater rates of activation). Moreover, BDNF transgenic embryos had higher number of GABAergic interneuron synapses, as shown by higher GAD67 mRNA (by 3-fold) and K(+)/Cl(-) KCC2 mRNA expression (by 4.3-fold) (responsible for the conversion of GABA responses from depolarizing to inhibitory), without altering the expression of GABA and glutamate ionotropic receptors. These data indicate that BDNF controls both GABAergic pre- and postsynaptic sites.

KEs proceeding the AO (decreased cognition), such as "Reduced BDNF Release" and "Decreased synaptogenesis" are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure, as a reference chemical, are reported. These studies do not refer to TH disruption; however, they provide empirical support for this KER (Reduced release of BDNF leads to decreased synaptogenesis).

Synaptic structural plasticity was shown to be modified by Pb treatment during early (pre-weaning) or late (post-weaning) brain development in rats exposed to 2 mM Pb in drinking water for 3 weeks (Xiao et al., 2014). An iron chelator (clioquinol) can rescue the Pb-induced impairment of synaptic plasticity in hippocampus (Chen et al., 2007), showing that Pb can affect synaptogenesis and synaptic plasticity. Primary hippocampal neurons obtained from ED18 rat pups and treated with Pb (1, 2 microM) for 5 days exhibited pre-synaptic deficits due to disruption of NMDAR-dependent BDNF signaling (Neal et al., 2010; Stansfield et al., 2012). A decrease in bdnf expression was observed in mouse embryonic stem cells differentiated into neurons, if they were exposed to Pb 0.1 microM throughout the whole differentiation process (Sanchez-Martin et al., 2013). Similar alterations in gene expression patterns of neural markers (synapsin 1), neurotrophins (bdnf), transcription factors and glutamate-related genes were found in mice, when their mothers were exposed to 0-3 ppm of Pb in drinking water from 8 weeks prior to mating, through gestation and until postnatal day 10 (Sanchez-Martin et al., 2013).

Alterations of BDNF signaling is probably not the only mechanism leading to impaired synaptogenesis and synaptic plasticity. Indeed NMDAR activity can also modulate nitric oxide (NO) signaling. Exogenous NO addition during Pb exposure results in complete recovery of whole-cell synaptophysin levels and partial recovery of synaptophysin and synaptobrevin in synapses in Pb-exposed neurons (Neal et al., 2012). In addition, in Wistar rats, the anti-oxidant and radical scavenger quercetin was able to relieve the impairment of synaptic plasticity induced by chronic Pb exposure (from parturition through adulthood (PND 60); 0.2% Pb in drinking water of mothers and post-weaning pups) (Hu et al., 2008), suggesting that oxidative stress can also interfere with synapse formation.

Additionally, while PTU (a TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in the study from Cortés and colleagues (Cortés et al., 2012), treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB protein, the BDNF receptor, resulted reduced at the PSD of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD. These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain. However, even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.

Results variability from study to study is due to different experimental study designs, accounting for differences in brain development stages (PND vs adult), times of exposures to chemicals, and regional brain differences.

There is a lack of studies directly linking BDNF levels (gene and/or protein) with the quantitative analysis of synaptogenesis induced by decreased TH levels, and therefore no robust quantitative information can be provided. However, in the AOP 13 ("Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" https://aopwiki.org/aops/13) direct associations between Pb-induced decrease of BDNF (protein and/or mRNA) and decrease of pre- and post-synaptic proteins are discussed, supported also by quantitative analyses of spines and dendrite morphology.

References

Aguado F, Carmona MA, Pozas E, Aguiló A, Martínez-Guijarro FJ, Alcantara S, Borrell V, Yuste R, Ibañez CF, SorianoE. (2003). BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl–co-transporter KCC2. Development 130:1267-1280.

Ambrogini P, Lattanzi D, Ciuffoli S, Betti M, Fanelli M, Cuppini R. (2013). Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: possible role of BDNF. Brain Res 1534: 1-12.

Buckmaster PS, Ingram EA, Wen X. (2009). Inhibition of the mammalian target of rapamycin signaling pathway suppresses dentate granule cell axon sprouting in a rodent model of temporal lobe epilepsy. J Neurosci. Jun 24; 29(25):8259-69.

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Chapleau CA, Larimore JL, Theibert A, Pozzo-Miller L. (2009). Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism. J Neurodev Disord;1:185–196.

Chen WH, Wang M, Yu SS, Su L, Zhu DM, She JQ, et al. (2007). Clioquinol and vitamin B12 (cobalamin) synergistically rescue the lead-induced impairments of synaptic plasticity in hippocampal dentate gyrus area of the anesthetized rats in vivo. Neuroscience 147(3): 853-864.

