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Endocrinology Vol. 144, No. 9 4195-4203
Copyright © 2003 by The Endocrine Society

Pre- and Postnatal Propylthiouracil-Induced Hypothyroidism Impairs Synaptic Transmission and Plasticity in Area CA1 of the Neonatal Rat Hippocampus

Li Sui and M. E. Gilbert

National Research Council (L.S.), Neurotoxicology Division (M.E.G.), United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and Department of Psychology, University of North Carolina, North Carolina 27599

Address all correspondence and requests for reprints to: M. E. Gilbert, Ph.D., Neurotoxicology Division (MD-B105-05), National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711. E-mail: gilbert.mary{at}epamail.epa.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones are essential for neonatal brain development. It is well established that insufficiency of thyroid hormone during critical periods of development can impair cognitive functions. The mechanisms that underlie learning deficits in hypothyroid animals, however, are not well understood. As impairments in synaptic function are likely to contribute to cognitive deficits, the current study tested whether thyroid hormone insufficiency during development would alter quantitative characteristics of synaptic function in the hippocampus. Developing rats were exposed in utero and postnatally to 0, 3, or 10 ppm propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, administered in the drinking water of dams from gestation d 6 until postnatal day (PN) 30. Excitatory postsynaptic potentials and population spikes were recorded from the stratum radiatum and the pyramidal cell layer, respectively, in area CA1 of hippocampal slices from offspring between PN21 and PN30. Baseline synaptic transmission was evaluated by comparing input-output relationships between groups. Paired-pulse facilitation, paired-pulse depression, long-term potentiation, and long-term depression were recorded to examine short- and long-term synaptic plasticity. PTU reduced thyroid hormones, reduced body weight gain, and delayed eye-opening in a dose-dependent manner. Excitatory synaptic transmission was increased by developmental exposure to PTU. Thyroid hormone insufficiency was also dose-dependently associated with a reduction paired-pulse facilitation and long-term potentiation of the excitatory postsynaptic potential and elimination of paired-pulse depression of the population spike. The results indicate that thyroid hormone insufficiency compromises the functional integrity of synaptic communication in area CA1 of developing rat hippocampus and suggest that these changes may contribute to learning deficits associated with developmental hypothyroidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, A MAJOR regulator in the developing central nervous system is thyroid hormone (1). Severe thyroid hormone insufficiency in man leads to cretinism, a syndrome associated with mental retardation and numerous neurological deficits (2). Animal models have shown that hypothyroidism during critical periods of development causes a vast array of abnormalities including incomplete maturation of neuronal and glial cells, reductions in the synaptic densities, myelin deficits, and changes in the number of specific cell populations (3). Even mild thyroid hormone insufficiency during specific developmental periods can result in permanent deficits in cognitive function (4).

The hippocampus is crucially involved in learning and memory in humans and other animals and expresses a very high degree of functional and structural plasticity (5, 6, 7). The deleterious effects of developmental hypothyroidism on hippocampal morphology have been well established (8, 9, 10). Although a variety of behavioral deficits have been described in hypothyroid animals, some of which implicate impairment in hippocampal function (11, 12), the mechanism whereby mild hypothyroidism impairs cognitive function has not been well studied.

Synaptic function in the hippocampus is often evaluated by short- and long-term activity-dependent modifications of synaptic efficacy as these measures likely reflect early intracellular events underlying some forms of learning and memory (7, 13, 14). Two measures of short-term changes in synaptic efficacy are known as paired-pulse facilitation and paired-pulse depression. Paired-pulse facilitation is measured by recording from the dendritic field of CA1 in the hippocampus in response to electrical stimulation of the neurons projecting to this field and appears as an increase in the excitatory postsynaptic potential (EPSP) when two stimulus pulses are delivered in close succession. Thus, the EPSP evoked by the second stimulus is quantitatively larger than the EPSP evoked by the first stimulus. In spite of its short duration (millisecond range), paired-pulse facilitation is believed to reflect mechanisms important for learning, in part because learning deficits have been correlated with deficits in this paradigm (14, 15).

Paired-pulse depression, in contrast to facilitation, is measured by recording from the cell soma layer following a stimulus presentation similar to that of paired-pulse facilitation, and occurs over a more limited range of interpulse intervals (IPIs). As the name implies, paired-pulse depression appears as a quantitatively smaller response evoked by the second stimulus relative to the first. Paired-pulse depression provides an index of the integrity of inhibitory synaptic transmission in the hippocampus (16, 17).

