Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates

Neurotoxicology and Teratology 33 (2011) 464–472

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Neurotoxicology and Teratology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / n e u t e r a

Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates S.M. Lasley a, M.E. Gilbert b,⁎ a b

Dept. of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, USA Toxicity Assessment Division, Neurotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA

a r t i c l e

i n f o

Article history: Received 7 January 2011 Received in revised form 8 April 2011 Accepted 8 April 2011 Available online 17 April 2011 Keywords: Brain-derived neurotrophic factor Hypothyroidism Hippocampus Cortex Cerebellum Developmental

a b s t r a c t Brain-derived neurotrophic factor (BDNF) is a neurotrophin critical for many developmental and physiological aspects of CNS function. Severe hypothyroidism in the early neonatal period results in developmental and cognitive impairments and reductions in mRNA and protein expression of BDNF in a number of brain regions. The present study examined the impact of modest levels of developmental thyroid hormone insufficiency on BDNF protein expression in the hippocampus, cortex and cerebellum in the neonatal and adult offspring of rat dams treated throughout pregnancy and lactation. Graded levels of hormone insufficiency were induced by adding propylthiouracil (PTU, 0, 1, 2, 3 and 10 ppm) to the drinking water of pregnant dams from early gestation (gestational day 6) until weaning of the pups. Pups were sacrificed on postnatal days (PN) 14 and 21, and ~ PN100, and trunk blood collected for thyroid hormone analysis. Hippocampus, cortex, and cerebellum were separated from dissected brains and assessed for BDNF protein. Dose-dependent reductions in serum hormones in dams and pups were produced by PTU. Consistent with previous findings, age and regional differences in BDNF concentrations were observed. However, no differences in BDNF expression were detected in the preweanling animals as a function of PTU exposure; yet dose-dependent alterations emerged in adulthood despite the return of thyroid hormone levels to control values. Males were more affected by PTU than females, BDNF levels in hippocampus and cortex were altered but not those in cerebellum, and biphasic dose–response functions were detected in both sexes. These findings indicate that BDNF may mediate some of the adverse effects accompanying developmental thyroid hormone insufficiency, and reflect the potential for delayed impact of modest reductions in thyroid hormones during critical periods of brain development on a protein important for normal synaptic function. Published by Elsevier Inc.

1. Introduction Thyroid hormones are critical for normal brain development with deficiencies during the gestational and early postnatal period resulting in severe neurological deficits. Even modest reductions in thyroid hormones during the critical period of brain development produce morphological alterations, synaptic dysfunction, and behavioral impairments (see Gilbert and Zoeller, 2010 for review). The action of thyroid hormones is primarily mediated by thyroid hormone receptors (TR) that exert transcriptional control over a number of target genes. Although many genes have been reported to be altered by perinatal hypothyroidism (e.g., Anderson et al., 2003; Bernal, 2002; Koibuchi and Chin, 2000; Oppenheimer and Schwartz, 1997; Royland et al., 2008; Takahashi et al., 2008; Thompson and Potter, 2000), the

⁎ Corresponding author at: Toxicity Assessment Division, Neurotoxicology Branch (MD-B105-05), National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA. Tel.: + 1 919 541 4394; fax: + 1 919 541 4849. E-mail address: [email protected] (M.E. Gilbert). 0892-0362/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.ntt.2011.04.001

subset of TR-regulated genes that is critical for normal brain development has not been elucidated. Brain-derived neurotrophic factor (BDNF) is a protein belonging to the neurotrophin family of growth factors. BDNF is the most functionally diverse neurotrophin, and several clinical reports have implicated this protein in neurodevelopmental disorders, neurodegenerative disease, psychiatric disorders, and depression (see Lewin and Barde, 1996; Sheikh et al., 2010; Yoshii and Constantine-Paton, 2010). The activity of BDNF is central to many aspects of CNS development, ranging from neuronal differentiation and survival to synaptogenesis (Lewin and Barde, 1996; Lu and Figurov, 1997). In the adult brain, BDNF also plays a significant role in neuroprotection, neurogenesis, activity-dependent synaptic plasticity, and learning (Alonso et al., 2002; Bath et al., in press; Heldt et al., 2007; Takei et al., 2011; Chen et al., 2007, 2005; Di Fausto et al., 2007; Messaoudi et al., 2002; Pérez-Navarro et al., 1999; Rex et al., 2007; Ying et al., 2002). A number of previous reports have documented increases in expression of BDNF in cerebellum, cortex, and hippocampus in response to thyroid hormone administration to induce hyperthyroidism in developing and adult rats (Sui et al., 2010; Camboni et al., 2003;