Cortés C, Eugenin E, Aliaga E, Carreño LJ, Bueno SM, Gonzalez PA, Gayol S, Naranjo D, Noches V, Marassi MP, Rosenthal D, Jadue C, Ibarra P, Keitel C, Wohllk N, Court F, Kalergis AM, Riedel CA. (2012). Hypothyroidism in the adult rat causes incremental changes in brain-derived neurotrophic factor, neuronal and astrocyte apoptosis, gliosis, and deterioration of postsynaptic density. Thyroid. Sep;22(9):951-63.

Danglot L, Triller A, Marty S. (2006). The development of hippocampal interneurons in rodents. Hippocampus. 16:1032-1060.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology, Feb;157(2):774-87

Hu Y, Russek SJ. (2008). BDNF and the diseased nervous system: a delicate balance between adaptive and pathological processes of gene regulation. J Neurochem. 105:1-17.

Hu P, Wang M, Chen WH, Liu J, Chen L, Yin ST, et al. (2008). Quercetin relieves chronic lead exposure-induced impairment of synaptic plasticity in rat dentate gyrus in vivo. Naunyn Schmiedebergs Arch Pharmacol. Jul;378(1):43-51.

Jin X, Hu H, Mathers PH, Agmon A. (2003). Brain-derived neurotrophic factor mediates activity-dependent dendritic growth in nonpyramidal neocortical interneurons in developing organotypic cultures. J Neurosci 23:5662–5673.

Koibuchi N, Yamaoka S, Chin WW. (2001). Effect of altered thyroid status on neurotrophin gene expression during postnatal development of the mouse cerebellum. Thyroid 11:205–210.

Leal G, Comprido D, Duarte CB. (2014). BDNF-induced local protein synthesis and synaptic plasticity. Neuropharmacology 76 Pt C: 639-656.

Lu B, Nagappan G, Guan X, Nathan PJ, Wren P. (2013). BDNF-based synaptic repair as a disease-modifying strategy for neurodegenerative diseases. Nat Rev Neurosci 14(6): 401-416.

Marty S, Wehrle R, Sotelo C. (2000). Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus. J Neurosci 20: 8087–8095.

Neal AP, Stansfield KH, Worley PF, Thompson RE, Guilarte TR. (2010). Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: potential role of NMDA receptor-dependent BDNF signaling. Toxicol Sci 116(1): 249-263.

Neal AP, Stansfield KH, Guilarte TR. (2012). Enhanced nitric oxide production during lead (Pb(2)(+)) exposure recovers protein expression but not presynaptic localization of synaptic proteins in developing hippocampal neurons. Brain Res 1439: 88-95.

Polleux F, Whitford KL, Dijkhuizen PA, Vitalis T, Ghosh A. (2002). Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling. Development 129:3147–60.

Rico B, Xu B, Reichardt LF. (2002). TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum. Nat Neurosci 5:225–233.

Sanchez-Martin FJ, Fan Y, Lindquist DM, Xia Y, Puga A. (2013). Lead induces similar gene expression changes in brains of gestationally exposed adult mice and in neurons differentiated from mouse embryonic stem cells. PLoS One 8(11): e80558.

Sato K, Akaishi T, Matsuki N, Ohno Y, Nakazawa K. (2007). beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells. Brain Res. May 30;1150:108-20.

Schildt S, Endres T, Lessmann V, Edelmann E. (2013). Acute and chronic interference with BDNF/TrkB-signaling impair LTP selectively at mossy fiber synapses in the CA3 region of mouse hippocampus. Neuropharmacology. Aug;71:247-54.

Schjetnan AG, Escobar ML. (2012). In vivo BDNF modulation of hippocampal mossy fiber plasticity induced by high frequency stimulation. Hippocampus. Jan;22(1):1-8.

Seil FJ, Drake-Baumann R. (2000). TrkB receptor ligands promote activity-dependent inhibitory synaptogenesis. J Neurosci 20: 5367–73.

Shafiee SM, Vafaei AA, Rashidy-Pour A. (2016). Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise. Neuroscience. Aug 4;329:151-61.

Stansfield KH, Pilsner JR, Lu Q, Wright RO, Guilarte TR. (2012). Dysregulation of BDNF-TrkB signaling in developing hippocampal neurons by Pb(2+): implications for an environmental basis of neurodevelopmental disorders. Toxicol Sci 127(1): 277-295.

Vicario-Abejon C, Collin C, McKay RD, Segal M. (1998). Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons. J Neurosci 18:7256–71

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Xiao Y, Fu H, Han X, Hu X, Gu H, Chen Y, et al. (2014). Role of synaptic structural plasticity in impairments of spatial learning and memory induced by developmental lead exposure in Wistar rats. PLoS One 9(12): e115556.

BDNF, Reduced leads to Impairment, Learning and memory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from in vivo studies with rodents.

