(BDNF) in Adults but Not in Neonates

Neurotoxicology and Teratology 33 (2011) 464–472

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Neurotoxicology and Teratology

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

Article history: Brain-derived neurotrophic factor (BDNF) is a neurotrophin critical for many developmental and Received 7 January 2011 physiological aspects of CNS function. Severe hypothyroidism in the early neonatal period results in Received in revised form 8 April 2011 developmental and cognitive impairments and reductions in mRNA and protein expression of BDNF in a Accepted 8 April 2011 number of brain regions. The present study examined the impact of modest levels of developmental thyroid Available online 17 April 2011 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 Keywords: fi Brain-derived neurotrophic factor hormone insuf ciency were induced by adding propylthiouracil (PTU, 0, 1, 2, 3 and 10 ppm) to the drinking Hypothyroidism water of pregnant dams from early gestation (gestational day 6) until weaning of the pups. Pups were Hippocampus sacrificed on postnatal days (PN) 14 and 21, and ~PN100, and trunk blood collected for thyroid hormone Cortex analysis. Hippocampus, cortex, and cerebellum were separated from dissected brains and assessed for BDNF Cerebellum protein. Dose-dependent reductions in serum hormones in dams and pups were produced by PTU. Consistent Developmental 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 subset of TR-regulated genes that is critical for normal brain development has not been elucidated. Thyroid hormones are critical for normal brain development with Brain-derived neurotrophic factor (BDNF) is a protein belonging to deficiencies during the gestational and early postnatal period the neurotrophin family of growth factors. BDNF is the most functionally resulting in severe neurological deficits. Even modest reductions in diverse neurotrophin, and several clinical reports have implicated this thyroid hormones during the critical period of brain development protein in neurodevelopmental disorders, neurodegenerative disease, produce morphological alterations, synaptic dysfunction, and behav- psychiatric disorders, and depression (see Lewin and Barde, 1996; ioral impairments (see Gilbert and Zoeller, 2010 for review). The Sheikh et al., 2010; Yoshii and Constantine-Paton, 2010). The activity of action of thyroid hormones is primarily mediated by thyroid hormone BDNF is central to many aspects of CNS development, ranging from receptors (TR) that exert transcriptional control over a number of neuronal differentiation and survival to synaptogenesis (Lewin and target genes. Although many genes have been reported to be altered Barde, 1996; Lu and Figurov, 1997). In the adult brain, BDNF also plays a by perinatal hypothyroidism (e.g., Anderson et al., 2003; Bernal, 2002; significant role in neuroprotection, neurogenesis, activity-dependent Koibuchi and Chin, 2000; Oppenheimer and Schwartz, 1997; Royland synaptic plasticity, and learning (Alonso et al., 2002; Bath et al., in press; et al., 2008; Takahashi et al., 2008; Thompson and Potter, 2000), the 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). ⁎ Corresponding author at: Toxicity Assessment Division, Neurotoxicology Branch A number of previous reports have documented increases in (MD-B105-05), National Health and Environmental Effects Research Laboratory, U.S. expression of BDNF in cerebellum, cortex, and hippocampus in Environmental Protection Agency, Research Triangle Park, NC 27711, USA. Tel.: +1 919 541 4394; fax: +1 919 541 4849. response to thyroid hormone administration to induce hyperthyroid- E-mail address: [email protected] (M.E. Gilbert). ism in developing and adult rats (Sui et al., 2010; Camboni et al., 2003;

0892-0362/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.ntt.2011.04.001 S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472 465