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Lüesse et al., 1998; Roskoden et al., 1999; Shulga et al., 2009). In contrast, studies examining the impact of thyroid hormone insufficiency on BDNF expression have not produced consistent results. Differences in degree, timing, and duration of hormone insufficiency, brain region sampled, age, species and strain of test animal, and endpoint assessed (protein or mRNA) have complicated the synthesis of findings across studies. For example, Koibuchi et al. (1999, 2001) reported reductions in mRNA and/or protein in neonatal rats and mice exposed to high doses of propylthiouracil (PTU) or a related goitrogen, methimazole (MMI), in cerebellum. No such changes were evident in cortical tissue from the same animals. Like Koibuchi et al. (2001), Sinha et al. (2009) reported reductions in BDNF mRNA in cerebellum in PN8 offspring exposed to MMI (250 ppm) beginning in early gestation. Similarly, in pups exposed to PTU beginning in late gestation (GD17), Neveu and Arenas (1996) reported reductions in cerebellar BDNF mRNA on PN15 and PN30. In contrast, AlvarezDolado et al. (1994) observed no change in BDNF in hippocampus, cortex, or cerebellum at PN15 or PN90 following high doses of MMI beginning on GD9. The latter observation in hippocampus is consistent with a recent report in adult offspring by Opazo et al. (2008) using a transient model of maternal hypothyroidism in which MMI was administered to dams between GD12 and 15 only. With the possible exception of Opazo et al. (2008), the bulk of these studies were conducted under conditions of severe maternal hypothyroidism, were focused on the cerebellum, and made assessments during the preweaning period when offspring were still experiencing thyroid hormone deficiency. In both humans and animal models it is well documented that neurological deficits are induced by developmental hypothyroidism (see Zoeller and Rovet, 2004). Previous work from our laboratory and others has revealed alterations in hippocampal physiology, brain structure, and cognitive function following developmental thyroid hormone insufficiency (e.g., Akaike et al., 1999; Sui and Gilbert, 2003; Sui et al., 2005; Opazo et al., 2008; Axelstad et al., 2008; Sharlin et al., 2008, 2010; Goodman and Gilbert, 2007). Many of these deficiencies were evident at relatively modest degrees of hormone reductions and persisted to adulthood despite termination of exposure and full recovery of circulating levels of thyroid hormone (Opazo et al., 2008; Auso et al., 2004; Axelstad et al., 2008; Sui and Gilbert, 2003). Given the well documented role of BDNF on brain development and in adult brain function and plasticity (e.g., Lewin and Barde, 1996; Lu and Figurov, 1997), we propose that alterations in BDNF may underlie some of the persistent neurological impairments associated with developmental hypothyroidism. To date, limited data are available for BDNF in animal models of moderate thyroid hormone insufficiency. The present study was conducted to examine the impact of graded levels of thyroid hormone insufficiency from early gestation to weaning on the pattern of BDNF protein expression in multiple brain regions in neonatal and adult offspring.

2. Methods 2.1. Subjects Pregnant Long–Evans rats were obtained from Charles River (Raleigh, NC) on gestational day (GD) 2 and housed individually in standard plastic hanging cages in an AAALAC-accredited animal facility. Body weights of dams were monitored throughout gestation and the postnatal period; body weights of offspring were monitored from PN5 to PN35. All animal treatments were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize the number of animals utilized. The housing rooms were maintained on a 12:12 light–dark cycle, and animals were permitted free access to standard laboratory chow and tap water.