How Does This Key Event Relationship Work

BDNF and its high-affinity receptor TrkB are widely expressed in the mammalian brain (Lewin and Barde, 1996). They play a crucial role in the development, maintenance and functioning of the CNS (Huang and Reichardt, 2003; Shafiee et al., 2016). BDNF is known to be directly regulated by thyroid hormones and plays essential roles during the critical period of fetal brain development (Wang et al., 2006), including cell proliferation, migration, differentiation, synaptogenesis and neuronal network formation. In addition, neuronal activity regulates BDNF transcription, transport of BDNF mRNA and protein into dendrites and the activity-dependent secretion of BDNF, which, in turn, modulate synaptic plasticity, synaptogenesis and memory formation (Bekinschtein et al., 2008).

Developmental thyroid hormone insufficiency is associated with reduced cognitive functions and lowered BDNF levels, as shown in both humans and animal models (Chakraborty et al., 2012). For instance, in rats, maternal thyroidectomy significantly reduces BDNF expression in the brain of developing pups (Liu et al., 2010), leading to learning and memory deficits. Prenatal exposure to PTU also leads to reduced hippocampal BDNF in neonatal rats (Chakraborty et al., 2012). This evidence supports the link between decrease of BDNF and learning and memory impairment described in this indirect KER.

Weight of Evidence

The BDNF gene is a key signal transduction element required for synaptic plasticity and many forms of associative learning (Lu et al., 2005; Park et al., 2013). Moreover, reduced function of BDNF leads to neurodevelopmental and learning disorders (Bienvenu et al., 2006). BDNF plays an important role in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development (Chapleau et al., 2009). Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, as well as in mouse models of these disorders (Chapleau et al., 2009).

Some studies have shown associations between decrease of BDNF (e.g., as a consequence of exposure to several pollutants, such as BPA) and reduction of memory and learning:

- Wang et al., 2016 Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on PND 21 with the object recognition task. Data revealed a decrease in BDNF (~ 38% decrease at 50 mg/kg BW/day vs control) in the hippocampus. BPA-exposed male offspring underwent memory and cognitive impairments: they not only spent more time (~ 43% more, at 1.5 hr after training) in exploring the familiar object at the highest dose than the control, but also displayed a significant decrease in the object recognition index (at 50 mg/kg BW/day, ~ 54% lower short term memory measured 1.5 hr after training).

- Jang et al., 2012 In this study, pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. Exposure of F0 mice to BPA (10 mg/kg) decreased hippocampal neurogenesis (~ 30% decrease of hippocampal BrdU⁺ cells vs control) in F2 female mice. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (causing inhibition of NIS function) in pregnant mothers could decrease hippocampal neurogenesis and cognitive function in future generations.

- Shafiee et al., 2016 This study investigated the effects of PTU (TPO inhibitor, 100 mg/L) in pregnant rats to evaluate the effects elicited by maternal hypothyroidism in the offspring. PTU was added to the drinking water from gestation day 6 to PND 21. Analysis of hippocampal BDNF levels, and learning and memory tests were performed on PNDs 45-52 on pups. These results indicated that hypothyroidism during the fetal period and the early postnatal period was associated with: (i) ~ 70% reduction of total serum T4, (ii) ~ 5-fold increase of total serum TSH levels (on PND 21; no significant differences could be found at the end of the behavioral testing, on PND 52), (iii) reduction of hippocampal BDNF protein levels (~ 8% decrease vs control), (iv) impairment of spatial learning and memory, in both male and female rat offspring.

- Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016: Long-term potentiation (LTP) is a model of activity-dependent synaptic plasticity critical for learning and memory. LTP is impaired in both sub-regions of hippocampus under conditions of TH deficiency, and these impairments persist in the adult offspring on recovery of euthyroid status. LTP induces activation of neurotrophins, particularly BDNF and related signaling molecules. Induction of these pathways underlies the persistence of experience-dependent plasticity. Offspring of hypothyroid animals are deficient in LTP and in the induction of neurotrophin gene changes in response to neuronal activation. Similar plasticity is operative during early brain development, influencing synapse formation and formation of neural networks.

- Gilbert et al., 2016 Exposure to PTU during development produced dose-dependent reductions in mRNA expression of nerve growth factor (Ngf) in whole hippocampus of neonates. These changes in basal expression persisted to adulthood despite the return to euthyroid conditions in blood. Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus (e.g., at PND14, rats treated with 3 ppm PTU, underwent reduction of Bdnft by ~25%, Bdnfiv by ~10%, Ngf by ~25%, and Pval by ~50%) and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2^nd trial resulted ~60% higher in rats treated with 10 ppm PTU).

