cImpairment in Short-Term but Enhanced Long-Term Synaptic Potentiation and ERK Activation in Adult Hippocampal Area CA1 Following Developmen
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《毒物学科学杂志》
National Research Council, Washington, DC 20001
Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Department of Psychology, University of North Carolina, North Carolina 27599
ABSTRACT
Thyroid hormones are critical for the development and maturation of the central nervous system. Insufficiency of thyroid hormones during development impairs performance on tasks of learning and memory that rely upon the hippocampus and impairs synaptic function in young hypothyroid animals. The present study was designed to determine if perturbations in synaptic function persist in adult euthyroid animals exposed developmentally to insufficient levels of hormone. Pre- and postnatal thyroid hormone insufficiency was induced by administration of 3 or 10 ppm propylthiouracil (PTU) to pregnant and lactating dams via the drinking water from gestation day (GD) 6 until postnatal day (PN) 30. This regimen produced a graded level of hormonal insufficiency in the dam and the offspring. Population spike and population excitatory postsynaptic potentials (EPSP) were recorded at the pyramidal cell layer and the stratum radiatum, respectively, in area CA1 of hippocampal slices from adult male offspring. PTU exposure increased baseline synaptic transmission, reduced paired-pulse facilitation, and increased the magnitude of the population spike long-term potentiation (LTP). Phosphorylation of the extracellular signal-regulated kinases (ERK1 and ERK2) was increased as a function of LTP stimulation in slices from PTU-exposed adult animals. On the other hand, no differences in the basal levels of synaptic proteins implicated in synaptic plasticity (total ERK, synapsin, growth-associated protein-43, and neurogranin) were detected. These results reinforce previous findings of persistent changes in synaptic function and, importantly extend these observations to moderate levels of thyroid hormone insufficiency that do not induce significant toxicity to the dams or the offspring. Such alterations in hippocampal synaptic function may contribute to persistent behavioral deficits associated with developmental hypothyroidism.
Key Words: hypothyroidism; hippocampus; paired-pulse facilitation; long-term potentiation; extracellular signal-regulated kinase; adult.
INTRODUCTION
Thyroid hormones play crucial roles in the development and maturation of the central nervous system (Anderson et al., 2003; Bernal, 2002; Morreale de Escobar et al., 2004). Severe thyroid hormone insufficiency during the perinatal period results in impaired brain development with diminished interneuronal connectivity, decreased myelination, defective cell migration, and alterations in levels of neurotransmitters (Bernal, 2002; Lavado-Autric et al., 2003; Thompson and Potter, 2000). Delay in restoring normal thyroid status in the neonate is associated with long-lasting behavioral impairments (Glinoer and Delange, 2000; Morreale de Escobar et al., 2004; Zoeller and Rovet, 2004), underscoring the persistence of neurological deficiencies induced by early thyroid hormone insufficiency. Recently, even mild perturbations in the hormonal status of pregnant women have been associated with permanent intellectual impairment in their children (Haddow et al., 1999; Morreale deEscobar et al., 2004; Pop et al., 1999; Zoeller and Rovet, 2004).
Until recently, few experimental studies have been conducted to model the effects of subtle thyroid hormone insufficiency on early brain development or to evaluate the dose-response relationships between altered serum hormones and brain insult. Berbel and colleagues (Auso et al., 2004; Lavado-Autric et al., 2003) have reported cortical and hippocampal neuronal misplacements in the offspring of dams experiencing brief and mild reductions in thyroid hormones during gestation and lactation. A number of genes critical for cell proliferation, migration, and myelination have been identified, which are regulated by thyroid hormone (Bernal, 2002; Bernal et al., 2003; Oppenheimer and Schwartz, 1997; Thompson and Potter, 2000). Most of these studies, designed to evaluate the role of thyroid hormones on gene expression, have utilized a model of severe hypothyroidism. What has been lacking is a link between perturbations in gene expression, structural abnormalities, and brain function and an evaluation of the dose-response relationships following mild disruptions of the thyroid axis (Bernal et al., 2003; Forrest, 2004; Thompson and Potter, 2000; Zoeller, 2003). The current study was performed to provide information on the functional consequences of early hormone insufficiency in a brain slice preparation well suited for investigation of physiological, morphological, and molecular substrates. Synaptic transmission and plasticity were examined in the hippocampus, a structure known to be sensitive to thyroid hormone disruption and to play a critical role in some types of learning and memory. Offspring of thyroid-deficient dams in which a graded level of hormone insufficiency was produced were evaluated to begin to define the long-term consequences of milder forms of thyroid hormone insufficiency.
The primary health outcome of concern resulting from early thyroid hormone insufficiency is intellectual impairment. The deleterious effects of severe developmental hypothyroidism on the hippocampal morphology have been well established (Madeira et al., 1992; Rami et al., 1986). Perturbations in thyroid hormone function are associated with behavioral impairments in tasks requiring integrity of the hippocampus (Akaike et al., 1991; Darbra et al., 2004; Guadano-Ferraz et al., 2003). Recently, electrophysiological studies have also demonstrated that hypothyroidism induced by propylthiouracil (PTU) or methimazole exposure alters synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus (Niemi et al., 1996; Sui and Gilbert, 2003; Vara et al., 2002). The best model system to investigate the synaptic basis of cognition is long-term potentiation (LTP) in the hippocampus (Malenka and Nicoll, 1999), and long-term changes in hippocampal synaptic strength are generally accepted to be involved in learning and memory (Bliss and Collingridge, 1993; Brown et al., 1990). More recently, short-term synaptic plasticity as measured by paired-pulse functions has also been proposed to play a functional role in temporal information processing and reflect mechanisms important for learning (Buonomano and Merzenich, 1995; Dobrunz et al., 1997; Lisman, 1997; Silva et al., 1996). LTP and paired-pulse facilitation are reduced in area CA1 of slices from neonatal rats that remained hormone deficient at the time of assessment (Sui and Gilbert, 2003; Vara et al., 2002). In the present study, the persistence of these impairments was investigated in adults following recovery from hormone insufficiencies endured during development.
Regulation of the expression and phosphorylation of a number of synaptic proteins are under the control of thyroid hormones (Bernal, 2002; Bernal et al., 2003; Davis et al., 2002; Lin et al., 1999; Thompson and Potter, 2000). Many of these same proteins are also necessary for normal synaptic transmission, short-term and long-term plasticity, and hippocampal-based learning (Atkins et al., 1998; Benowitz and Routtenberg, 1997; Blum et al., 1999; English and Sweatt, 1996, 1997; Gerendasy et al., 1994; Impey et al., 1999; Klann et al., 1992; Oestreicher et al., 1997; Rosahl et al., 1993; 1995; Selcher et al., 1999; Winder et al., 1999). Thus, in addition to physiological assessments, levels of synapsin, growth associated protein 43 (GAP-43), neurogranin (RC3), and mitogen-activated protein kinase (MAPK) were examined in area CA1 of the hippocampus. A second aim of the present study was to determine if perturbations in the expression or activation of these proteins persist upon recovery of normal thyroid hormone status and could underlie alterations in synaptic function and behavior that are maintained in adult animals.
MATERIALS AND METHODS
Animal treatment.
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-approved animal facility. All animal treatments were in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals and their suffering. Animal rooms were maintained on a 12:12 light:dark schedule, and animals were permitted free access to food (Purina rat chow) and tap water. From GD 6 to postnatal day (PN) 30, dams were administered 3 or 10 ppm of the antithyroid agent, propylthiouracil (PTU, Sigma, St. Louis, MO) through the drinking water; control rats
Slice preparation and electrophysiological recording.