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2.2. Hormone insufficiency during development Beginning on GD6 and continuing until postnatal day (PN) 21, dams were rendered hypothyroid by addition of 0, 1, 2 or 3 ppm of the thyroid hormone synthesis inhibitor PTU (Sigma, St. Louis, MO) to the drinking water (0–0.0003% solutions). The day of birth was designated PN0 and all litters were culled to 10 pups on PN4. Exposure to PTU terminated when pups were weaned on PN21. A second experiment was performed to determine if failure to replicate previously reported effects of hypothyroidism on BDNF at young ages was due to the moderate degree of hypothyroidism induced, or a sex-dependent effect that was missed by limiting the initial assessment to neonatal females. Pregnant dams were exposed to 0 and 10 ppm (0 and 0.001%) PTU and one male and one female pup were harvested from each litter on PN21 and ~PN90. Weaning of pups from the 10 ppm dose group was delayed until PN28 due to developmental delays that impacted pup viability. This dose of PTU, despite producing a more severe state of hypothyroidism than that of the 3 ppm group (based on serum hormones, body weight gains, and profound developmental delays), still fell an order of magnitude below the standard dose used in the published literature (e.g., 200 ppm, 0.02%). At weaning offspring were transferred to plastic hanging cages (two animals of same sex/cage) and were permitted free access to food and tap water. 2.3. Thyroid hormones One female pup per litter was sacrificed by decapitation on PN14 and PN21. Trunk blood was collected for thyroid hormones, and brain was harvested for BDNF determinations. These ages were chosen as they represent ages typically evaluated in investigations of BDNF ontogeny (e.g., Friedman et al., 1998; Katoh-Semba et al., 1997; Viberg et al., 2008; Das et al., 2001) and developmental studies of thyroid hormone insufficiency (Axelstad et al., 2008; Gilbert and Sui, 2006; Sharlin et al., 2010; Koibuchi et al., 2001). We sampled females as no gender-related differences in serum T3 and T4 have been evident in preweaning animals (Gilbert and Sui, 2006; Axelstad et al., 2008). Dams were sacrificed at weaning of the pups and blood collected in the same manner. Serum was separated via centrifugation and stored at -80 °C for later analysis. One male and one female adult offspring were sacrificed from each litter between PN97 and 107, blood collected for serum hormone analysis, and the brain harvested from the same animal. Serum concentrations of total thyroxine (T4) and total triiodothyronine (T3) were analyzed by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). All determinations of total T4 and total T3 were performed in duplicate and the intra- and inter-assay variability ranged from 9 to 12%. The lowest calibrator used for hormone analysis was 10 ng/dl and 5 ng/ml for the T3 and T4 assays, respectively. The minimum detectable concentration (MDC) for each assay was determined statistically (3 standard deviations above background levels). For all T3 assays the MDC was 7.8 ng/dl, and for the T4 assay the MDC was 4.9 ng/ml. In the majority of cases, values fell between the standards used to calibrate the assay and serum hormones were interpolated from these standard curves. In cases where the sample result was below the level of detection, i.e., a large proportion of 10 ppm serum samples from pups, the result was set by default to the MDC for statistical purposes. 2.4. BDNF ELISA In animals sacrificed for serum hormone analysis brains were quickly removed and cortex, hippocampus, and cerebellum dissected, frozen in liquid nitrogen, and stored at − 80 °C until analysis for BDNF protein expression. BDNF expression was determined with an Emax Immunoassay System (Promega) according to the manufacturer's

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instructions with minor modifications. Microplates were treated with a monoclonal anti-BDNF antibody in carbonate coating buffer (pH 9.7), covered and incubated without shaking for 36–48 h at 4 °C. Plates were then emptied, washed once with Tris-buffered saline (20 mM Tris–HCl, pH 7.6, 150 mM NaCl) with 0.05% Tween-20 (TBST; ELX50 Plate Washer, Bio-tek Instruments), and blocked without shaking for 1 h at room temperature. After washing a standard curve was prepared with authentic BDNF, and appropriately diluted samples (in blocking buffer) were added to other wells (total volume = 100 μl). Plates were then incubated with shaking for 2 h at room temperature and washed 5 times with TBST. A polyclonal anti-human BDNF was then diluted in blocking buffer and added to the wells followed by shaking for 2 h at room temperature. After 5 washes with TBST the HRP-conjugated anti-IgY secondary antibody was diluted in blocking buffer and added to the wells with shaking for 1 h at room temperature. After 5 more washes with TBST a peroxidase substrate was mixed with 3,3′,5,5′-tetramethylbenzidine (TMB, Thermo Scientific) solution and added to the wells, and the plates were shaken for 15 min at room temperature. The reaction was stopped with 2 M sulfuric acid, and absorbance was read in a microplate reader (SpectraMax Plus, Molecular Devices) at 450 nm using Softmax Pro software. Tissue samples for the immunoassay were sonicated in lysis buffer with the following components (in mM): NaCl 137, Tris–HCl (pH 8.0) 20, phenylmethylsulfonylfluoride 1, NaVO4 0.5, with 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1% NP40 and 10% glycerol. Aliquots of each sample lysate were analyzed in triplicate for total protein by the bicinchoninic acid assay (Thermo Scientific) and used to normalize BDNF values. Sample BDNF protein expression was