Lüesse et al., 1998; Roskoden et al., 1999; Shulga et al., 2009). In 2.2. Hormone insufficiency during development contrast, studies examining the impact of thyroid hormone insufficiency on BDNF expression have not produced consistent results. Beginning on GD6 and continuing until postnatal day (PN) 21, Differences in degree, timing, and duration of hormone insufficiency, dams were rendered hypothyroid by addition of 0, 1, 2 or 3 ppm of the brain region sampled, age, species and strain of test animal, and thyroid hormone synthesis inhibitor PTU (Sigma, St. Louis, MO) to endpoint assessed (protein or mRNA) have complicated the synthesis the drinking water (0–0.0003% solutions). The day of birth was of findings across studies. For example, Koibuchi et al. (1999, 2001) designated PN0 and all litters were culled to 10 pups on PN4. Exposure reported reductions in mRNA and/or protein in neonatal rats and mice to PTU terminated when pups were weaned on PN21. A second exposed to high doses of propylthiouracil (PTU) or a related experiment was performed to determine if failure to replicate goitrogen, methimazole (MMI), in cerebellum. No such changes previously reported effects of hypothyroidism on BDNF at young were evident in cortical tissue from the same animals. Like Koibuchi et ages was due to the moderate degree of hypothyroidism induced, or a al. (2001), Sinha et al. (2009) reported reductions in BDNF mRNA in sex-dependent effect that was missed by limiting the initial cerebellum in PN8 offspring exposed to MMI (250 ppm) beginning in assessment to neonatal females. Pregnant dams were exposed to early gestation. Similarly, in pups exposed to PTU beginning in late 0 and 10 ppm (0 and 0.001%) PTU and one male and one female pup gestation (GD17), Neveu and Arenas (1996) reported reductions in were harvested from each litter on PN21 and ~PN90. Weaning of pups cerebellar BDNF mRNA on PN15 and PN30. In contrast, Alvarez- from the 10 ppm dose group was delayed until PN28 due to Dolado et al. (1994) observed no change in BDNF in hippocampus, developmental delays that impacted pup viability. This dose of PTU, cortex, or cerebellum at PN15 or PN90 following high doses of MMI despite producing a more severe state of hypothyroidism than that of beginning on GD9. The latter observation in hippocampus is the 3 ppm group (based on serum hormones, body weight gains, and consistent with a recent report in adult offspring by Opazo et al. profound developmental delays), still fell an order of magnitude (2008) using a transient model of maternal hypothyroidism in which below the standard dose used in the published literature (e.g., MMI was administered to dams between GD12 and 15 only. With the 200 ppm, 0.02%). At weaning offspring were transferred to plastic possible exception of Opazo et al. (2008), the bulk of these studies hanging cages (two animals of same sex/cage) and were permitted were conducted under conditions of severe maternal hypothyroidism, free access to food and tap water. were focused on the cerebellum, and made assessments during the preweaning period when offspring were still experiencing thyroid 2.3. Thyroid hormones hormone deficiency. In both humans and animal models it is well documented that One female pup per litter was sacrificed by decapitation on PN14 neurological deficits are induced by developmental hypothyroidism and PN21. Trunk blood was collected for thyroid hormones, and brain (see Zoeller and Rovet, 2004). Previous work from our laboratory and was harvested for BDNF determinations. These ages were chosen as others has revealed alterations in hippocampal physiology, brain they represent ages typically evaluated in investigations of BDNF structure, and cognitive function following developmental thyroid ontogeny (e.g., Friedman et al., 1998; Katoh-Semba et al., 1997; Viberg hormone insufficiency (e.g., Akaike et al., 1999; Sui and Gilbert, 2003; et al., 2008; Das et al., 2001) and developmental studies of thyroid Sui et al., 2005; Opazo et al., 2008; Axelstad et al., 2008; Sharlin et al., hormone insufficiency (Axelstad et al., 2008; Gilbert and Sui, 2006; 2008, 2010; Goodman and Gilbert, 2007). Many of these deficiencies Sharlin et al., 2010; Koibuchi et al., 2001). We sampled females as no were evident at relatively modest degrees of hormone reductions and gender-related differences in serum T3 and T4 have been evident in persisted to adulthood despite termination of exposure and full preweaning animals (Gilbert and Sui, 2006; Axelstad et al., 2008). recovery of circulating levels of thyroid hormone (Opazo et al., 2008; Dams were sacrificed at weaning of the pups and blood collected Auso et al., 2004; Axelstad et al., 2008; Sui and Gilbert, 2003). Given the in the same manner. Serum was separated via centrifugation and well documented role of BDNF on brain development and in adult stored at -80 °C for later analysis. One male and one female adult brain function and plasticity (e.g., Lewin and Barde, 1996; Lu and offspring were sacrificed from each litter between PN97 and Figurov, 1997), we propose that alterations in BDNF may underlie 107, blood collected for serum hormone analysis, and the brain some of the persistent neurological impairments associated with harvested from the same animal. Serum concentrations of total developmental hypothyroidism. To date, limited data are available for thyroxine (T4) and total triiodothyronine (T3) were analyzed by BDNF in animal models of moderate thyroid hormone insufficiency. radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). All The present study was conducted to examine the impact of graded determinations of total T4 and total T3 were performed in duplicate levels of thyroid hormone insufficiency from early gestation to and the intra- and inter-assay variability ranged from 9 to 12%. weaning on the pattern of BDNF protein expression in multiple brain The lowest calibrator used for hormone analysis was 10 ng/dl and regions in neonatal and adult offspring. 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 2. Methods 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 2.1. Subjects used to calibrate the assay and serum hormones were interpolated from these standard curves. In cases where the sample result was Pregnant Long–Evans rats were obtained from Charles River below the level of detection, i.e., a large proportion of 10 ppm serum (Raleigh, NC) on gestational day (GD) 2 and housed individually in samples from pups, the result was set by default to the MDC for standard plastic hanging cages in an AAALAC-accredited animal statistical purposes. facility. Body weights of dams were monitored throughout gestation and the postnatal period; body weights of offspring were monitored 2.4. BDNF ELISA from PN5 to PN35. All animal treatments were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and In animals sacrificed for serum hormone analysis brains were every effort was made to minimize the number of animals utilized. quickly removed and cortex, hippocampus, and cerebellum dissected, The housing rooms were maintained on a 12:12 light–dark cycle, and frozen in liquid nitrogen, and stored at −80 °C until analysis for BDNF animals were permitted free access to standard laboratory chow and protein expression. BDNF expression was determined with an Emax tap water. Immunoassay System (Promega) according to the manufacturer's 466 S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472 instructions with minor modifications. Microplates were treated determined in triplicate in each experimental set of plates, and each with a monoclonal anti-BDNF antibody in carbonate coating buffer run was repeated three times. Samples were counterbalanced so that (pH 9.7), covered and incubated without shaking for 36–48 h at 4 °C. all treatments, ages, and genders were represented on each plate. Plates were then emptied, washed once with Tris-buffered saline Preliminary testing established the appropriate lysate dilutions to (20 mM Tris–HCl, pH 7.6, 150 mM NaCl) with 0.05% Tween-20 (TBST; employ so that sample values fell within the optimum ranges of the