At the age of 11–14 week (PN77–PN96), one male offspring was randomly selected from each litter (0 ppm, n = 12 litters; 3 ppm, n = 15 litters; 10 ppm, n = 8 litters), sacrificed by decapitation, and prepared for slice electrophysiology as described previously (Sui and Gilbert, 2003). Trunk blood was collected at the time of sacrifice and stored for hormone analysis. The brain was removed, and the hippocampus was dissected on ice. Transverse hippocampus slices (400 μm) were cut using a McIlwain tissue chopper and placed in ice-cold, oxygenated artificial cerebrospinal fluid (ACSF, 124 mM NaCl, 3 mM KCl, 2.0 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, pH 7.4). Slices were immediately transferred to an interface recording chamber containing warmed, oxygenated (95% O2/5% CO2) ACSF and incubated at 33°C for a minimum of 1 h prior to recording. Biphasic squarewave pulses were delivered through a stainless steel electrode placed in the stratum radiatum, and stimulation-evoked extracellular field potentials were recorded from both the pyramidal cell layer and the stratum radiatum of CA1 through glass micropipettes (2–4 μm tip diameter) filled with ACSF. Stability of baseline recordings was established by delivering single pulses (1/min, 0.1 ms pulse width at an intensity yielding 70–80% of maximal population spike amplitude for a given slice) for 15–20 min prior to recording. When the variation in field potential amplitude was less then ±10%, the baseline was considered stable. Slices with unstable baseline responses were excluded from further experimentation.
Input-output (I/O) functions were collected to examine baseline synaptic transmission by delivering an ascending series of 14 stimulus intensities (20–150 μA) that ranged from subthreshold for elicitation of an excitatory postsynaptic potential (EPSP) to those eliciting maximal responses. Five pulses were delivered at each stimulus intensity at a frequency of 0.1 Hz and averaged at each recording site. Following collection of the I/O function, paired-pulse facilitation of the dendritic EPSP was measured at ten interpulse intervals (20–1000 ms) at maximal (150 μA) and submaximal (100–110 μA) stimulus strengths. Finally, LTP of somatic population spike and dendritic EPSP was induced. A test stimulus was chosen of intensity sufficient to produce a population spike approximately 70–80% of maximum. Pretrain responses were recorded for 20 min, followed by theta burst stimulation (25 4-pulse bursts at 100 Hz, 200 ms between bursts at the same stimulus intensity as the pretrain pulse). Single pulse recording resumed immediately following the train delivery and continued for 60 min.
Waveform scoring.
Action potentials in pyramidal cell neurons are reflected in field potentials recordings from the pyramidal cell layer as a large negative going potential, the population spike (Bliss and Richards, 1971). The dendritic responses recorded from the stratum radiatum provide an index of synaptic activity comprising the summed population excitatory postsynaptic potentials (EPSP) (Bliss and Richards, 1971). Population spike amplitude was estimated by calculating the voltage difference between the most negative point of the spike and a tangent connecting the onset of the spike and the next positive peak on the waveform. At the dendritic site, EPSP slope was calculated as the rate of amplitude change for the initial negative deflection to the peak. The EPSP peak amplitude was the most negative point on the waveform, and the EPSP area measure was initiated at the point of the initial negative deflection and ended with the return of the EPSP to the baseline. For I/O functions, response amplitude was normalized to the percentage of maximal population spike amplitude and EPSP slope. Paired-pulse facilitation and depression were expressed as the ratio of the mean amplitude of the second response relative to the first (pulse 2/pulse 1 x 100). LTP was expressed as percent change from the mean of ten pretrain recordings taken just prior to train delivery.
Sample preparation for ERK1 and ERK2 analysis.
LTP-induced phosphorylation of the extracellular signal regulated kinases (ERK1 and ERK2) of the MAPK family of kinases was investigated in a different set of hippocampal slices harvested from the same animals used for electrophysiological assessments. To prepare these samples, slices were maintained in the same interface chamber as those used for electrophysiological recordings and were allowed to incubate at 33°C for at least 1 h. Pairs of slices either were left unstimulated or
Sample preparation for synapsin, GAP-43, and RC3 analysis.
Levels of synaptic proteins synapsin, GAP-43, and neurogranin (RC3) were examined in area CA1 of hippocampus of adult male littermates of animals used for electrophysiology. Animals were sacrificed by decapitation, the hippocampus was removed, and the midtemporal region dissected. Tissue consisting primarily of area CA1 was extracted by cutting along the hippocampal fissure and was stored at –80°C for later analyses.
Western blot analysis.
For Western blot analyses, tissue was homogenized by sonication in ice-cold solubilizing buffer containing 1% Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, and 0.5% protease inhibitors (Protease Inhibitor Cocktail III, Calbiochem, La Jolla, CA). The insoluble material was removed by centrifugation at 10,000 x g for 10 min at 4°C. An aliquot of the supernatant was taken for protein determination, and the remaining supernatant was added to an equal volume of Laemmli's sample buffer (BioRad, Hercules, CA), and samples were boiled at 100°C for 5 min. Samples were resolved by SDS–PAGE followed by electrophoretic transfer onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The blots were blocked for 1 h with 5% nonfat dried milk at room temperature, then incubated overnight at 4°C with commercially available specific antibodies to the proteins of interest. After three short washes, the blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (1:20,000, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The blots were visualized using a chemiluminescence substrate (ECL, Bio-Rad, Hercules, CA), and the light images were collected and analyzed photometrically with a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA). For all Western blots, on each gel 2 or 3 lanes were reserved for a quality control (QC) sample. This sample was taken from a pool of a large number of hippocampal slices from nave animals. Aliquots of the pool were maintained at –70°C. One aliquot was used for each gel or series of gels run at one time. After correction for background chemiluminescence, the signals from target bands on a gel were normalized to the average signal for the QC sample bands to simplify comparison across gels and reduce inter-gel variability. The coefficient of variation for the QC values across gels was typically below 10%.
To investigate phosphorylation of ERK, mouse monoclonal anti-phospho ERK1/ERK2 (diluted 1:2500, Cell Signaling, Beverly, MA) was used for the primary antibody incubation. Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Chemical Co, Rockford IL) for 5 min, reblocked for 15 min with 5% nonfat dried milk at room temperature, then probed for total ERK using anti-total ERK1/ERK2 (diluted 1:5000, Cell Signaling, Beverly, MA). For each experiment, both total-ERK1 and total-ERK2 and phospho-ERK1 and phospho-ERK2 signals were normalized relative to those seen in unstimulated slices from the same animal. In addition, active ERK1 and ERK2 signals were normalized to total ERK1 and total ERK2 band intensities.
Rabbit polyclonal antibodies against synapsin (diluted 1:25000, Chemicon, Temecula, CA), and one recognizing a common site on both GAP-43 and RC3 (diluted 1:1000, Upstate, Lake Placid, NY) were used to probe for levels of these neuronal markers. For each blot, synapsin, GAP-43, and RC3 levels in the two PTU-exposed groups were normalized to QC standards and expressed as a percentage of the levels observed in control animals.
Thyroid hormones.