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3. Results 3.1. Thyroid hormones Thyroid hormones were reduced in a dose-dependent manner in dams and pups in response to 0, 1, 2 and 3 ppm of PTU. T4 was dose-dependently reduced at all dose levels on PN14 [F(4,44) = 12.91, p b 0.001] and PN21 [F(4,52) = 33.40, p b 0.001] and in dams at

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Body weight for dams and pups were assessed by a repeated measure ANOVA with litter as the unit of analysis. Serum hormones were analyzed by one-way ANOVAs at each age and for the dams. In the event of a significant main effect or interaction, mean contrast tests were performed using Dunnett's t-test with alphas set at p b 0.05. BDNF protein expression was evaluated using two-way ANOVAs in each brain region with either Age and Dose or Sex and Dose as the main factors. In adults, when the main effect of Sex was significant, one-way step down ANOVAs were performed across PTU doses for each sex. Where appropriate, mean contrast tests were performed using Dunnett's ttest and an alpha level set to 0.05.

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determined in triplicate in each experimental set of plates, and each run was repeated three times. Samples were counterbalanced so that all treatments, ages, and genders were represented on each plate. Preliminary testing established the appropriate lysate dilutions to employ so that sample values fell within the optimum ranges of the total protein and BDNF standard curves.

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Fig. 1. Thyroid hormones are dose-dependently reduced by PTU in dams and pups during the postnatal period. (A) Serum (mean ± SEM) T4 was reduced in a graded fashion as a function of PTU dose in pups and dams (n = 5–15/group). (B) More modest reductions in circulating T3 were seen at 3 and 10 ppm PTU concentrations in pups and dams. Nonetheless, serum T4 (C) and T3 (D) exhibited full recovery by PN90. Significant main effects for Dose at each age with ANOVA were further evaluated with Dunnett's t-test,; *p b 0.05 compared to controls.

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weaning [F(4,53) = 7.73, p b 0.001] (Fig. 1A). T3 was also reduced on PN14 [F(4,46) = 166.37, p b 0.001)] and PN21 [F(4,54) = 114.37, p b 0.001] but to a lesser degree than T4. Dam T3 values were altered only in the highest dose group [F(4,55) = 32.04, p b 0.001] (Fig. 1B). Both T4 (Fig. 1C) and T3 (Fig. 1D) had fully recovered in adult male and female offspring when assessed ~ PN90 (all p's N 0.15). 3.2. Body weight in dams and pups No significant alterations in dam or pup weights were detected in response to 1–3 ppm PTU through PN22 (Fig. 2A, all p's N 0.22). At 10 ppm PTU small reductions in body weight were observed in dams [Dose × Age interaction, F(12,96) = 5.02, p b 0.001] beginning in late gestation (GD20) and extending to the early postnatal period (Fig. 2B, inset). Offspring maintained on 10 ppm displayed a slower growth rate (Fig. 2B), and significant reductions in body weight were evident by PN14 and became more profound with age [Dose × Age interaction, F(6,42) = 24.06, p b 0.001]. 3.3. BDNF expression varies by brain region and age Control levels of BDNF expression were highest in hippocampus (Fig. 3A) and much lower in cerebral cortex (Fig. 3B) and cerebellum

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Postnatal Day Fig. 2. (A) Mean ± SEM body weight of dams (inset) and pups in the low dose cohort, which were comparable to controls (n = 10–15/group). (B) Slight reductions in body weight were seen in dams in the last few days of pregnancy in response to 10 ppm PTU (inset, n = 5/group). Body weight deficits (mean ± SEM) were evident by PN14 in the offspring that became more apparent with age and did not fully recover by adulthood (data not shown) despite termination of PTU exposure at weaning. The broken line for controls reflects the break in the y-axis necessary to show the large increase in body weight that occurs between PN21 and PN35 (almost 100 g increase), which is not seen in the 10 ppm animals (~ 15 g). Significant main effects of Dose and Age in repeated measures ANOVA (see Section 3.2) were further evaluated by Dunnetts t. *p b 0.05 compared to 0 ppm group values at the same age.