Liu et al., 2010 This study assessed the effects of hypothyroidism in 60 female rats who were divided into three groups: (i) maternal subclinical hypothyroidism (total thyroidectomy with T4 infusion), (ii) maternal hypothyroidism (total thyroidectomy without T4 infusion), and (iii) control (sham operated). Data showed that rat pups born from subclinical hypothyroidism dams had lower BDNF mRNA expression (on PND 3) and protein level (on PND 3 and PND 7) in the hippocampus. Moreover, the Morris water maze tests revealed that pups from the subclinical hypothyroidism group showed long-term memory deficits, and a trend toward short-term memory deficits.

- Wang et al., 2012: This study showed that maternal subclinical hypothyroidism impairs spatial learning in the offspring, as well as the efficacy and optimal time of T4 treatment in pregnancy. Female adult Wistar rats were randomly divided into six groups: control, hypothyroid (H), subclinical hypothyroid (SCH) and SCH treated with T4, starting from GD10, GD13 and GD17, respectively, to restore normal TH levels. Results indicate that progenies of both SCH and H groups had lower levels of BDNF protein (~35% lower in both groups) and also of Egr1, Arc, and p-ERK compared to controls. T4 treatment ameliorated these protein expression changes in the progeny of rats with subclinical hypothyroidism. Moreover, the SCH and H groups demonstrated significantly longer mean latency in the water maze test (on the 2^nd training day, latency was ~83% higher in H group, and ~50% higher in SCH), and a lower amplification percentage of the amplitude (~15% lower in H group, and 12% lower in SCH), and slope of the field excitatory postsynaptic potential (fEPSP) recording (~20% lower in H group, and 17% lower in SCH), compared to control group. T4 treatment at GD10 and GD13 significantly shortened mean latency and increased the amplification percentage of the amplitude and slope of the fEPSPs of the progeny of rats with subclinical hypothyroidism. However, T4 treatment at GD17 showed only minimal effects on spatial learning in the offspring. Altogether these data indicate direct correlation between decrease of BDNF and learning and memory deficits.

There are no inconsistencies in this KER; however, alterations of BDNF signalling is reliably not the only mechanism leading to impaired learning and memory. Additional studies are required to better correlate BDNF levels, TH brain levels with learning and memory tests performed simultaneously.

Despite some studies reporting strong associations between decrease of BDNF and learning and memory deficits (e.g., Liu et al., 2010; Wang et al., 2012; Shafiee et al., 2016), it is still not defined how much decrease in BDNF release is needed to observe learning and memory impairment. This highlights the need to produce empirical data based models for this KER.

References

Bekinschtein P, Cammarota M, Katche C, Slipczuk L, Rossato JI, Goldin A, Izquierdo I, Medina JH. (2008). BDNF is essential to promote persistence of long-term memory storage. Proc Natl Acad Sci U S A, 105, pp. 2711–2716.

Bienvenu T, Chelly J. (2006). Molecular genetics of Rett syndrome: When DNA methylation goes unrecognized. Nat. Rev. Genet; 7:415–426.

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Chapleau CA, Larimore JL, Theibert A, Pozzo-Miller L. (2009). Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism. J Neurodev Disord;1:185–196.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Sanchez-Huerta K, Wood C. (2016). Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Gilbert ME, Sui L. (2006). Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Huang EJ, Reichardt LF. (2003). Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem, 72, pp. 609–642.

Jang YJ, Park HR, Kim TH, Yang WJ, Lee JJ, Choi SY, Oh SB, Lee E, Park JH, Kim HP, Kim HS, Lee J. (2012). High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations. Toxicology. Jun 14;296(1-3):73-82.

Lewin GR, Barde YA. (1996) Physiology of the neurotrophins. Annu Rev Neurosci, 19, pp. 289–317.

Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H. (2010). The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring. Thyroid 20:909–915.

Lu B, Pang TP, Woo NH (2005). The Yin and Yang of neurotrophin action. Nat. Rev. Neurosci. 2005;6:603–614.

Park H, Poo MM. Neurotrophin regulation of neural circuit development and function. (2013). Nat. Rev. Neurosci;14:7–23.

Shafiee SM, Vafaei AA, Rashidy-Pour A. (2016). Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise. Neuroscience. Aug 4;329:151-61.

Wang Y, Su B, Xia Z. (2006). Brain-derived neurotrophic factor activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade. J Biol Chem, 281, pp. 35965–35974.

Wang S, Teng W, Gao Y, Fan C, Zhang H, Shan Z. (2012). Early levothyroxine treatment on maternal subclinical hypothyroidism improves spatial learning of offspring in rats. J Neuroendocrinol 24:841–848.

Wang C, Li Z, Han H, Luo G, Zhou B, Wang S, Wang J. (2016). Impairment of object recognition memory by maternal bisphenol A exposure is associated with inhibition of Akt and ERK/CREB/BDNF pathway in the male offspring hippocampus. Toxicology. Feb 3;341-343:56-64.