At the time of sacrifice for electrophysiological testing, trunk blood was collected and allowed to clot on ice for a minimum of 30 min. Serum was separated via centrifugation and stored at –80°C for later analyses by radioimmunoassay. Serum concentrations of total thyroxine (T4) and total triiodothyronine (T3) were analyzed by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). Thyroid stimulating hormone (TSH) was measured using standard double antibody assay as described by Thibodeaux et al. (2003). All samples for total T4 and total T3 were run in duplicate and the intra- and interassay variations ranged from 9 to 12%. The minimum detectable concentration (MDC) for each assay was determined statistically (3 standard deviations above background levels). For all T3 assays (n = 4), the MDC was 7.8 ng/ml, and for the T4 assay (n = 4), the MDC was 4.9 ng/ml. The lowest calibrator for each assay was 10 ng/ml. For statistical purposes, in those cases where the sample result was below the level of this calibrator, the result was set by default to the MDC.
Statistical analysis.
Group statistics were calculated as the mean ± SEM. Data from electrophysiological studies were evaluated using repeated measures analysis of variance (ANOVA). Where significant interactions were found, step-down ANOVAs and mean contrast tests using Tukey's t-test were performed. Thyroid hormone data were evaluated using one-way ANOVAs. Protein data were evaluated using nonparametric chi-square analysis. Probabilities less than 0.05 were considered significant.
Results
PTU Treatment on Offspring Body Weight and Thyroid Hormone Levels
There were no significant PTU treatment-related differences in dam body weights throughout gestation (data not shown). There were no apparent PTU treatment-related effects on offspring body weight over the first 10 postnatal days. Dose-dependent deficits in offspring body weight became apparent from PN13, and these deficits persisted to PN33 (119.8 ± 3.77 g, 89.0 ± 6.54 g, and 28.85 ± 1.70 g, in the controls, 3 ppm PTU-treated, and 10 ppm PTU-treated groups, respectively) (see Sui and Gilbert, 2003). In the adult offspring (PN77), the body weights were comparable between the control and the 3 ppm PTU-treatment group (475.6 ± 15.59 g and 429.9 ± 22.70 g, respectively), but remained significantly reduced in the 10 ppm PTU-treatment group (296.2 ± 14.32 g).
Dose-dependent reductions in thyroid hormones with concomitant elevations in TSH were observed in offspring at weaning and were reported in Sui and Gilbert (2003) and are summarized in Table 1. Dams displayed hypothyroxinemia at the low dose (reduced T4 with no significant reduction in T3), but hypothyroidism in the high-dose group (T3 and T4 reduced by 45 and 65%, respectively). Offspring were more severely affected; T4 in the low-dose group was reduced by 70% at weaning, and the majority of high dose animals fell below the level of detection of the assay. T3 was reduced by 35% and 70% in low and high dose animals, respectively. Although significant changes in thyroid hormones were observed in dams and young animals, complete recovery of T3 and T4 was evident in adult males at the time of testing (see Table 1). No significant differences were apparent in T4, T3, or TSH across dose groups (all p values > 0.1).
PTU Treatment on Baseline Synaptic Transmission
Baseline synaptic transmission, assessed by the EPSP slope and population spike amplitude in response to electrical stimulation of varying current amplitudes were recorded by two glass electrodes in area CA1, one placed in the stratum radiatum to assess dendritic activation, the other in stratum pyramidale to assess cell firing at the somatic site. Measurement of baseline synaptic transmission revealed that although maximal responses recorded at 150 μA stimulus intensity did not differ between the groups (p > 0.05), a leftward shift in normalized I/O functions was evident, indicative of an enhancement of baseline synaptic transmission (Fig. 1). Repeated measures ANOVAs of the normalized I/O functions revealed that EPSP slope (Fig. 1A; Dose x Intensity interaction [F(26, 416) = 2.22, p < 0.0006]) and population spike amplitude (Fig. 1B; Dose x Intensity interaction [F(26, 416) = 1.63, p < 0.027] were increased in PTU-exposed animals. Step-down ANOVAs revealed that these increases in EPSP and population spike relative to controls were restricted to the high-dose group (both step-down ANOVAs p values < 0.02).
As both EPSP slope and population spike amplitudes were increased in high dose PTU animals, it was of interest to determine if the relationship of synaptic activation and cell firing was disrupted. Figure 2 reveals that, although the high dose of PTU enhanced EPSP and population spike amplitude, coupling between dendritic and somatic response amplitudes was left unchanged.
PTU Treatment on Paired-Pulse Facilitation of EPSP
In slices from control animals, paired-pulse ratios of dendritic responses increased with interval, became maximal at mid-range, then declined at longer intervals (Fig. 3). Although it appeared as though the high dose of PTU reduced paired-pulse facilitation of the EPSP slope at the briefer intervals (Fig. 3A), variability was high, and ANOVA revealed no significant differences among the groups (p > 0.50). However, a similar pattern was observed in EPSP peak amplitude (Fig. 3B), and clear reductions in the 10 ppm group were apparent in the statistical analysis. A significant main effects of Dose [F(2, 30) = 3.58, p < 0.0404] and Dose x Interval interaction [F(18, 270) = 3.79, p < 0.0001] were obtained, but this reduction in EPSP amplitude measures was restricted in the high-dose group (Step-down ANOVA of 0 vs. 10 ppm, p < 0.03). In contrast, dose-dependent reductions in paired-pulse ratios of EPSP area were apparent from PTU-exposed animals (Figure 3C). Significant main effects of Dose [F(2, 31) = 11.95, p < 0.0001] and Dose x Interval interaction [F(18, 279) = 2.65, p < 0.0004] confirmed that PTU treatment impaired paired-pulse facilitation of dendritic synaptic responses. Step-down ANOVAs revealed that the 3 ppm and the 10 ppm groups were both significantly reduced relative to the controls (both p values < 0.03). Results of mean contrast comparisons at each IPI using Tukey's t-test are indicated by asterisks. A similar pattern was observed for all three measures at maximal and submaximal stimulus strengths. As with findings in younger animals, EPSP area appeared to represent the most sensitive marker of altered paired-pulse facilitation.
PTU Treatment Alters Long-Term Potentiation and ERK1 and ERK2 Activation
After train delivery, all groups displayed enhancement of dendritic EPSP slope and somatic population spike. No significant differences were obtained in dendritic EPSP slope (Fig. 4A) measures between the three treatment groups [main effect of Dose: F(2, 23) = 1.01, p > 0.38], whereas LTP of somatic population spike (Fig. 4B) was significantly increased in PTU-exposed groups relative to the controls [main effect of Dose: F(2, 26) = 3.623, p < 0.041]. Step-down ANOVAs confirmed that population spike LTP significantly elevated above the control values in both dose groups (both p values < 0.05).
The levels of the MAPK proteins, ERK1 and ERK2, were examined in LTP-stimulated and nonstimulated slices from the control and PTU-exposed animals (Fig. 5A). Western blots revealed that total ERK2 and total ERK1 levels were not affected by PTU treatment in either stimulated or nonstimulated slices (left panel Figs. 5B and 5C). Neither did basal levels of phospho-ERKs differ across groups in nonstimulated slices. However, increases in phospho-ERK2 (right panel Fig. 5B) and phospho-ERK1 (right panel Fig. 5C) were observed in response to LTP-inducing stimulation in PTU-exposed groups. Expressed as a percentage of the nonstimulated slices in each group, the data show PTU treatment did not interfere with total ERK2 [2 (2) = 4.49, p > 0.105, Fig. 5B] or total ERK1 levels [2 (2) = 0.611, p > 0.737, Fig. 5C]. PTU treatment dose-dependently elevated the phospho-ERK2 [2 (2) = 6.899, p < 0.032, Fig. 5B] and phospho-ERK1 [2 (2) = 7.00, p < 0.030, Fig. 4C] induced by LTP stimulation.