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(Fig. 3C — note differences in y-axis scales). Brains were harvested from female neonates in the low dose cohort, so assessment of BDNF expression as a function of age was limited to a comparison of female animals only on PN14, PN21, PN~90. A significant effect of Age was observed in each brain region, with younger animals having greater expression of BDNF than adults (Fig. 3). In hippocampus and cerebellum, BDNF declined monotonically from PN14 to adulthood [F(2,96) = 112.76, p b 0.001, and F(2,96) = 28.12, p b 0.001, respectively] with the value at each age significantly different from values at other ages (all p's b 0.001). In cortex, BDNF levels were higher on PN21 than PN14, but declined to lowest levels in adulthood [F(2,94) = 24.30, p b 0.001]. 3.4. BDNF expression varies by sex and dose of PTU 3.4.1. Effects in neonates No effects of PTU were observed in hippocampus, cortex, or cerebellum from neonatal female pups on PN14 or PN21 in the low dose cohort (Fig. 3). In the high dose cohort, despite the 10 ppm dose of PTU inducing a more severe state of hypothyroidism, BDNF protein expression was not reduced in male or female offspring on PN21 (data not shown); BDNF expression on PN14 was not assessed in the high dose cohort. 3.4.2. Effects in adult offspring In contrast to the results in neonates, absolute BDNF values were altered in adult offspring as a function of exposure to PTU. Although all animals had returned to control levels of circulating thyroid hormones at the time of sacrifice ~PN90, significant differences in BDNF were detected in hippocampus (Fig. 4A). Statistical analyses revealed significant main effects of Dose [F(3,65) = 5.46, p b 0.002] and Sex [F(1,65) = 67.61, p b 0.001]. Step-down ANOVAs for each sex showed significant reductions in adult male [F(3,34) = 4.52, p b 0.009] but not in adult female [F(3,31) = 1.92, p N 0.14] offspring. In males BDNF reductions were restricted to the two lowest dose groups (Dunnett's t, p b 0.05) with a return to control levels at 3 ppm PTU. Similar trends were evident in cortex (Fig. 4B) with significant main effects observed for Dose [F(3,65) = 2.79, p b 0.047] and Sex [F(1,65) = 5.53, p b 0.022]. Step-down ANOVAs revealed a significant reduction in cortical BDNF in adult male [F(3,35), p b 0.004] but not in female offspring [F(3,33) = 0.91, p N 0.44]. In male cortex this reduction was evident only at the lowest dose (Dunnett's t, p b 0.05). No significant differences in BDNF expression in adult offspring were detected in cerebellum (Fig. 4C) as a function of Dose [F(3,66) = 0.15, p N 0.98] or Sex [F(1,66) = 0.15, p N 0.69]. As the high dose cohort was evaluated at a different time than the low dose cohort using different lots of ELISA plates and BDNF antibodies, a direct comparison of absolute levels of protein expression between the low and high dose cohorts was not possible (Elfving et al., 2010). However, data across all dose levels can be compared by expressing each value as a percent of the appropriate control (Fig. 5). The results of this analysis revealed significant main effects of Sex [F(1,92) = 11.24, p b 0.001] and Dose [F(4,92) = 8.70, p b 0.001] in hippocampus, and a marginal Sex × Dose interaction [F(4,92) = 2.43, p b 0.053]. The U-shaped dose–response function evident in the absolute neurotrophin levels in the low dose cohort described above was further reinforced in the present analysis (Fig. 5A). Step-down one-way ANOVAs by Sex indicated that in males [F(4,47) = 6.10, p b 0.001] low doses of PTU (1 ppm and 2 ppm) reduced hippocampal BDNF by ~ 40–45% (Dunnett's t, p b 0.05), and a return to control levels was seen at the two highest doses (3 and 10 ppm). In females [F(4,45) = 4.95, p b 0.002] significant reductions were limited to the 2 ppm dose group (Dunnett's t, p b 0.05) with a trend towards an increase above control levels at the highest PTU dose level.

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cortical BDNF levels in male offspring [step down F(4,50) = 3.63, p b 0.011] were limited to the lowest dose level (Dunnett's t, p b 0.05) with return to control levels at the higher doses. In females a significant effect of PTU on cortical BDNF levels [step down F(4,45) = 4.39, p b 0.004] was driven by an augmentation in BDNF protein expression in the 10 ppm dose group (Dunnett's t, p b 0.05).