Both paired-pulse facilitation and depression in area CA1 undergo significant maturation between postnatal days (PN) 15 and 30 (18). Specifically, the magnitude of paired-pulse facilitation of the EPSP at brief intervals (IPIs < 30 msec) is smaller on PN15 than on PN30. The first appearance of paired-pulse facilitation at longer IPIs (~200 msec) occurs on PN18 and is maximal on PN30. Similarly, age-dependent changes in paired-pulse depression of the population spike have been described, with a peak in paired-pulse depression at the 20-msec IPI evident on PN30. Thus, examination of hypothyroid-induced disruption of paired-pulse facilitation and depression at particular stimulus frequencies may be used to characterize impairments and to identify mechanisms of altered synaptic function.

In addition to short-term changes in synaptic behavior, longer lasting forms of synaptic plasticity, long-term potentiation (LTP), and long-term depression (LTD), have been widely studied in the hippocampus as models of memory storage at the synaptic level (7, 19). These measures, LTP and LTD, appear as enhancements or diminutions of synaptic strength, respectively, that persist for hours. Despite their opposing effects, LTP and LTD share some common mechanisms, including N-methyl-D-aspartate receptor and calcium dependence (20, 21). Repetitive high-frequency bursts of stimulation delivered at the {theta} frequency (5–10 Hz) mimic the typical firing mode of hippocampal pyramidal cells during learning (22). Stimulation of inputs to area CA1 by {theta} burst stimulation preferentially induces LTP, whereas prolonged low frequency stimulation reliably elicits LTD in area CA1 of hippocampal slices, especially in relatively young animals (7, 13, 19).

Previous work has shown that LTP is reduced in area CA1 of animals ranging in age from 2–6 wk following the induction of severe hypothyroidism (23). Recent work from our laboratory has demonstrated impairments in synaptic transmission and LTP in the dentate gyrus of the adult hippocampus following neonatal hypothyroidism (24). The goal of the present study was to test the hypothesis that milder forms of hypothyroidism would exert deleterious effects on synaptic function in area CA1. Specifically, we used two low doses of propylthiouracil (PTU) to induce different intensities of thyroid hormone insufficiency. We observed that even mild hypothyroidism produces measurable effects on synaptic activity in area CA1 of the hippocampus. These disruptions in synaptic function may underlie cognitive deficits seen in animals experiencing a brief period of hormone insufficiency during critical phases of hippocampal development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment
Pregnant Long-Evans rats (Charles River, Raleigh, NC) were obtained on gestational day (GD) 2 and housed individually in standard plastic hanging cages with sterilized pine shavings as bedding in an Association for Assessment and Accredidation of Laboratory Animal Care (AAALAC)-approved animal facility. All animal treatments were in strict accordance with the National Institutes 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-h light, 12-h dark photoperiod, and all animals were permitted free access to food (Purina rat chow, Purina Mills, St. Louis, MO) and tap water. Beginning on GD6 and continuing until PN30, dams were given 3 or 10 ppm of PTU (Sigma, St. Louis, MO), a thyroid hormone synthesis inhibitor, via drinking water, control animals received deionized water. Administration of PTU in drinking water blocks the oxidation of iodide to iodine reducing the formation of T4 and T3 in the maternal thyroid (25). In addition to blocking synthesis, PTU also inhibits the peripheral deiodination of T4 to T3 (26). Because PTU readily passes the placental barrier and is transmitted to the suckling pups in the mother’s milk, the fetus and neonate also become hypothyroid (25). The day of birth was designated PN1. All litters were culled to 10 pups on PN4, keeping the maximal number of males, and were weaned on PN30. Dam weights were monitored frequently throughout pregnancy, and male pup weights were recorded during the first postnatal month. Eye opening was examined by daily observation between PN15 and PN19 and determining the ratio of pups within a litter with both eyes open.