ELX50 Plate Washer, Bio-tek Instruments), and blocked without total protein and BDNF standard curves. shaking for 1 h at room temperature. After washing a standard curve was prepared with authentic BDNF, and appropriately diluted 2.5. Statistical analyses samples (in blocking buffer) were added to other wells (total volume=100 μl). Plates were then incubated with shaking for 2 h Body weight for dams and pups were assessed by a repeated at room temperature and washed 5 times with TBST. A polyclonal measure ANOVA with litter as the unit of analysis. Serum hormones anti-human BDNF was then diluted in blocking buffer and added to were analyzed by one-way ANOVAs at each age and for the dams. In the the wells followed by shaking for 2 h at room temperature. After 5 event of a significant main effect or interaction, mean contrast tests were washes with TBST the HRP-conjugated anti-IgY secondary antibody performed using Dunnett's t-test with alphas set at pb0.05. BDNF was diluted in blocking buffer and added to the wells with shaking for protein expression was evaluated using two-way ANOVAs in each brain 1 h at room temperature. After 5 more washes with TBST a peroxidase region with either Age and Dose or Sex and Dose as the main factors. In substrate was mixed with 3,3′,5,5′-tetramethylbenzidine (TMB, adults, when the main effect of Sex was significant, one-way step down Thermo Scientific) solution and added to the wells, and the plates ANOVAs were performed across PTU doses for each sex. Where were shaken for 15 min at room temperature. The reaction was appropriate, mean contrast tests were performed using Dunnett's t- stopped with 2 M sulfuric acid, and absorbance was read in a test and an alpha level set to 0.05. microplate reader (SpectraMax Plus, Molecular Devices) at 450 nm using Softmax Pro software. Tissue samples for the immunoassay 3. Results were sonicated in lysis buffer with the following components (in mM): NaCl 137, Tris–HCl (pH 8.0) 20, phenylmethylsulfonylfluoride 1, 3.1. Thyroid hormones