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Age Fig. 3. BDNF protein expression as a function of age, PTU dose, and brain region in neonatal and adult female offspring. A significant effect of Age was detected in ANOVA for hippocampus (A), cortex (B), and cerebellum (C), see Section 3.3. Values are expressed as mean + SEM in pg/mg protein with n = 8–13 for each PTU dose at each age. ‡ p b 0.001 compared to the PND14 value; #p b 0.001 compared to the PND21 value.

The effect of PTU on cortical expression of BDNF in adult offspring was not as striking as that seen in hippocampus, however a similar biphasic dose–response pattern was evident (Fig. 5B). Results of ANOVA revealed significant main effects of Sex [F(1,95) = 4.89, p b 0.030] and Dose [F(4,95) = 5.48, p b 0.001] with a marginal Sex × Dose interaction [F(4,95) = 2.44, p b 0.052]. Reductions in

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Fig. 4. BDNF protein expression as a function of PTU dose and brain region in adult male and female offspring. Significant main effects of Sex and Dose, and a Sex × Dose interaction were evident in ANOVA, see Section 3.4. Values are expressed as mean + SEM in pg/mg protein with N = 8–11 for each PTU dose in each sex. Bracketed groups depict sex differences with step-down ANOVA: ‡ p b 0.01; & p b 0.05 compared to the values in adult males; *p b 0.05 compared to the 0 ppm value in adult males with Dunnett's t-test. No significant effect of PTU was detected in adult female offspring as a function of dose.

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PTU, ppm Fig. 5. Dose–effect relationships for BDNF protein expression and PTU dose in adult male and female offspring in each brain region. BDNF values are normalized to the 0 ppm value (= 100) for the respective gender in that brain area. Values are expressed as mean ± SEM in percent control with N = 5–11 for each PTU dose in each gender; *p b 0.05 compared to the 0 ppm value of the same gender. Marginal effects seen in female hippocampus at 10 ppm and cortex at 2 ppm are depicted as + p b 0.06 when contrasted to 0 ppm value in females.

Distinct from the effects observed in hippocampus and cortex, developmental exposure to PTU had no effect on cerebellar expression of BDNF at any Dose [F(4,94) = 0.12, p N 0.97] in either Sex [F(1,94) = 0.13, p N 0.71)] (Fig. 5C).

A number of novel observations resulted from this investigation of the impact of moderate thyroid hormone insufficiency on BDNF protein expression in three rat brain regions. PTU did not affect BDNF expression in the neonate, but surprisingly, altered BDNF expression profiles present in the hippocampus and cortex of adult offspring. Adult males displayed higher levels of BDNF than adult females, and males displayed greater reductions in BDNF than females in response to developmental thyroid hormone insufficiency. Biphasic dose– response functions were evident in both male and female adult offspring exposed to PTU in early life. BDNF was higher in all three brain regions studied during the neonatal period than in adulthood. This neurotrophin is widely expressed in the CNS, and distinct age- and region-specific profiles of protein expression have been previously reported (Friedman et al., 1991; 1998; Das et al., 2001; Kim et al., 2007; Maisonpierre et al., 1990; Numan et al., 2005; Viberg et al., 2008). The regional- and agedependent patterns of BDNF levels in control animals observed in the present study are consistent with many of these reports, both in terms of distribution and ontogeny (Kim et al., 2007; Das et al., 2001; Friedman et al., 1998; Viberg et al., 2008; Katoh-Semba et al., 1997). The hippocampus had the highest levels, with much lower concentrations of BDNF detected in the cortex and cerebellum, and younger animals had higher concentrations of BDNF than adults. Thyroid hormone insufficiency during development did not impact BDNF protein expression during the preweaning period in any of the three brain regions assessed despite significant reductions in circulating thyroid hormones in dams and offspring. These findings are consistent with some reports on transient gestational (GD15–19) (Opazo et al., 2008) or perinatal hormone deprivation (Alvarez-Dolado et al., 1994) on BDNF protein or mRNA expression (see Introduction). However, the current findings stand in contrast to other observations (e.g., Koibuchi et al., 1999, 2001; Neveu and Arenas, 1996; Sinha et al., 2009) in which severe hypothyroidism in rats and mice led to alterations in BDNF mRNA in the cerebellum. BDNF mRNA changes not accompanied by changes in BDNF protein have been reported by numerous investigators (Alonso et al., 2002; Pollock et al., 2001; Nanda and Mack, 1998). Lack of protein changes may reflect increased turnover of BDNF protein, lack of translation of newly synthesized BDNF mRNA, or treatment-related alterations in protein trafficking (see Pollock et al., 2001). More recently, Liu et al. (2010) induced a state of subclinical hypothyroidism by surgical removal of the dam's thyroid gland and partial replacement of hormone with subcutaneous injections to the dams and pups during gestation and lactation. Thyroidectomy without exogenous hormone supplementation produced a severe state of hypothyroidism and reduced BDNF mRNA and protein in the early (PN3 and PN7) and late (PN21) neonatal period. Subclinical hypothyroidism was accomplished by supplementing thyroidectomized dams with T4 to model a modest reduction in thyroid hormone levels that is perhaps more analogous to the graded degrees of thyroid hormone insufficiency induced in the present study. The subclinical model also reduced BDNF mRNA and protein expression in hippocampus in the very young pup on PN3 and PN7, but not on PN21. The latter finding in the subclinical model is consistent with our findings of no effect of PTU at any dose level tested in the preweanling rat (PN14 or PN21); unfortunately, animals in the current study were not assessed prior to PN14. To our knowledge, sex differences in basal levels of BDNF and the late emergence of effects of developmental thyroid hormone insufficiency on BDNF protein expression have not been previously reported. In terms of absolute levels of the neurotrophin, we observed higher levels in adult males than in adult females in hippocampus and cortex with no gender difference evident in cerebellum. Adult male offspring also exhibited a greater proportional reduction in BDNF levels than females in these regions in response to PTU, but males did not exhibit