Thyroid hormones
Trunk blood from dams on PN30 and pups at the time they were killed for electrophysiology recording was collected to clot on ice for a minimum of 30 min. Serum was separated via centrifugation of clotted samples and stored at -80 C for later analyses by RIA (Diagnostic Products Corp., Los Angeles, CA). Serum concentrations of total T4, total T3, and TSH were assayed as previously described by Thibodeaux et al. (27). All samples for total T4 and total T3 measurements were run in duplicate, and the intra- and interassay variations were less than 10%. Based on greater than 90% specific binding, the sensitivity of the RIA for total T4 was 10 ng/ml. Results below this limit of quantification were recorded at 10 ng/ml for statistical purposes. The level of detection in the TSH assay was 0.195 ng/ml, and values falling below this cutoff were excluded from the analysis.

Slice preparation
Between PN21 and PN30, one male offspring was randomly selected from each litter within each treatment group. As only a single animal could be examined each day, care was taken to systematically sample litters from each dose group such that age of testing was equated across dose groups. Only one animal from each litter was sampled for electrophysiological testing. Animals were killed by decapitation, the brain removed, and the hippocampus dissected on ice. Transverse hippocampus slices (400 µm) were prepared using a McIllwain tissue chopper (Stoelting, Wood Dale, IL) 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)]. Following chopping, slices were immediately transferred to an interface recording chamber containing warmed, oxygenated (95%O2/5%CO2) ACSF, and were incubated at 33 C for a minimum of 1 h before recording. Biphasic squarewave pulses were delivered through a stainless steel electrode placed in the stratum radiatum. Stimulation-evoked extracellular field potentials were recorded from both the stratum radiatum and the pyramidal cell layer of CA1 through glass micropipettes (2–4 µm tip diameter) filled with ACSF. The dendritic responses recorded from the stratum radiatum provide an index of synaptic activity comprising the summed EPSP. Action potentials in pyramidal cell neurons are reflected in field potential recordings from the pyramidal cell layer as a large negative going potential, the population spike (Fig. 1Go) (28). Stability of baseline recordings was established by delivering single pulses (1/min, 0.1 msec pulse width) for 15–20 min before recording. Data acquisition (33 kHz sampling rate) and rescoring were performed using a PC-based LabWindows (National Instruments Inc., Austin, TX) interface and custom-designed software.



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  		FIG. 1. Schematic illustration of a hippocampal slice with the position of recording electrodes and the waveforms recorded simultaneously from the somatic site, stratum pyramidal (population spike), and the dendritic site, stratum radiatum (EPSP) in response to the stimulation of Schaeffer collaterals of the stratum radiatum. Inhibitory interneurons are also displayed in the pyramidal cell layer.

 
Electrophysiological recording
To examine baseline synaptic transmission, an input-output (I/O) function was determined by delivering an ascending series of 14 stimulus intensities (20–150 µA) that ranged from subthreshold for elicitation of an EPSP to those eliciting maximal responses. Five pulses were averaged at each stimulus intensity at a frequency of 0.1 Hz. Following collection of the I/O function, paired-pulse facilitation of the EPSP was measured at ten intervals (IPIs 20–1000 msec) at maximal and submaximal stimulus intensities. Paired-pulse depression of the population spike was examined in the somatic site at an IPI of 20 msec. Five stimulus pairs delivered at 10-sec intervals were averaged at each IPI. All waveform scoring was performed on the averaged evoked responses.

For induction of LTP, a probe stimulus was chosen at an intensity sufficient to produce a population spike approximately 70–80% of maximum. Pretrain baseline responses (1/min) were recorded for 20 min followed by high-frequency stimulus bursts (25 four-pulse bursts at 100 Hz, 200 msec between bursts) delivered at the same stimulus intensity as the pretrain probe pulse. Following train stimulation, single pulse recording resumed for 60 min. LTD induction was carried out on a different slice from the same animal in a similar fashion. Instead of high frequency stimulation for LTP induction, LTD was induced by applying 900 pulses delivered at 1 Hz. Probing with single pulses was continued for at least 45 min after low-frequency train delivery.

Waveform scoring
Measurements were made of the slope, area, and peak amplitude of the EPSP in the dendritic recording site, and of the population spike amplitude in the somatic recording site using customized software. The slope of the EPSP was calculated as the rate of amplitude change for the initial negative deflection to the peak at the dendritic site. The EPSP peak was the most negative point on the waveform and EPSP area was derived by calculating area under the curve. The population spike amplitude was estimated in recordings from the somatic site by calculating the voltage difference between the most negative point of the spike and a tangent connecting the beginning of the spike and the next positive peak on the waveform (see Fig. 1Go). For I/O functions, response amplitude was normalized to the percentage of maximal EPSP slope and population spike amplitude. 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 and LTD were expressed as the percent change from the mean of 10 pretrain recordings taken just before train delivery.