NaVO4 0.5, with 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1% NP40 and 10% glycerol. Thyroid hormones were reduced in a dose-dependent manner Aliquots of each sample lysate were analyzed in triplicate for total in dams and pups in response to 0, 1, 2 and 3 ppm of PTU. T4 was protein by the bicinchoninic acid assay (Thermo Scientiﬁc) and used dose-dependently reduced at all dose levels on PN14 [F(4,44)=12.91, to normalize BDNF values. Sample BDNF protein expression was pb0.001] and PN21 [F(4,52) =33.40, pb0.001] and in dams at

AB 50 Pup and Dam Serum T4 0 ppm Pup and Dam Serum T3 0 ppm 1 ppm 100 1 ppm 2 ppm 2 ppm 40 3 ppm 3 ppm 10 ppm 80 10 ppm

30 * * 60 * * * 20 40 * * * Total T3 (ng/dl) Total T4 (ng/ml) * * 10 * 20 * * * * * 0 0 PN14 PN21 Dam PN14 PN21 Dam

CD 60 0 ppm 1 ppm Adult Offspring Serum T4 100 Adult Offspring Serum T3 2 ppm 0 ppm 50 3 ppm 1 ppm 10 ppm 2 ppm 80 3 ppm 40 10 ppm

60 30

20 40 Total T3 (ng/dl) Total T4 (ng/ml)

10 20

0 0 PN90-F PN90-M PN90-F PN90-M

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,; *pb0.05 compared to controls. S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472 467 weaning [F(4,53)=7.73, pb0.001] (Fig. 1A). T3 was also reduced on (Fig. 3C — note differences in y-axis scales). Brains were harvested PN14 [F(4,46)=166.37, pb0.001)] and PN21 [F(4,54)=114.37, from female neonates in the low dose cohort, so assessment of BDNF pb0.001] but to a lesser degree than T4. Dam T3 values were altered expression as a function of age was limited to a comparison of female only in the highest dose group [F(4,55)=32.04, pb0.001] (Fig. 1B). animals only on PN14, PN21, PN~90. A significant effect of Age was Both T4 (Fig. 1C) and T3 (Fig. 1D) had fully recovered in adult male observed in each brain region, with younger animals having greater and female offspring when assessed ~PN90 (all p'sN0.15). expression of BDNF than adults (Fig. 3). In hippocampus and cerebellum, BDNF declined monotonically from PN14 to adulthood b b 3.2. Body weight in dams and pups [F(2,96)=112.76, p 0.001, and F(2,96)=28.12, p 0.001, respectively] with the value at each age significantly different from values b No significant alterations in dam or pup weights were detected in at other ages (all p's 0.001). In cortex, BDNF levels were higher on PN21 than PN14, but declined to lowest levels in adulthood response to 1–3 ppm PTU through PN22 (Fig. 2A, all p'sN0.22). At b 10 ppm PTU small reductions in body weight were observed in dams [F(2,94)=24.30, p 0.001]. [Dose×Age interaction, F(12,96)=5.02, pb0.001] beginning in late gestation (GD20) and extending to the early postnatal period (Fig. 2B, 3.4. BDNF expression varies by sex and dose of PTU inset). Offspring maintained on 10 ppm displayed a slower growth rate (Fig. 2B), and significant reductions in body weight were evident 3.4.1. Effects in neonates by PN14 and became more profound with age [Dose×Age interaction, No effects of PTU were observed in hippocampus, cortex, or F(6,42)=24.06, pb0.001]. 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 3.3. BDNF expression varies by brain region and age of PTU inducing a more severe state of hypothyroidism, BDNF protein expression was not reduced in male or female offspring on PN21 (data Control levels of BDNF expression were highest in hippocampus not shown); BDNF expression on PN14 was not assessed in the high (Fig. 3A) and much lower in cerebral cortex (Fig. 3B) and cerebellum dose cohort.