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the elevation in BDNF expression seen in adult females at the highest PTU dose. This sex-dependent differential sensitivity to the effects of developmental thyroid hormone insufficiency on BDNF protein may be due to estrogenic modulation of BDNF expression or to interactions of estrogen and thyroid hormones in the brain (Graupner et al., 1991; Kia et al., 2001; Pfaff et al., 1994; Tang et al., 2004). Estrogen has proven to be a potent regulator of BDNF in rat brain (Gibbs, 1999; Sohrabji and Lewis, 2006; Zhu et al., 1996), and administration of exogenous estrogen increases BDNF expression in hippocampus (Aguirre and Baudry, 2009). Furthermore, hippocampal BDNF expression is more robustly augmented in female rats following traumatic brain injury than in males (Chen et al., 2005). Moreover, estrogen and thyroid receptors share a common consensus site on their hormone response elements (EREs and TREs) and thus interact to regulate gene expression (Graupner et al., 1991; Hirst et al., 1992; Zhu et al., 1996). Consequently, the presence of estrogen in adult female offspring may attenuate the effect of developmental thyroid hormone insufficiency on BDNF expression at low PTU doses and underlie the elevation in BDNF observed at the highest PTU dose. In contrast to the lack of effect during the preweanling period in this model of thyroid hormone insufficiency, BDNF protein expression in the cortex and hippocampus of adult offspring of PTU-exposed dams was disrupted. The observation of this late emergence of effect on neurotrophin expression in response to developmental thyroid hormone insufficiency is novel. Neither has the biphasic dose– response function evident in the current study been previously described. BNDF protein levels were reduced in adult offspring despite full return of the animals to euthyroid conditions at the time of testing. This reduction in BDNF was limited to the lowest doses of PTU and to the most modest reductions in circulating hormones in dams and pups during the neonatal period. As the dose of PTU and consequently the level of hormone insufficiency increased, BDNF expression recovered toward control levels. The reduction in BDNF values at 1–2 ppm PTU was most striking in the hippocampus of adult male offspring, and no differences were seen when moderate (3 ppm) or more severe (10 ppm) hypothyroid conditions were imposed. Females displayed an attenuated response at low doses of PTU with the degree of suppression of BDNF smaller than in males and limited to a single dose. In the presence of a more severe hypothyroid state induced by 10 ppm PTU, increases above control BDNF levels were observed in females in cortex and marginally in hippocampus. It is not at all clear what may underlie the delayed emergence of altered BDNF protein expression following developmental thyroid hormone insufficiency, or what this may mean functionally to the animal. Neither is there any simple explanation for the nonmonotonic dose–response relationships. Circulating levels of thyroid hormones typically return to control levels within a few weeks of termination of PTU exposure (Axelstad et al., 2008; Cooper et al., 1983), and no differences were seen in the present study at the adult time point. Therefore, a chronic reduction in circulating levels of thyroid hormone cannot account for the present findings. Elevated brain levels of BDNF and other neurotrophins accompany brain trauma and injury in adult and neonatal models where they serve a neuroprotective role and also are necessary for recovery and repair (Di Fausto et al., 2007; Chen et al., 2005). It is possible that hypothyroidism-induced reductions of BDNF evident at low doses of PTU are countered by the activation of protective processes that augment BDNF expression when more severe hypothyroid conditions prevailed (e.g. 3 and 10 ppm). This neuroprotective induction of BDNF serves to mask the effects of thyroid hormone insufficiency evident at lower doses and results in a biphasic dose–response pattern. Epigenetic modifications are often invoked to account for delayed manifestations of disease in response to early developmental insults (e.g., Barker, 2007; Gluckman et al., 2008). Two mechanisms of epigenetic modification of gene transcription – gene silencing by DNA methylation and gene activation by histone acetylation – have been