PTU Treatment on the Levels of Synapsin, GAP-43 and RC3
The levels of synapsin, GAP-43, and RC3 were examined in area CA1 of the adult hippocampus from the control and the PTU-exposed animals (Fig. 6). Western blots revealed that synapsin, GAP-43, and RC3 levels were not affected by PTU treatment (all p values > 0.05).
Discussion
The present study identified persistent perturbations in adult hippocampal synaptic functions following thyroid hormone insufficiency induced during early development. Despite recovery of thyroid hormone to control levels, increases in baseline synaptic transmission, reductions in paired-pulse facilitation, and augmentation in population spike LTP were observed in the high-dose group. LTP-induced increase in the phosphorylation of ERK1 and ERK2 was enhanced in slices from PTU-exposed animals, whereas the levels of synaptic proteins in CA1 homogenate did not differ among groups.
Less severe thyroid hormone reductions in the low-dose group were without impact on baseline measures of synaptic transmission, but did reduce paired-pulse facilitation of the EPSP area, augmented population spike LTP, and enhanced stimulation-induced phosphorylation of ERKs. These results indicate that long-lasting disruptions in hippocampal synaptic function remain following moderate thyroid hormone insufficiency during early brain development. Persistent perturbations in synaptic function and cell signaling may contribute to cognitive dysfunction in adult animals suffering from a temporary thyroid hormone insufficiency during critical periods of brain development (Akaike et al., 1991; Darbra et al., 2004; Guadano-Ferrez et al., 2003).
Previous studies have reported that synaptic proteins or mRNA levels of these proteins are altered by developmental (e.g., Iniguez et al., 1993, 1996; Vara et al., 2002; Wong and Leung, 2001) and adult-onset hypothyroidism (Gerges and Alkadhi, 2004; Iniguez et al., 1992). Augmentation in basal levels of phosphorylated ERK was also recently reported in the hippocampus of newborn rat pups born to hypothyroid dams (Calloni et al., 2004). In this study, the protein levels of synapsin, GAP-43, RC3, and basal levels of total or phosphorylated ERKs in area CA1 of rat hippocampus were not affected by prior PTU exposure. These findings indicate that recovery of these biochemical indices correlates with the animal's return to a euthyroid state. Thus, it is unlikely that the observed functional deficits are attributable to permanent reductions in the basal levels of these neurochemical substrates. Rather, we postulate that the interference with thyroid hormone regulation of the expression of these proteins induced by early hormonal insufficiencies resulted in subtle aberrations in synaptic structure, connectivity, and network wiring and is reflected in the persistent impairments in synaptic function observed in the adult.
We do not know what is responsible for the PTU-induced dissociation observed in LTP of the dendritic versus somatic cell region. This observation is consistent with previous reports from our laboratory in slices from adult animals exposed in a primarily postnatal hypothyroid paradigm (Gilbert, 2003), and in population spike LTP assessed in vivo in the dentate gyrus following recovery from developmental hypothyroidism (Gilbert and Paczkowski, 2003). It is unlikely that augmentations in population spike LTP are secondary to the observed enhancements in baseline synaptic transmission. Increases in population spike LTP of comparable magnitude were evident in both dose groups, whereas increased cell excitability as evidenced in input-output functions (Fig. 1) was limited to the high-dose group. Furthermore, EPSP-population spike coupling presented in Figure 2 revealed that, despite significant increases in these parameters in high-dose animals, a normal ratio between the two components of the synaptic field potentials was maintained. Collectively, these observations indicate that augmented LTP of the population spike is not a simple consequence of increased excitability, but reflects a change in the network response to activity-induced plasticity.
Subtle perturbations in synaptic architecture or expression and localization of receptor- and voltage-gated ion channels induced by thyroid hormone insufficiencies may account for augmented population spike LTP. Calcium influx through N-methyl-d-aspartate (NMDA) receptors localized on dendritic spines is critical for LTP induction of the EPSP (Bliss and Collingridge, 1993; Zador et al., 1990). Voltage-dependent calcium channels (VDCC) localized on the cell soma and proximal dendrites mediate Ca2+ increases at the somatic level and contribute to synaptic plasticity of the population spike (Dudek and Fields, 2001, 2002; Muller and Connor, 1991; Nicoll and Malenka, 1995). Thus, differential expression or localization of NMDA and VDCC on pyramidal cell soma versus dendrites induced by inadequate hormone supplies during brain development may be reflected in an enhanced population spike LTP in the absence of changes in the dendritically-derived EPSP measure.
This hypothesis is consistent with our observations that developmental PTU treatment augments the phosphorylation of ERKs induced by LTP. Recent reports indicate that theta burst stimulation, as used in the present study, induces activation of the MAPK cascade and phosphorylation of ERK2 via calcium influx channels coupled to NMDA-receptors (Dudek and Fields, 2001). As is the case in the present study, LTP in slices from control animals was associated with phosphorylation of ERK2, with no change in ERK1 (Figs. 5B and 5C). However, calcium influx through L-type calcium channels in response to somatic action potentials and NMDA-receptor independent LTP are correlated with activation of ERK1 in addition to ERK2 (Aniksztejn and Ben-Ari, 1991; Dudek and Fields, 2001, 2002; Kanterewicz et al., 2000). Thus in the present study, the LTP-induced increase in phosphorylation of ERK2 above the levels achieved in controls, with the added phosphorylation of ERK1 that was restricted to PTU-exposed animals, is consistent with the observed augmentation in the amplitude of population spike LTP. As no difference in LTP magnitude was seen in the EPSP slope, it is tempting to speculate that augmented population spike LTP reflects enhanced calcium influx through L-type calcium channels at the cell soma in slices from developmentally thyroid-compromised animals.
The functional significance of enhanced LTP, protein phosphorylation, and perhaps ion channel properties to the behavior of the animal are unclear. Certainly the coordinated synchronization between synaptic depolarization and cell discharge, and the complex cell signaling pathways put into motion by activity-dependent synaptic plasticity in the hippocampus have been permanently disturbed as a consequence of early thyroid hormone insufficiency. This is likely to lead to information processing impairments within the hippocampal network and perhaps contribute to the learning deficits observed in animal models and subtle cognitive impairments in children suffering compromised thyroid status early in development.
In summary, the present study reported the persistent effects of moderate developmental thyroid hormone insufficiency on adult hippocampal synaptic functions. The degree of thyroid hormone suppression in treated dams at the time of weaning was indicative of hypothyroxinemia (i.e., no change in T3 but reduced T4 and concomitant rise in TSH). The perturbations in hippocampal synaptic function persist despite return to normal thyroid status. These observations replicate and expand upon recent work from our laboratory (Gilbert, 2004; Gilbert and Paczkowski, 2003), but significantly extend the range of observation of functional deficits to levels of hormone disruption that do not impart significant toxicity to the dam or offspring (i.e., 3 ppm-dose group). In addition, results of neurochemical analyses reveal that the physiological perturbations in synaptic plasticity observed in the euthyroid adult animal are associated with alterations in MAPK signaling, and further implicate this signaling cascade in the neuropsychological impairments that accompany developmental thyroid hormone insufficiency.
NOTES
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.
ACKNOWLEDGMENTS
This work was performed while the first author (L.S.) held a National Research Council Research Associateship Award at the U.S. Environmental Protection Agency. The authors wish to thank to Dr. Christopher Lau and Julie Thibodeaux for assistance in hormone analyses and Drs. Kevin Crofton and Diane Miller for insightful comments on an earlier version of this manuscript as well as constructive guidance provided by anonymous journal reviewers.