Statistical analysis
Body weight, eye opening, and hormone data, I/O, and paired-pulse facilitation and depression data were subjected to repeated measures ANOVA. Repeated measures ANOVAs were also conducted on the 5-min averages taken 15, 30, and 45 min post train to evaluate the magnitude of LTP and LTD of EPSP slope and population spike amplitudes. Where significant interactions were found, step-down ANOVAs and mean contrast tests using Tukey’s t test were performed. All values were taken as the mean ± SEM. Probabilities less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTU effects on offspring development
There were no significant PTU treatment-related differences in dam body weights throughout gestation (Fig. 2AGo). ANOVA failed to reveal any main effect of dose or any dose x age interaction (both P values > 0.05). Weight gain of the dams was significantly reduced in the postnatal period, however, and this reduction was limited to the PN4–PN9 in the high-dose group [Fig. 2AGo, dose F (2, 42) = 3.57, P < 0.037; dose x age interaction F (14, 294) = 2.07, P < 0.013]. There were no apparent PTU treatment-related effects on the number of pups born per litter or on body weight over PN1–PN10 (all P values >0.05). Dose-dependent deficits in offspring body weight became apparent from PN13, and these deficits persisted to PN33 (Fig. 2BGo). These findings were supported by a significant main effect of dose [F (2, 40) = 67.32, P < 0.0001] and a significant dose x age interaction [F (12, 240) = 92.75, P < 0.0001].



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  		FIG. 2. PTU treatment did not affect maternal body weight gain throughout gestation but reduced weight gain in the high dose group in the postnatal period (A). Body weight gain was also reduced in developing pups in the high dose group (B). Mean contrast tests (Tukey, P < 0.05) are indicated where the 3 ppm (*) or 10 ppm (#) group were significantly reduced relative to controls.

 
Eye opening was delayed in PTU-treated animals in a dose-dependent fashion (Fig. 3Go). The 3-ppm treatment group showed a moderate delay, whereas a longer delay in the age of eye opening was seen in the 10-ppm treatment group. These findings were supported by a significant main effect of dose [F (2, 30) = 16.70, P < 0.001] and a significant age x dose interaction [F (8, 120) = 6.53, P < 0.001].



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  		FIG. 3. PTU exposure delayed the eye opening in offspring. *, Significantly different from control; #, significantly different from 3 ppm (Tukey, P < 0.05).

 
PTU treatment on thyroid hormone levels
Maternal exposure to PTU caused alterations in circulating levels of thyroid hormones and TSH levels in the dams (Fig. 4A) and offspring (Fig. 4BGo). At the termination of exposure on PN30, maternal total T4 was decreased 43.9% in the 3-ppm and 65.0% in the 10-ppm group relative to controls [dose F (2, 35) = 19.71, P < 0.001], with no concomitant change in total T3 [F (2, 35) = 0.61, P > 0.551]. Maternal TSH was increased above control levels in a dose-dependent manner [dose F (2, 33) = 9.21, P < 0.001].



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  		FIG. 4. A, Dam hormonal profile. PTU exposure reduced circulating levels of total T4 in dams at the termination of exposure on PN30 in a dose-dependent manner. No significant differences in total T3 were evident in dams on PN30. TSH levels were elevated in a dose-dependent manner. B, Pup hormonal profile. PTU treatment reduced total T4 and total T3 in blood of offspring collected on the day of testing. TSH was increased in the 3-ppm dose group, relative to controls but was not significantly different in the 10-ppm group. Results of mean contrast tests (Tukey, P < 0.05) indicate *, significantly different from 0 ppm; #, significantly different from 3 ppm.