implicated in such nonheritable modifications of gene transcription (Dobosy and Selker, 2001; Fang and Lu, 2002; Santos et al., 2005; Miller et al., 2010). Up-regulation of DNA methyltransferase and down-regulation of histone acetylases have been recently reported for the hippocampus in a perinatal hypothyoid model and associated with decreases in BDNF gene and protein expression (Sui and Li, 2010). It is unknown if these mechanisms are engaged with more modest perturbation of the thyroid axis that is limited to the fetal and early neonatal period. If so, it is conceivable that such modifications in gene transcription could contribute to the altered BDNF protein expression in adulthood. Activation of opposing mechanisms with different responsiveness to variable degrees of hormone insufficiency could also plausibly constitute the basis for biphasic PTU dose–effect relationships. In summary, a late emergent impact of perinatal thyroid hormone insufficiency on BDNF protein expression was observed at a time when circulating levels of thyroid hormone had fully recovered. These persistent effects on the expression of this critical brain protein were seen following relatively modest and transient perturbations of the thyroid axis restricted to the early developmental period. BDNF in the adult brain has been implicated in adult neurogenesis, synaptic plasticity, learning, and recovery from injury (Shulga et al., 2008, 2009; Pérez-Navarro et al., 1999; Lu and Figurov, 1997; Lemkine et al., 2005; Desouza et al., 2005; Ambrogini et al., 2005), and its disruption may contribute to the neurological impairments associated with moderate levels of developmental thyroid hormone insufficiency (e.g., Gilbert et al., 2007; Gilbert and Sui, 2006; Lavado-Autric et al., 2003., Auso et al., 2004; Opazo et al., 2008; Sharlin et al., 2008). If BDNF and BDNF-induced signaling do mediate some of the adverse effects on brain development and function accompanying developmental thyroid hormone insufficiency, the factors influencing its protein expression are complex. It will be important to unravel this complexity if we are to elucidate the role of thyroid hormone in brain development and the functional impairments that persist as a consequence of its insufficiency. Conflict of interest No competing interests to report. Acknowledgments This document has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The authors thank Drs. Tom Zoeller and William Mundy for comments on an earlier version of this manuscript, and Willard Anderson, Meena Shanmugasundaram, and Brittney Hanerhoff for technical assistance. References Aguirre CC, Baudry M. Progesterone reverses 17beta-estradiol-mediated neuroprotection and BDNF induction in cultured hippocampal slices. Eur J Neurosci 2009;29(3): 447–54 Feb. Akaike M, Kato N, Ohno H, Kobayashi T. Hyperactivity and spatial maze learning impairment of adult rats with temporary neonatal hypothyroidism. Neurotoxicol Teratol 1999;13(3):317–22. Alonso M, Vianna MR, Depino AM, Mello e Souza T, Pereira P, Szapiro G, et al. BDNFtriggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 2002;12(4):551–60. Alvarez-Dolado M, Iglesias T, Rodríguez-Peña A, Bernal J, Muñoz A. Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain. Brain Res Mol Brain Res 1994;27(2):249–57 Dec. Ambrogini P, Cuppini R, Ferri P, Mancini C, Ciaroni S, Voci A, et al. Thyroid hormones affect neurogenesis in the dentate gyrus of adult rat. Neuroendocrinology 2005;81: 244–53. Anderson GW, Schoonover CM, Jones SA. Control of thyroid hormone action in the developing rat brain. Thyroid 2003;13:1039–56.

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