A 70 3.4.2. Effects in adult offspring 425 Dam Body Weights In contrast to the results in neonates, absolute BDNF values were 400 60 375 altered in adult offspring as a function of exposure to PTU. Although all 350 animals had returned to control levels of circulating thyroid hormones 50 325 300 at the time of sacrifice ~PN90, significant differences in BDNF were 275 Weight in gms 250 0 ppm 1 ppm detected in hippocampus (Fig. 4A). Statistical analyses revealed 40 225 2 ppm 3 ppm 200 significant main effects of Dose [F(3,65)=5.46, pb0.002] and Sex GD2 GD13 GD20 PN5 PN9 PN16 PN22 30 Age [F(1,65)=67.61, pb0.001]. Step-down ANOVAs for each sex showed significant reductions in adult male [F(3,34)=4.52, pb0.009] but not

Weight in Grams 20 0 ppm in adult female [F(3,31)=1.92, pN0.14] offspring. In males BDNF 1 ppm 2 ppm reductions were restricted to the two lowest dose groups (Dunnett's t, 10 3 ppm pb0.05) with a return to control levels at 3 ppm PTU. fi 0 Similar trends were evident in cortex (Fig. 4B) with signi cant 3 5 7 9 13 16 19 22 main effects observed for Dose [F(3,65)=2.79, pb0.047] and Sex Postnatal Day [F(1,65)=5.53, pb0.022]. Step-down ANOVAs revealed a significant B reduction in cortical BDNF in adult male [F(3,35), pb0.004] but not in 160 female offspring [F(3,33)=0.91, pN0.44]. In male cortex this 450 Dam Body Weights reduction was evident only at the lowest dose (Dunnett's t, pb0.05). 140 400 fi 350 No signi cant differences in BDNF expression in adult off-

300 spring were detected in cerebellum (Fig. 4C) as a function of Dose 120 250 N N

Weight in gms [F(3,66)=0.15, p 0.98] or Sex [F(1,66)=0.15, p 0.69]. 0 ppm 200 10ppm As the high dose cohort was evaluated at a different time than the 100 150 GD2 GD9 GD17 GD21 PN9 PN14 PN20 low dose cohort using different lots of ELISA plates and BDNF Age 60 antibodies, a direct comparison of absolute levels of protein expression between the low and high dose cohorts was not possible

Weight in Grams 40 (Elfving et al., 2010). However, data across all dose levels can be 0 ppm compared by expressing each value as a percent of the appropriate 20 10 ppm control (Fig. 5). The results of this analysis revealed significant main 0 effects of Sex [F(1,92)=11.24, pb0.001] and Dose [F(4,92)=8.70, 5 101520253035 p b0.001] in hippocampus, and a marginal Sex×Dose interaction Postnatal Day [F(4,92)=2.43, pb0.053]. The U-shaped dose–response function evident in the absolute neurotrophin levels in the low dose cohort Fig. 2. (A) Mean±SEM body weight of dams (inset) and pups in the low dose cohort, described above was further reinforced in the present analysis – which were comparable to controls (n=10 15/group). (B) Slight reductions in body (Fig. 5A). Step-down one-way ANOVAs by Sex indicated that in males weight were seen in dams in the last few days of pregnancy in response to 10 ppm PTU b (inset, n=5/group). Body weight deficits (mean±SEM) were evident by PN14 in the [F(4,47)=6.10, p 0.001] low doses of PTU (1 ppm and 2 ppm) offspring that became more apparent with age and did not fully recover by adulthood reduced hippocampal BDNF by ~40–45% (Dunnett's t, pb0.05), and a (data not shown) despite termination of PTU exposure at weaning. The broken line for return to control levels was seen at the two highest doses (3 and controls reflects the break in the y-axis necessary to show the large increase in body 10 ppm). In females [F(4,45)=4.95, pb0.002] significant reductions weight that occurs between PN21 and PN35 (almost 100 g increase), which is not seen were limited to the 2 ppm dose group (Dunnett's t, p b0.05) with a 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. *pb0.05 trend towards an increase above control levels at the highest PTU compared to 0 ppm group values at the same age. dose level. 468 S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472