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Zoeller, R. T. (2003). Thyroid toxicology and brain development: Should we think differently Environ. Health Perspect. 111, A628.(L. Sui, W. L. Anderson an)
Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Department of Psychology, University of North Carolina, North Carolina 27599
ABSTRACT
Thyroid hormones are critical for the development and maturation of the central nervous system. Insufficiency of thyroid hormones during development impairs performance on tasks of learning and memory that rely upon the hippocampus and impairs synaptic function in young hypothyroid animals. The present study was designed to determine if perturbations in synaptic function persist in adult euthyroid animals exposed developmentally to insufficient levels of hormone. Pre- and postnatal thyroid hormone insufficiency was induced by administration of 3 or 10 ppm propylthiouracil (PTU) to pregnant and lactating dams via the drinking water from gestation day (GD) 6 until postnatal day (PN) 30. This regimen produced a graded level of hormonal insufficiency in the dam and the offspring. Population spike and population excitatory postsynaptic potentials (EPSP) were recorded at the pyramidal cell layer and the stratum radiatum, respectively, in area CA1 of hippocampal slices from adult male offspring. PTU exposure increased baseline synaptic transmission, reduced paired-pulse facilitation, and increased the magnitude of the population spike long-term potentiation (LTP). Phosphorylation of the extracellular signal-regulated kinases (ERK1 and ERK2) was increased as a function of LTP stimulation in slices from PTU-exposed adult animals. On the other hand, no differences in the basal levels of synaptic proteins implicated in synaptic plasticity (total ERK, synapsin, growth-associated protein-43, and neurogranin) were detected. These results reinforce previous findings of persistent changes in synaptic function and, importantly extend these observations to moderate levels of thyroid hormone insufficiency that do not induce significant toxicity to the dams or the offspring. Such alterations in hippocampal synaptic function may contribute to persistent behavioral deficits associated with developmental hypothyroidism.
Key Words: hypothyroidism; hippocampus; paired-pulse facilitation; long-term potentiation; extracellular signal-regulated kinase; adult.
INTRODUCTION
Thyroid hormones play crucial roles in the development and maturation of the central nervous system (Anderson et al., 2003; Bernal, 2002; Morreale de Escobar et al., 2004). Severe thyroid hormone insufficiency during the perinatal period results in impaired brain development with diminished interneuronal connectivity, decreased myelination, defective cell migration, and alterations in levels of neurotransmitters (Bernal, 2002; Lavado-Autric et al., 2003; Thompson and Potter, 2000). Delay in restoring normal thyroid status in the neonate is associated with long-lasting behavioral impairments (Glinoer and Delange, 2000; Morreale de Escobar et al., 2004; Zoeller and Rovet, 2004), underscoring the persistence of neurological deficiencies induced by early thyroid hormone insufficiency. Recently, even mild perturbations in the hormonal status of pregnant women have been associated with permanent intellectual impairment in their children (Haddow et al., 1999; Morreale deEscobar et al., 2004; Pop et al., 1999; Zoeller and Rovet, 2004).
Until recently, few experimental studies have been conducted to model the effects of subtle thyroid hormone insufficiency on early brain development or to evaluate the dose-response relationships between altered serum hormones and brain insult. Berbel and colleagues (Auso et al., 2004; Lavado-Autric et al., 2003) have reported cortical and hippocampal neuronal misplacements in the offspring of dams experiencing brief and mild reductions in thyroid hormones during gestation and lactation. A number of genes critical for cell proliferation, migration, and myelination have been identified, which are regulated by thyroid hormone (Bernal, 2002; Bernal et al., 2003; Oppenheimer and Schwartz, 1997; Thompson and Potter, 2000). Most of these studies, designed to evaluate the role of thyroid hormones on gene expression, have utilized a model of severe hypothyroidism. What has been lacking is a link between perturbations in gene expression, structural abnormalities, and brain function and an evaluation of the dose-response relationships following mild disruptions of the thyroid axis (Bernal et al., 2003; Forrest, 2004; Thompson and Potter, 2000; Zoeller, 2003). The current study was performed to provide information on the functional consequences of early hormone insufficiency in a brain slice preparation well suited for investigation of physiological, morphological, and molecular substrates. Synaptic transmission and plasticity were examined in the hippocampus, a structure known to be sensitive to thyroid hormone disruption and to play a critical role in some types of learning and memory. Offspring of thyroid-deficient dams in which a graded level of hormone insufficiency was produced were evaluated to begin to define the long-term consequences of milder forms of thyroid hormone insufficiency.
The primary health outcome of concern resulting from early thyroid hormone insufficiency is intellectual impairment. The deleterious effects of severe developmental hypothyroidism on the hippocampal morphology have been well established (Madeira et al., 1992; Rami et al., 1986). Perturbations in thyroid hormone function are associated with behavioral impairments in tasks requiring integrity of the hippocampus (Akaike et al., 1991; Darbra et al., 2004; Guadano-Ferraz et al., 2003). Recently, electrophysiological studies have also demonstrated that hypothyroidism induced by propylthiouracil (PTU) or methimazole exposure alters synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus (Niemi et al., 1996; Sui and Gilbert, 2003; Vara et al., 2002). The best model system to investigate the synaptic basis of cognition is long-term potentiation (LTP) in the hippocampus (Malenka and Nicoll, 1999), and long-term changes in hippocampal synaptic strength are generally accepted to be involved in learning and memory (Bliss and Collingridge, 1993; Brown et al., 1990). More recently, short-term synaptic plasticity as measured by paired-pulse functions has also been proposed to play a functional role in temporal information processing and reflect mechanisms important for learning (Buonomano and Merzenich, 1995; Dobrunz et al., 1997; Lisman, 1997; Silva et al., 1996). LTP and paired-pulse facilitation are reduced in area CA1 of slices from neonatal rats that remained hormone deficient at the time of assessment (Sui and Gilbert, 2003; Vara et al., 2002). In the present study, the persistence of these impairments was investigated in adults following recovery from hormone insufficiencies endured during development.
Regulation of the expression and phosphorylation of a number of synaptic proteins are under the control of thyroid hormones (Bernal, 2002; Bernal et al., 2003; Davis et al., 2002; Lin et al., 1999; Thompson and Potter, 2000). Many of these same proteins are also necessary for normal synaptic transmission, short-term and long-term plasticity, and hippocampal-based learning (Atkins et al., 1998; Benowitz and Routtenberg, 1997; Blum et al., 1999; English and Sweatt, 1996, 1997; Gerendasy et al., 1994; Impey et al., 1999; Klann et al., 1992; Oestreicher et al., 1997; Rosahl et al., 1993; 1995; Selcher et al., 1999; Winder et al., 1999). Thus, in addition to physiological assessments, levels of synapsin, growth associated protein 43 (GAP-43), neurogranin (RC3), and mitogen-activated protein kinase (MAPK) were examined in area CA1 of the hippocampus. A second aim of the present study was to determine if perturbations in the expression or activation of these proteins persist upon recovery of normal thyroid hormone status and could underlie alterations in synaptic function and behavior that are maintained in adult animals.
MATERIALS AND METHODS
Animal treatment.
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-approved animal facility. All animal treatments were in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals and their suffering. Animal rooms were maintained on a 12:12 light:dark schedule, and animals were permitted free access to food (Purina rat chow) and tap water. From GD 6 to postnatal day (PN) 30, dams were administered 3 or 10 ppm of the antithyroid agent, propylthiouracil (PTU, Sigma, St. Louis, MO) through the drinking water; control rats
Slice preparation and electrophysiological recording.