 
In pups at the time of testing (PN21–PN30), total T4 was depressed approximately 75% in both groups relative to the controls (Fig. 4AGo). A large proportion of samples from the 10-ppm group (12/14) were below the level of sensitivity of the assay and a value of 10 ng/ml was substituted for statistical purposes. This also occurred in a much smaller set of samples from the 3-ppm group (3/14) but all control values were well within the range of detection of the assay. ANOVAs revealed a significant main effect of dose [F (2, 36) = 99.86, P < 0.001], and mean contrast comparisons supported a significant difference between controls and both PTU- exposure groups (both P values < 0.05). PTU also suppressed the circulating levels of total T3 in offspring and the degree of suppression was dependent on administered dose (Fig. 4BGo). Total T3 was depressed 35.8% in the 3-ppm group and 66.5% in the 10-ppm group relative to the controls and a significant main effect of dose [F (2, 31) = 80.76, P < 0.001] was apparent. PTU also significantly increased TSH levels, ANOVA revealed a significant main effect of dose [F (2, 33) = 4.24, P < 0.02]. However, this increase was restricted to the 3-ppm group. A more blunted TSH response was seen in the high-dose group, and mean values were not significantly different from controls.

PTU treatment on baseline synaptic transmission
Baseline synaptic transmission, represented by the EPSP slope and population spike amplitude in response to electrical stimulation of varying current intensities, was examined in the dendritic and somatic sites of area CA1. Measurement of baseline synaptic transmission showed that although maximal responses recorded at 150 µA stimulus intensity did not differ between the groups (Table 1Go, all P values > 0.05), a shift in normalized I/O functions was evident (Fig. 5Go). Both EPSP slope and population spike amplitudes were enhanced relative to controls as a function of PTU treatment. Repeated measures ANOVAs of the normalized I/O functions confirmed that EPSP slope and population spike amplitude increased as a function of stimulus intensity (both P values < 0.001). For EPSP slope (Fig. 5AGo), significant main effects of dose [F (2, 34) = 5.68, P < 0.007] and dose x intensity interaction [F (26, 442) = 3.36, P < 0.001] demonstrated that PTU treatment increased dendritic synaptic output. Similarly, a significant main effect of dose [F (2, 34) = 4.85, P < 0.014] and dose x intensity interaction [F (26, 442) = 2.33, P < 0.001] for normalized population spike amplitude (Fig. 5BGo) indicated that PTU treatment also increased cell excitability.


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  		TABLE 1. Mean (±SEM) EPSP slope and population spike (PS) amplitude values in I/O function at maximal stimulus intensity of 150 µA

 


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  		FIG. 5. Normalized I/O functions. Mean (±SEM) EPSP slope (A) and population spike amplitude (B) for the control, 3 ppm and 10 ppm PTU-exposed rats as a function of stimulus intensity. PTU treatment dose dependently increased EPSP slope and population spike amplitude. Mean contrast comparisons (Tukey, P < 0.05) demonstrated the 10-ppm group (#) was significantly increased relative to controls at a number of intensity levels for both EPSP slope and population spike. The 3-ppm group appeared elevated relative to control values but differed significantly at only one intensity level in the EPSP slope measure.

 
PTU treatment on paired-pulse facilitation of dendritic responses
When pairs of stimulus pulses are delivered in close succession, paired-pulse facilitation represents an increase of the second postsynaptic response (pulse 2) relative to the first (pulse 1). In control slices, paired pulse ratios increased with interval, became maximal at IPIs 30–70 msec, and then declined at longer intervals (Fig. 6Go). Similar patterns were seen at submaximal (80–90 µA) and maximal stimulus (150 µA, data not shown) strengths. Area measures of paired-pulse facilitation (Fig. 6AGo) were the most sensitive to PTU-induced alterations, demonstrating significant reductions at all intervals less than 500 msec at both dose levels [dose F (2, 28) = 28.22, P < 0.001; dose x IPI interaction F (18, 252) = 8.68, P < 0.0001]. Significant reductions in paired-pulse facilitation were also evident in EPSP peak and EPSP slope measures and were most prominent in the high-dose group.



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  		FIG. 6. Mean (±SEM) paired-pulse ratios of EPSP area (A) and EPSP peak (B) and EPSP slope (C) were reduced in PTU-treated animals at a submaximal stimulus intensity. Mean contrast tests (Tukey, P < 0.05) indicated the 3-ppm (*) and the 10-ppm (#) groups were significantly reduced relative to controls. The high-dose group demonstrated a dramatic reduction in paired-pulse facilitation of all measures and at most intervals. EPSP area measures (A) appeared to be the most sensitive to disruption by low doses of PTU.