A cortical BDNF levels in male offspring [step down F(4,50)=3.63, 110 b b Hippocampus 0 ppm p 0.011] were limited to the lowest dose level (Dunnett's t, p 0.05) 100 1 ppm with return to control levels at the higher doses. In females a signiﬁ- 2 ppm 90 cant effect of PTU on cortical BDNF levels [step down F(4,45)=4.39, 3 ppm pb0.004] was driven by an augmentation in BDNF protein expression 80 ‡ in the 10 ppm dose group (Dunnett's t, pb0.05). 70 60 50 A 40 80 Hippocampus ‡ 30 #

BDNF, pg/mg protein 70 20 60 0 ppm 10 1 ppm 2 ppm 0 50 PND 14 PND 21 Adult Female 3 ppm Age 40 * * B 30 ‡ Cortex

BDNF, pg/mg protein 20 14 ‡ 10 12

0 10 Adult Male Adult Female ‡ # 8 B 12 Cortex 6

4 10 BDNF, pg/mg protein

2 8 & 0 PND 14 PND 21 Adult Female 6 Age *

4 C 20 Cerebellum BDNF, pg/mg protein 2 18

16 0 Adult Male Adult Female 14 ‡ 12 C 10 10 Cerebellum 8 ‡ # 6 8 BDNF, pg/mg protein 4 2 6 0 PND 14 PND 21 Adult Female Age 4 BDNF, pg/mg protein Fig. 3. BDNF protein expression as a function of age, PTU dose, and brain region in 2 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 0 age. ‡pb0.001 compared to the PND14 value; #pb0.001 compared to the PND21 value. Adult Male Adult Female

Fig. 4. BDNF protein expression as a function of PTU dose and brain region in adult male The effect of PTU on cortical expression of BDNF in adult offspring and female offspring. Significant main effects of Sex and Dose, and a Sex×Dose was not as striking as that seen in hippocampus, however a similar interaction were evident in ANOVA, see Section 3.4. Values are expressed as mean+ biphasic dose–response pattern was evident (Fig. 5B). Results of 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: ‡pb0.01; & pb0.05 compared to the ANOVA revealed significant main effects of Sex [F(1,95) =4.89, values in adult males; *pb0.05 compared to the 0 ppm value in adult males with b b p 0.030] and Dose [F(4,95)=5.48, p 0.001] with a marginal Dunnett's t-test. No significant effect of PTU was detected in adult female offspring as a Sex×Dose interaction [F(4,95)=2.44, pb0.052]. Reductions in function of dose. S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472 469

4. Discussion A Hippocampus + 140 A number of novel observations resulted from this investigation of the impact of moderate thyroid hormone insufficiency on BDNF 120 protein expression in three rat brain regions. PTU did not affect BDNF expression in the neonate, but surprisingly, altered BDNF expression 100 profiles present in the hippocampus and cortex of adult offspring. Adult males displayed higher levels of BDNF than adult females, and 80 * males displayed greater reductions in BDNF than females in response to developmental thyroid hormone insufficiency. Biphasic dose–

BDNF, % control response functions were evident in both male and female adult 60 Adult Male offspring exposed to PTU in early life. * Adult Female BDNF was higher in all three brain regions studied during the 40 * neonatal period than in adulthood. This neurotrophin is widely expressed in the CNS, and distinct age- and region-specific profiles of 0 1 2 3 10 protein expression have been previously reported (Friedman et al., PTU, ppm 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 age- B Cortex dependent patterns of BDNF levels in control animals observed in the present study are consistent with many of these reports, both in terms 140 * 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). 120 The hippocampus had the highest levels, with much lower concentrations of BDNF detected in the cortex and cerebellum, and younger 100 animals had higher concentrations of BDNF than adults. + Thyroid hormone insufficiency during development did not impact 80 BDNF protein expression during the preweaning period in any of the three brain regions assessed despite significant reductions in circulat-