At the age of 11–14 week (PN77–PN96), one male offspring was randomly selected from each litter (0 ppm, n = 12 litters; 3 ppm, n = 15 litters; 10 ppm, n = 8 litters), sacrificed by decapitation, and prepared for slice electrophysiology as described previously (Sui and Gilbert, 2003). Trunk blood was collected at the time of sacrifice and stored for hormone analysis. The brain was removed, and the hippocampus was dissected on ice. Transverse hippocampus slices (400 μm) were cut using a McIlwain tissue chopper and placed in ice-cold, oxygenated artificial cerebrospinal fluid (ACSF, 124 mM NaCl, 3 mM KCl, 2.0 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, pH 7.4). Slices were immediately transferred to an interface recording chamber containing warmed, oxygenated (95% O2/5% CO2) ACSF and incubated at 33°C for a minimum of 1 h prior to recording. Biphasic squarewave pulses were delivered through a stainless steel electrode placed in the stratum radiatum, and stimulation-evoked extracellular field potentials were recorded from both the pyramidal cell layer and the stratum radiatum of CA1 through glass micropipettes (2–4 μm tip diameter) filled with ACSF. Stability of baseline recordings was established by delivering single pulses (1/min, 0.1 ms pulse width at an intensity yielding 70–80% of maximal population spike amplitude for a given slice) for 15–20 min prior to recording. When the variation in field potential amplitude was less then ±10%, the baseline was considered stable. Slices with unstable baseline responses were excluded from further experimentation.
Input-output (I/O) functions were collected to examine baseline synaptic transmission by delivering an ascending series of 14 stimulus intensities (20–150 μA) that ranged from subthreshold for elicitation of an excitatory postsynaptic potential (EPSP) to those eliciting maximal responses. Five pulses were delivered at each stimulus intensity at a frequency of 0.1 Hz and averaged at each recording site. Following collection of the I/O function, paired-pulse facilitation of the dendritic EPSP was measured at ten interpulse intervals (20–1000 ms) at maximal (150 μA) and submaximal (100–110 μA) stimulus strengths. Finally, LTP of somatic population spike and dendritic EPSP was induced. A test stimulus was chosen of intensity sufficient to produce a population spike approximately 70–80% of maximum. Pretrain responses were recorded for 20 min, followed by theta burst stimulation (25 4-pulse bursts at 100 Hz, 200 ms between bursts at the same stimulus intensity as the pretrain pulse). Single pulse recording resumed immediately following the train delivery and continued for 60 min.
Waveform scoring.
Action potentials in pyramidal cell neurons are reflected in field potentials recordings from the pyramidal cell layer as a large negative going potential, the population spike (Bliss and Richards, 1971). The dendritic responses recorded from the stratum radiatum provide an index of synaptic activity comprising the summed population excitatory postsynaptic potentials (EPSP) (Bliss and Richards, 1971). Population spike amplitude was estimated by calculating the voltage difference between the most negative point of the spike and a tangent connecting the onset of the spike and the next positive peak on the waveform. At the dendritic site, EPSP slope was calculated as the rate of amplitude change for the initial negative deflection to the peak. The EPSP peak amplitude was the most negative point on the waveform, and the EPSP area measure was initiated at the point of the initial negative deflection and ended with the return of the EPSP to the baseline. For I/O functions, response amplitude was normalized to the percentage of maximal population spike amplitude and EPSP slope. Paired-pulse facilitation and depression were expressed as the ratio of the mean amplitude of the second response relative to the first (pulse 2/pulse 1 x 100). LTP was expressed as percent change from the mean of ten pretrain recordings taken just prior to train delivery.
Sample preparation for ERK1 and ERK2 analysis.
LTP-induced phosphorylation of the extracellular signal regulated kinases (ERK1 and ERK2) of the MAPK family of kinases was investigated in a different set of hippocampal slices harvested from the same animals used for electrophysiological assessments. To prepare these samples, slices were maintained in the same interface chamber as those used for electrophysiological recordings and were allowed to incubate at 33°C for at least 1 h. Pairs of slices either were left unstimulated or
Sample preparation for synapsin, GAP-43, and RC3 analysis.
Levels of synaptic proteins synapsin, GAP-43, and neurogranin (RC3) were examined in area CA1 of hippocampus of adult male littermates of animals used for electrophysiology. Animals were sacrificed by decapitation, the hippocampus was removed, and the midtemporal region dissected. Tissue consisting primarily of area CA1 was extracted by cutting along the hippocampal fissure and was stored at –80°C for later analyses.
Western blot analysis.
For Western blot analyses, tissue was homogenized by sonication in ice-cold solubilizing buffer containing 1% Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, and 0.5% protease inhibitors (Protease Inhibitor Cocktail III, Calbiochem, La Jolla, CA). The insoluble material was removed by centrifugation at 10,000 x g for 10 min at 4°C. An aliquot of the supernatant was taken for protein determination, and the remaining supernatant was added to an equal volume of Laemmli's sample buffer (BioRad, Hercules, CA), and samples were boiled at 100°C for 5 min. Samples were resolved by SDS–PAGE followed by electrophoretic transfer onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The blots were blocked for 1 h with 5% nonfat dried milk at room temperature, then incubated overnight at 4°C with commercially available specific antibodies to the proteins of interest. After three short washes, the blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (1:20,000, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The blots were visualized using a chemiluminescence substrate (ECL, Bio-Rad, Hercules, CA), and the light images were collected and analyzed photometrically with a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA). For all Western blots, on each gel 2 or 3 lanes were reserved for a quality control (QC) sample. This sample was taken from a pool of a large number of hippocampal slices from nave animals. Aliquots of the pool were maintained at –70°C. One aliquot was used for each gel or series of gels run at one time. After correction for background chemiluminescence, the signals from target bands on a gel were normalized to the average signal for the QC sample bands to simplify comparison across gels and reduce inter-gel variability. The coefficient of variation for the QC values across gels was typically below 10%.
To investigate phosphorylation of ERK, mouse monoclonal anti-phospho ERK1/ERK2 (diluted 1:2500, Cell Signaling, Beverly, MA) was used for the primary antibody incubation. Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Chemical Co, Rockford IL) for 5 min, reblocked for 15 min with 5% nonfat dried milk at room temperature, then probed for total ERK using anti-total ERK1/ERK2 (diluted 1:5000, Cell Signaling, Beverly, MA). For each experiment, both total-ERK1 and total-ERK2 and phospho-ERK1 and phospho-ERK2 signals were normalized relative to those seen in unstimulated slices from the same animal. In addition, active ERK1 and ERK2 signals were normalized to total ERK1 and total ERK2 band intensities.
Rabbit polyclonal antibodies against synapsin (diluted 1:25000, Chemicon, Temecula, CA), and one recognizing a common site on both GAP-43 and RC3 (diluted 1:1000, Upstate, Lake Placid, NY) were used to probe for levels of these neuronal markers. For each blot, synapsin, GAP-43, and RC3 levels in the two PTU-exposed groups were normalized to QC standards and expressed as a percentage of the levels observed in control animals.
Thyroid hormones.