 
PTU treatment on paired-pulse depression of somatic responses
Paired pulse ratios of somatic population spike amplitude were also recorded at a brief IPI of 20 msec. In contrast to the facilitation that is characteristic of dendritic recordings, somatic responses are typically predominated by inhibitory influences at brief IPIs so that the second response of the pair is suppressed relative to the first. This is indicated by a mean percent ratio of less than 100, the dotted line in Fig. 7Go. In slices from control animals, the paired-pulse depression of the population spike was approximately 35%, whereas both PTU treatment groups displayed no depression, but rather clear facilitation of the population spike at both stimulus strengths (Fig. 7Go). ANOVA revealed a significant main effect of dose for the submaximal [F (2, 33) = 9.22, P < 0.0007] and maximal stimulus strengths [F (2, 33) = 4.20, P < 0.023].



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  		FIG. 7. Mean (±SEM) paired pulse ratios of population spike amplitude at 20 msec IPI at submaximal and maximal stimulus intensities were increased in PTU-exposed animals. Depression of the population spike in slices from control animals was replaced by facilitation of the population spike in slices from PTU-treated animals (mean values > 100% indicated by dotted line). Mean contrast tests (Tukey, P < 0.05) indicated the 3-ppm (*) and the 10-ppm (#) groups were significantly different from controls.

 
PTU treatment on LTP
Robust LTP of EPSP slope and population spike was evident in slices from control subjects (Fig. 8Go). An initial reduction in response amplitude was consistently observed in the first minute post train, but clear augmentation was evident over the next several minutes. It is possible that the relatively high stimulus strength of the train and the test pulse coupled with the age of the animal may account for this suppression, which has not been observed in similar recordings from adult animals in our laboratory. LTP of the dendritic EPSP slope was significantly smaller in both PTU treatment groups (Fig. 8AGo). Repeated measures of ANOVAs of the posttrain responses revealed a significant main effect of dose [F (2, 27) = 5.11, P < 0.013] with no interaction. Step-down ANOVAs indicated that LTP of EPSP slope of the 3-ppm and 10-ppm PTU-exposed groups were significantly suppressed relative to the controls (both P values < 0.05). Unlike EPSP slope LTP, no differences were seen in population spike LTP (Fig. 8BGo), and this observation is supported by the ANOVA results [dose F (2, 31) = 0.24, P > 0.787].



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  		FIG. 8. The percent of baseline (±SEM) of EPSP slope (A) and population spike amplitude (B) after high-frequency train delivery to induce LTP in the control and PTU-exposed rats. EPSP slope LTP was reduced in both PTU-exposed groups relative to the controls. Population spike LTP was unaffected by PTU treatment.

 
PTU treatment on LTD
A long-lasting depression of EPSP slope and population spike amplitude was observed in slices from the control and PTU-exposed animals administered low-frequency stimulation. PTU treatment did not interfere with LTD induction (data not shown). Repeated measures of ANOVAs of the posttrain responses indicated comparable LTD of EPSP slope [dose F (2, 31) = 0.38, P > 0.68] and of population spike [dose F (2, 28) = 0.08, P > 0.92] in all groups.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study examined the role of mild thyroid hormone insufficiency in the development of synaptic functions in area CA1 of the hippocampus. Consistent with previous findings, the extent to which circulating thyroid hormones were reduced in the dams and in the pups was directly related to the administered dose of PTU, with a preferential sensitivity for T4 over T3 (29, 30, 31). The degree of thyroid hormone insufficiency in treated dams at the time of weaning was indicative of hypothyroxinemia (i.e. low T4 without a change in T3), whereas pups were more severely impacted. A dose-dependent elevation of TSH in dams was apparent, whereas the mean concentration of TSH in offspring revealed a dramatic increase in the low-dose group that curiously, was not evident in pups from the high-dose group. The reason for the lack of TSH increase in the high-dose animals is unclear. Circulating levels of T4 and TSH can be reduced by low food intake (32), and the high-dose animals were of smaller body size and were developmentally delayed. These pups suckled for a longer period of time relative to their control and low dose counterparts who were acquiring sustenance from the dam in addition to ingestion of chow during the period of testing. The possibility exists that in the high-dose group with nondetectable serum T4 concentrations, an apparent blunted TSH response may arise from a compromised nutritional status.