BDNF, % control fi 60 ing thyroid hormones in dams and offspring. These ndings are Adult Male consistent with some reports on transient gestational (GD15–19) * Adult Female (Opazo et al., 2008) or perinatal hormone deprivation (Alvarez-Dolado 40 et al., 1994) on BDNF protein or mRNA expression (see Introduction). 0 1 2 3 10 However, the current findings stand in contrast to other observations PTU, ppm (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 C alterations in BDNF mRNA in the cerebellum. BDNF mRNA changes not Cerebellum accompanied by changes in BDNF protein have been reported by 140 numerous investigators (Alonso et al., 2002; Pollock et al., 2001; Nanda and Mack, 1998). Lack of protein changes may reflect increased 120 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 100 state of subclinical hypothyroidism by surgical removal of the dam's thyroid gland and partial replacement of hormone with subcutaneous 80 injections to the dams and pups during gestation and lactation. Thyroidectomy without exogenous hormone supplementation pro- BDNF, % control 60 duced a severe state of hypothyroidism and reduced BDNF mRNA and Adult Male Adult Female protein in the early (PN3 and PN7) and late (PN21) neonatal period. 40 Subclinical hypothyroidism was accomplished by supplementing thyroidectomized dams with T4 to model a modest reduction in 0 1 2 3 10 thyroid hormone levels that is perhaps more analogous to the graded PTU, ppm degrees of thyroid hormone insufficiency induced in the present study. The subclinical model also reduced BDNF mRNA and protein Fig. 5. Dose–effect relationships for BDNF protein expression and PTU dose in adult expression in hippocampus in the very young pup on PN3 and PN7, male and female offspring in each brain region. BDNF values are normalized to the but not on PN21. The latter finding in the subclinical model is 0 ppm value (=100) for the respective gender in that brain area. Values are expressed consistent with our findings of no effect of PTU at any dose level tested as mean ± SEM in percent control with N=5–11 for each PTU dose in each gender; in the preweanling rat (PN14 or PN21); unfortunately, animals in the *pb0.05 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 +pb0.06 when current study were not assessed prior to PN14. contrasted to 0 ppm value in females. 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 Distinct from the effects observed in hippocampus and cortex, levels in adult males than in adult females in hippocampus and cortex developmental exposure to PTU had no effect on cerebellar expression with no gender difference evident in cerebellum. Adult male offspring of BDNF at any Dose [F(4,94)=0.12, pN0.97] in either Sex [F(1,94)= also exhibited a greater proportional reduction in BDNF levels than 0.13, pN0.71)] (Fig. 5C). females in these regions in response to PTU, but males did not exhibit 470 S.M. Lasley, M.E. Gilbert / Neurotoxicology and Teratology 33 (2011) 464–472 the elevation in BDNF expression seen in adult females at the highest implicated in such nonheritable modifications of gene transcription PTU dose. This sex-dependent differential sensitivity to the effects of (Dobosy and Selker, 2001; Fang and Lu, 2002; Santos et al., 2005; developmental thyroid hormone insufficiency on BDNF protein may Miller et al., 2010). Up-regulation of DNA methyltransferase and be due to estrogenic modulation of BDNF expression or to interactions down-regulation of histone acetylases have been recently reported for of estrogen and thyroid hormones in the brain (Graupner et al., 1991; the hippocampus in a perinatal hypothyoid model and associated Kia et al., 2001; Pfaff et al., 1994; Tang et al., 2004). Estrogen has with decreases in BDNF gene and protein expression (Sui and Li, proven to be a potent regulator of BDNF in rat brain (Gibbs, 1999; 2010). It is unknown if these mechanisms are engaged with more Sohrabji and Lewis, 2006; Zhu et al., 1996), and administration of modest perturbation of the thyroid axis that is limited to the fetal and exogenous estrogen increases BDNF expression in hippocampus early neonatal period. If so, it is conceivable that such modifications in (Aguirre and Baudry, 2009). Furthermore, hippocampal BDNF expres- gene transcription could contribute to the altered BDNF protein sion is more robustly augmented in female rats following traumatic expression in adulthood. Activation of opposing mechanisms with brain injury than in males (Chen et al., 2005). Moreover, estrogen and different responsiveness to variable degrees of hormone insufficiency thyroid receptors share a common consensus site on their hormone could also plausibly constitute the basis for biphasic PTU dose–effect response elements (EREs and TREs) and thus interact to regulate gene relationships. expression (Graupner et al., 1991; Hirst et al., 1992; Zhu