At the time of sacrifice for electrophysiological testing, trunk blood was collected and allowed to clot on ice for a minimum of 30 min. Serum was separated via centrifugation and stored at –80°C for later analyses by radioimmunoassay. Serum concentrations of total thyroxine (T4) and total triiodothyronine (T3) were analyzed by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). Thyroid stimulating hormone (TSH) was measured using standard double antibody assay as described by Thibodeaux et al. (2003). All samples for total T4 and total T3 were run in duplicate and the intra- and interassay variations ranged from 9 to 12%. The minimum detectable concentration (MDC) for each assay was determined statistically (3 standard deviations above background levels). For all T3 assays (n = 4), the MDC was 7.8 ng/ml, and for the T4 assay (n = 4), the MDC was 4.9 ng/ml. The lowest calibrator for each assay was 10 ng/ml. For statistical purposes, in those cases where the sample result was below the level of this calibrator, the result was set by default to the MDC.
Statistical analysis.
Group statistics were calculated as the mean ± SEM. Data from electrophysiological studies were evaluated using repeated measures analysis of variance (ANOVA). Where significant interactions were found, step-down ANOVAs and mean contrast tests using Tukey's t-test were performed. Thyroid hormone data were evaluated using one-way ANOVAs. Protein data were evaluated using nonparametric chi-square analysis. Probabilities less than 0.05 were considered significant.
Results
PTU Treatment on Offspring Body Weight and Thyroid Hormone Levels
There were no significant PTU treatment-related differences in dam body weights throughout gestation (data not shown). There were no apparent PTU treatment-related effects on offspring body weight over the first 10 postnatal days. Dose-dependent deficits in offspring body weight became apparent from PN13, and these deficits persisted to PN33 (119.8 ± 3.77 g, 89.0 ± 6.54 g, and 28.85 ± 1.70 g, in the controls, 3 ppm PTU-treated, and 10 ppm PTU-treated groups, respectively) (see Sui and Gilbert, 2003). In the adult offspring (PN77), the body weights were comparable between the control and the 3 ppm PTU-treatment group (475.6 ± 15.59 g and 429.9 ± 22.70 g, respectively), but remained significantly reduced in the 10 ppm PTU-treatment group (296.2 ± 14.32 g).
Dose-dependent reductions in thyroid hormones with concomitant elevations in TSH were observed in offspring at weaning and were reported in Sui and Gilbert (2003) and are summarized in Table 1. Dams displayed hypothyroxinemia at the low dose (reduced T4 with no significant reduction in T3), but hypothyroidism in the high-dose group (T3 and T4 reduced by 45 and 65%, respectively). Offspring were more severely affected; T4 in the low-dose group was reduced by 70% at weaning, and the majority of high dose animals fell below the level of detection of the assay. T3 was reduced by 35% and 70% in low and high dose animals, respectively. Although significant changes in thyroid hormones were observed in dams and young animals, complete recovery of T3 and T4 was evident in adult males at the time of testing (see Table 1). No significant differences were apparent in T4, T3, or TSH across dose groups (all p values > 0.1).
PTU Treatment on Baseline Synaptic Transmission
Baseline synaptic transmission, assessed by the EPSP slope and population spike amplitude in response to electrical stimulation of varying current amplitudes were recorded by two glass electrodes in area CA1, one placed in the stratum radiatum to assess dendritic activation, the other in stratum pyramidale to assess cell firing at the somatic site. Measurement of baseline synaptic transmission revealed that although maximal responses recorded at 150 μA stimulus intensity did not differ between the groups (p > 0.05), a leftward shift in normalized I/O functions was evident, indicative of an enhancement of baseline synaptic transmission (Fig. 1). Repeated measures ANOVAs of the normalized I/O functions revealed that EPSP slope (Fig. 1A; Dose x Intensity interaction [F(26, 416) = 2.22, p < 0.0006]) and population spike amplitude (Fig. 1B; Dose x Intensity interaction [F(26, 416) = 1.63, p < 0.027] were increased in PTU-exposed animals. Step-down ANOVAs revealed that these increases in EPSP and population spike relative to controls were restricted to the high-dose group (both step-down ANOVAs p values < 0.02).
As both EPSP slope and population spike amplitudes were increased in high dose PTU animals, it was of interest to determine if the relationship of synaptic activation and cell firing was disrupted. Figure 2 reveals that, although the high dose of PTU enhanced EPSP and population spike amplitude, coupling between dendritic and somatic response amplitudes was left unchanged.
PTU Treatment on Paired-Pulse Facilitation of EPSP
In slices from control animals, paired-pulse ratios of dendritic responses increased with interval, became maximal at mid-range, then declined at longer intervals (Fig. 3). Although it appeared as though the high dose of PTU reduced paired-pulse facilitation of the EPSP slope at the briefer intervals (Fig. 3A), variability was high, and ANOVA revealed no significant differences among the groups (p > 0.50). However, a similar pattern was observed in EPSP peak amplitude (Fig. 3B), and clear reductions in the 10 ppm group were apparent in the statistical analysis. A significant main effects of Dose [F(2, 30) = 3.58, p < 0.0404] and Dose x Interval interaction [F(18, 270) = 3.79, p < 0.0001] were obtained, but this reduction in EPSP amplitude measures was restricted in the high-dose group (Step-down ANOVA of 0 vs. 10 ppm, p < 0.03). In contrast, dose-dependent reductions in paired-pulse ratios of EPSP area were apparent from PTU-exposed animals (Figure 3C). Significant main effects of Dose [F(2, 31) = 11.95, p < 0.0001] and Dose x Interval interaction [F(18, 279) = 2.65, p < 0.0004] confirmed that PTU treatment impaired paired-pulse facilitation of dendritic synaptic responses. Step-down ANOVAs revealed that the 3 ppm and the 10 ppm groups were both significantly reduced relative to the controls (both p values < 0.03). Results of mean contrast comparisons at each IPI using Tukey's t-test are indicated by asterisks. A similar pattern was observed for all three measures at maximal and submaximal stimulus strengths. As with findings in younger animals, EPSP area appeared to represent the most sensitive marker of altered paired-pulse facilitation.
PTU Treatment Alters Long-Term Potentiation and ERK1 and ERK2 Activation
After train delivery, all groups displayed enhancement of dendritic EPSP slope and somatic population spike. No significant differences were obtained in dendritic EPSP slope (Fig. 4A) measures between the three treatment groups [main effect of Dose: F(2, 23) = 1.01, p > 0.38], whereas LTP of somatic population spike (Fig. 4B) was significantly increased in PTU-exposed groups relative to the controls [main effect of Dose: F(2, 26) = 3.623, p < 0.041]. Step-down ANOVAs confirmed that population spike LTP significantly elevated above the control values in both dose groups (both p values < 0.05).
The levels of the MAPK proteins, ERK1 and ERK2, were examined in LTP-stimulated and nonstimulated slices from the control and PTU-exposed animals (Fig. 5A). Western blots revealed that total ERK2 and total ERK1 levels were not affected by PTU treatment in either stimulated or nonstimulated slices (left panel Figs. 5B and 5C). Neither did basal levels of phospho-ERKs differ across groups in nonstimulated slices. However, increases in phospho-ERK2 (right panel Fig. 5B) and phospho-ERK1 (right panel Fig. 5C) were observed in response to LTP-inducing stimulation in PTU-exposed groups. Expressed as a percentage of the nonstimulated slices in each group, the data show PTU treatment did not interfere with total ERK2 [2 (2) = 4.49, p > 0.105, Fig. 5B] or total ERK1 levels [2 (2) = 0.611, p > 0.737, Fig. 5C]. PTU treatment dose-dependently elevated the phospho-ERK2 [2 (2) = 6.899, p < 0.032, Fig. 5B] and phospho-ERK1 [2 (2) = 7.00, p < 0.030, Fig. 4C] induced by LTP stimulation.