The results of neurophysiological assessments showed that baseline synaptic transmission in area CA1 was increased, whereas paired-pulse facilitation and LTP of the dendritic synaptic potentials, and paired-pulse depression of the somatic population spike were reduced in young animals experiencing thyroid hormone insufficiency in the early developmental period. Increased excitatory transmission in hypothyroid animals as revealed in I/O functions is consistent with augmented transmitter release and reduced inhibitory tone suggested by the results of paired pulse tests. LTP deficits were evident and indicate that hormone deprivation interferes with one of the basic mechanisms of information storage in the brain. Together, these alterations in synaptic function may underlie cognitive impairments in animals deprived of thyroid hormone early in life. Furthermore, these data reveal dose-dependent dysfunction in synaptic transmission in response to modest degrees of thyroid hormone insufficiency. Additional work is required to determine if thyroid hormone supplements may reverse or diminish the observed impairment in synaptic function.

I/O functions and paired pulse tests
Significant enhancements in both dendritic (EPSP slope) and somatic (population spike) measures of synaptic transmission were observed in I/O functions of slices from PTU-exposed animals. Paired-pulse facilitation and depression were also reduced. Under control conditions, augmentation of the second response relative to the first results from an increased probability of transmitter release (33, 34). Reductions in paired-pulse facilitation in the present study are indicative of increases in the probability of transmitter release in response to the first pulse such that the second pulse of the pair arrives at a time of diminished supply of vesicles from the releasable pool (33, 34). Increases in EPSP slope and population spike amplitude revealed in I/O functions could stem from these changes in transmitter release properties. These observations are consistent with hypothyroid-induced disruptions in neurotransmitter release in hippocampal pyramidal cells recently reported by Vara et al. (35).

In addition to presynaptic contributions, components of paired-pulse facilitation of the EPSP are derived from disinhibition of {gamma}-aminobutyric acid (GABA)-mediated inhibitory postsynaptic potentials (IPSPs) (17, 36). Disinhibition results from activation of presynaptic GABAB autoreceptors located on interneurons that serve to limit further release of inhibitory transmitter (37, 38). Thus, reductions in paired-pulse facilitation of the EPSP in hypothyroid animals may not only reflect increases in excitatory transmitter release, but also reductions in inhibition mediated by interneurons synapsing on the dendrites proximal to the synaptic site (Fig. 1Go). At the cell soma level, paired-pulse depression of the population spike was also reduced indicative of diminished synaptic inhibition mediated by interneurons synapsing close to the cell body (Fig. 1Go) (36). Augmentations in I/O estimates of EPSP slope and population spike amplitudes are consistent with PTU-induced perturbations in transmitter release and inhibitory properties of hippocampal neurons. Such disturbances in basic communication of synaptic circuits would be anticipated to disrupt information processing within the hippocampal network.

Deficits in synaptic plasticity
Two forms of activity-dependent synaptic modification, LTP and LTD, assessed in the present study have been implicated in memory function in adult animals (7) and the fine tuning of precise synaptic connections in many brain regions during ontogeny (39). LTP of dendritic EPSP slope amplitudes was significantly reduced by developmental thyroid hormone insufficiency. Dendritic spines represent the specialized postsynaptic contact points and the primary locus for synaptic communication and postsynaptic modifications associated with experience-dependent plasticity (40, 41). As the number of synaptic spines on hippocampal neurons are modulated by thyroid hormone, and reductions in spine density are evident in hypothyroid animals, reductions in spine density and diminished synaptic numbers could account for the observed decrements in LTP (5, 8, 9, 10).

EPSP slope deficits were not mirrored in population spike measures of LTP at the cell soma layer. Dissociation between dendritic and somatic indices of potentiation is modulated by GABAergic and calcium channel activity (42, 43, 44). Disruptions in both GABA- and calcium-mediated functions are suggested by the results of paired-pulse facilitation and depression. Thus, perturbations revealed in tests of short-term synaptic plasticity may contribute to the selective failure of EPSP over population spike LTP.