et al., 1996). In summary, a late emergent impact of perinatal thyroid hormone Consequently, the presence of estrogen in adult female offspring may insufficiency on BDNF protein expression was observed at a time attenuate the effect of developmental thyroid hormone insufficiency when circulating levels of thyroid hormone had fully recovered. These on BDNF expression at low PTU doses and underlie the elevation in persistent effects on the expression of this critical brain protein were BDNF observed at the highest PTU dose. seen following relatively modest and transient perturbations of the In contrast to the lack of effect during the preweanling period in thyroid axis restricted to the early developmental period. BDNF in the this model of thyroid hormone insufficiency, BDNF protein expression adult brain has been implicated in adult neurogenesis, synaptic in the cortex and hippocampus of adult offspring of PTU-exposed plasticity, learning, and recovery from injury (Shulga et al., 2008, dams was disrupted. The observation of this late emergence of effect 2009; Pérez-Navarro et al., 1999; Lu and Figurov, 1997; Lemkine et al., on neurotrophin expression in response to developmental thyroid 2005; Desouza et al., 2005; Ambrogini et al., 2005), and its disruption hormone insufficiency is novel. Neither has the biphasic dose– may contribute to the neurological impairments associated with response function evident in the current study been previously moderate levels of developmental thyroid hormone insufficiency described. BNDF protein levels were reduced in adult offspring despite (e.g., Gilbert et al., 2007; Gilbert and Sui, 2006; Lavado-Autric et al., full return of the animals to euthyroid conditions at the time of 2003., Auso et al., 2004; Opazo et al., 2008; Sharlin et al., 2008). If testing. This reduction in BDNF was limited to the lowest doses of PTU BDNF and BDNF-induced signaling do mediate some of the adverse and to the most modest reductions in circulating hormones in dams effects on brain development and function accompanying develop- and pups during the neonatal period. As the dose of PTU and mental thyroid hormone insufficiency, the factors influencing its consequently the level of hormone insufficiency increased, BDNF protein expression are complex. It will be important to unravel this expression recovered toward control levels. The reduction in BDNF complexity if we are to elucidate the role of thyroid hormone in brain values at 1–2 ppm PTU was most striking in the hippocampus of adult development and the functional impairments that persist as a male offspring, and no differences were seen when moderate (3 ppm) consequence of its insufficiency. or more severe (10 ppm) hypothyroid conditions were imposed. Females displayed an attenuated response at low doses of PTU with Conflict of interest 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 No competing interests to report. induced by 10 ppm PTU, increases above control BDNF levels were observed in females in cortex and marginally in hippocampus. Acknowledgments It is not at all clear what may underlie the delayed emergence of altered BDNF protein expression following developmental thyroid This document has been subjected to review by the National hormone insufficiency, or what this may mean functionally to the Health and Environmental Effects Research Laboratory and approved animal. Neither is there any simple explanation for the non- for publication. Approval does not signify that the contents reflect the monotonic dose–response relationships. Circulating levels of thyroid views of the agency, nor does mention of trade names or commercial hormones typically return to control levels within a few weeks of products constitute endorsement or recommendation for use. The termination of PTU exposure (Axelstad et al., 2008; Cooper et al., authors thank Drs. Tom Zoeller and William Mundy for comments on 1983), and no differences were seen in the present study at the adult an earlier version of this manuscript, and Willard Anderson, Meena time point. Therefore, a chronic reduction in circulating levels of Shanmugasundaram, and Brittney Hanerhoff for technical assistance. thyroid hormone cannot account for the present findings. Elevated brain levels of BDNF and other neurotrophins accompany brain References trauma and injury in adult and neonatal models where they serve a neuroprotective role and also are necessary for recovery and repair Aguirre CC, Baudry M. Progesterone reverses 17beta-estradiol-mediated neuroprotection and BDNF induction in cultured hippocampal slices. Eur J Neurosci 2009;29(3): (Di Fausto et al., 2007; Chen et al., 2005). It is possible that 447–54 Feb. hypothyroidism-induced reductions of BDNF evident at low doses of Akaike M, Kato N, Ohno H, Kobayashi T. 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