PTU Treatment on the Levels of Synapsin, GAP-43 and RC3
The levels of synapsin, GAP-43, and RC3 were examined in area CA1 of the adult hippocampus from the control and the PTU-exposed animals (Fig. 6). Western blots revealed that synapsin, GAP-43, and RC3 levels were not affected by PTU treatment (all p values > 0.05).
Discussion
The present study identified persistent perturbations in adult hippocampal synaptic functions following thyroid hormone insufficiency induced during early development. Despite recovery of thyroid hormone to control levels, increases in baseline synaptic transmission, reductions in paired-pulse facilitation, and augmentation in population spike LTP were observed in the high-dose group. LTP-induced increase in the phosphorylation of ERK1 and ERK2 was enhanced in slices from PTU-exposed animals, whereas the levels of synaptic proteins in CA1 homogenate did not differ among groups.
Less severe thyroid hormone reductions in the low-dose group were without impact on baseline measures of synaptic transmission, but did reduce paired-pulse facilitation of the EPSP area, augmented population spike LTP, and enhanced stimulation-induced phosphorylation of ERKs. These results indicate that long-lasting disruptions in hippocampal synaptic function remain following moderate thyroid hormone insufficiency during early brain development. Persistent perturbations in synaptic function and cell signaling may contribute to cognitive dysfunction in adult animals suffering from a temporary thyroid hormone insufficiency during critical periods of brain development (Akaike et al., 1991; Darbra et al., 2004; Guadano-Ferrez et al., 2003).
Previous studies have reported that synaptic proteins or mRNA levels of these proteins are altered by developmental (e.g., Iniguez et al., 1993, 1996; Vara et al., 2002; Wong and Leung, 2001) and adult-onset hypothyroidism (Gerges and Alkadhi, 2004; Iniguez et al., 1992). Augmentation in basal levels of phosphorylated ERK was also recently reported in the hippocampus of newborn rat pups born to hypothyroid dams (Calloni et al., 2004). In this study, the protein levels of synapsin, GAP-43, RC3, and basal levels of total or phosphorylated ERKs in area CA1 of rat hippocampus were not affected by prior PTU exposure. These findings indicate that recovery of these biochemical indices correlates with the animal's return to a euthyroid state. Thus, it is unlikely that the observed functional deficits are attributable to permanent reductions in the basal levels of these neurochemical substrates. Rather, we postulate that the interference with thyroid hormone regulation of the expression of these proteins induced by early hormonal insufficiencies resulted in subtle aberrations in synaptic structure, connectivity, and network wiring and is reflected in the persistent impairments in synaptic function observed in the adult.
We do not know what is responsible for the PTU-induced dissociation observed in LTP of the dendritic versus somatic cell region. This observation is consistent with previous reports from our laboratory in slices from adult animals exposed in a primarily postnatal hypothyroid paradigm (Gilbert, 2003), and in population spike LTP assessed in vivo in the dentate gyrus following recovery from developmental hypothyroidism (Gilbert and Paczkowski, 2003). It is unlikely that augmentations in population spike LTP are secondary to the observed enhancements in baseline synaptic transmission. Increases in population spike LTP of comparable magnitude were evident in both dose groups, whereas increased cell excitability as evidenced in input-output functions (Fig. 1) was limited to the high-dose group. Furthermore, EPSP-population spike coupling presented in Figure 2 revealed that, despite significant increases in these parameters in high-dose animals, a normal ratio between the two components of the synaptic field potentials was maintained. Collectively, these observations indicate that augmented LTP of the population spike is not a simple consequence of increased excitability, but reflects a change in the network response to activity-induced plasticity.
Subtle perturbations in synaptic architecture or expression and localization of receptor- and voltage-gated ion channels induced by thyroid hormone insufficiencies may account for augmented population spike LTP. Calcium influx through N-methyl-d-aspartate (NMDA) receptors localized on dendritic spines is critical for LTP induction of the EPSP (Bliss and Collingridge, 1993; Zador et al., 1990). Voltage-dependent calcium channels (VDCC) localized on the cell soma and proximal dendrites mediate Ca2+ increases at the somatic level and contribute to synaptic plasticity of the population spike (Dudek and Fields, 2001, 2002; Muller and Connor, 1991; Nicoll and Malenka, 1995). Thus, differential expression or localization of NMDA and VDCC on pyramidal cell soma versus dendrites induced by inadequate hormone supplies during brain development may be reflected in an enhanced population spike LTP in the absence of changes in the dendritically-derived EPSP measure.
This hypothesis is consistent with our observations that developmental PTU treatment augments the phosphorylation of ERKs induced by LTP. Recent reports indicate that theta burst stimulation, as used in the present study, induces activation of the MAPK cascade and phosphorylation of ERK2 via calcium influx channels coupled to NMDA-receptors (Dudek and Fields, 2001). As is the case in the present study, LTP in slices from control animals was associated with phosphorylation of ERK2, with no change in ERK1 (Figs. 5B and 5C). However, calcium influx through L-type calcium channels in response to somatic action potentials and NMDA-receptor independent LTP are correlated with activation of ERK1 in addition to ERK2 (Aniksztejn and Ben-Ari, 1991; Dudek and Fields, 2001, 2002; Kanterewicz et al., 2000). Thus in the present study, the LTP-induced increase in phosphorylation of ERK2 above the levels achieved in controls, with the added phosphorylation of ERK1 that was restricted to PTU-exposed animals, is consistent with the observed augmentation in the amplitude of population spike LTP. As no difference in LTP magnitude was seen in the EPSP slope, it is tempting to speculate that augmented population spike LTP reflects enhanced calcium influx through L-type calcium channels at the cell soma in slices from developmentally thyroid-compromised animals.
The functional significance of enhanced LTP, protein phosphorylation, and perhaps ion channel properties to the behavior of the animal are unclear. Certainly the coordinated synchronization between synaptic depolarization and cell discharge, and the complex cell signaling pathways put into motion by activity-dependent synaptic plasticity in the hippocampus have been permanently disturbed as a consequence of early thyroid hormone insufficiency. This is likely to lead to information processing impairments within the hippocampal network and perhaps contribute to the learning deficits observed in animal models and subtle cognitive impairments in children suffering compromised thyroid status early in development.
In summary, the present study reported the persistent effects of moderate developmental thyroid hormone insufficiency on adult hippocampal synaptic functions. The degree of thyroid hormone suppression in treated dams at the time of weaning was indicative of hypothyroxinemia (i.e., no change in T3 but reduced T4 and concomitant rise in TSH). The perturbations in hippocampal synaptic function persist despite return to normal thyroid status. These observations replicate and expand upon recent work from our laboratory (Gilbert, 2004; Gilbert and Paczkowski, 2003), but significantly extend the range of observation of functional deficits to levels of hormone disruption that do not impart significant toxicity to the dam or offspring (i.e., 3 ppm-dose group). In addition, results of neurochemical analyses reveal that the physiological perturbations in synaptic plasticity observed in the euthyroid adult animal are associated with alterations in MAPK signaling, and further implicate this signaling cascade in the neuropsychological impairments that accompany developmental thyroid hormone insufficiency.
NOTES
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.
ACKNOWLEDGMENTS
This work was performed while the first author (L.S.) held a National Research Council Research Associateship Award at the U.S. Environmental Protection Agency. The authors wish to thank to Dr. Christopher Lau and Julie Thibodeaux for assistance in hormone analyses and Drs. Kevin Crofton and Diane Miller for insightful comments on an earlier version of this manuscript as well as constructive guidance provided by anonymous journal reviewers.
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