In contrast to LTP, a related form of long-lasting synaptic plasticity, LTD, was not affected by hormone insufficiency early in life. Based on the findings from tests of paired pulse depression and facilitation, it is postulated that shifts in the developmental status of excitatory and inhibitory networks in hypothyroid animals may serve to favor frequency domains that impart LTD over LTP. For example, LTP was induced using 100 Hz trains (IPI within the train burst = 10 msec) delivered at {theta} frequency (inter-train interval = 200 msec), LTD with 1 Hz trains (IPI = 1000 msec). The magnitude of inhibitory components that modulate the population spike amplitude progressively increase from PN9 to PN30; thus, somatic responses to high frequency stimulation are under less inhibitory control in younger animals (18). Consistent with a hypothyroid-induced developmental delay, paired-pulse depression of the inhibitory components were reduced in PTU-exposed animals relative to controls. Therefore, it is possible that during LTP induction, control slices are stimulated at frequencies where inhibitory influences over population spike amplitude are more pronounced than in their developmentally delayed hypothyroid counterparts. In contrast, the level of LTP of the EPSP is enhanced in control slices due to the maximal disinhibition at 200 msec IPI, the {theta} frequency at which train bursts were delivered. Thus, in hypothyroid animals the reduction of EPSP paired-pulse facilitation may contribute to LTP impairment at the synaptic site, whereas the relative state of disinhibition of the population spike facilitates expression of population spike LTP. The slow stimulation pattern necessary to induce LTD, being outside the frequency range at which the maturity of inhibitory systems would have much effect, levels a degree of protection to this form of synaptic plasticity.

Acute effects of thyroid hormone
In addition to neurochemical, morphological, and maturational perturbations in hippocampus associated with developmental hypothyroidism, it is important to also consider the acute pharmacological actions of thyroid hormone on physiological responses (45). Recently, T3 activation of MAPK, a signaling pathway critical for LTP induction, has been demonstrated (46, 47, 48). Furthermore, Tang et al. (49) have reported increased expression of thyroid hormone-responsive protein mRNA following LTP in vivo, and the induction of potentiation by intrahippocampal infusion of T3. If T3 contributes to the degree of LTP in euthyroid animals, its absence in the hypothyroid state might be anticipated to impair LTP induction, independent of classic morphological and genomic perturbations associated with developmental hormonal deprivation. That such acute effects of thyroid hormone depletion contribute to impairments accompanying developmental hypothyroidism is indirectly supported by preliminary observations that LTP deficits in CA1 are not evident in assessments made of adult offspring following perinatal hypothyroidism (50, 51). On the other hand, LTP impairments are evident in euthyroid adults in a different subregion of the hippocampus, the dentate gyrus, following a brief period of neonatal hypothyroidism (24). This stands in contrast to observations made when hormone reductions are induced in the adult. Under these circumstances CA1 LTP is impaired and dentate gyrus LTP remains intact (52). Thus, the role thyroid hormones play in hippocampal physiology are complex and appear to vary with developmental state of the specific subregion at the time of deprivation, hormonal status at the time of testing, and hippocampal subregion under study.

In summary, this study characterizes alterations in synaptic transmission and plasticity in area CA1 of developing rat hippocampus induced by thyroid hormone insufficiency in the pre- and postnatal periods. The results demonstrate that synaptic dysfunction occurs following moderate degrees of hormone perturbation. Relative to previous reports (23, 35), hippocampal functional deficiencies were evident at relatively modest perturbations in thyroid function in the dams (<50% reductions in T4 with no concomitant change in T3), and displayed a clear dose dependency. Developmental hypothyroidism modulates the expression of a variety of genes that interfere with cell migration, myelination, synaptic connectivity, and dendritic arborization (53). Perturbation in the timing of expression of these genes critical for these developmental events is likely to impact upon synaptic function. Our results confirm that modest degrees of thyroid hormone insufficiency impair neurological development in neonatal rat hippocampus, and it is proposed that these functional changes in synaptic transmission and plasticity may contribute to learning deficits associated with developmental hypothyroidism.


   Acknowledgments
 
The authors wish to express sincere gratitude to Drs. Thomas Zoeller, Jeffrey Goodman, and Christopher Lau for insightful comments on an earlier version of this manuscript. The excellent technical assistance of Willard L. Anderson, Joan Hedge, and Julie Thibodeaux is also gratefully acknowledged.


   Footnotes
 
This work was performed while the author held a National Research Council Research Associateship Award at the United States Environmental Protection Agency. 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.

Abbreviations: ACSF, Artificial cerebrospinal fluid; EPSP, excitatory postsynaptic potential; GABA, {gamma}-aminobutyric acid; GD, gestation day; I/O, input-output; IPI, interpulse interval; LTD, long-term depression; LTP, long-term potentiation; PN, postnatal day; PTU, propylthiouracil.

Received March 28, 2003.

Accepted for publication May 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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