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Interdisciplinary Toxicology Program (M.A.T., J.J.W., J.W.F.) and Department of Physiology and Pharmacology (M.A.T., J.S., J.J.W.), University of Georgia, Athens, Georgia 30602; and Department of Veterinary Biosciences (D.C.F.), University of Illinois Urbana-Champaign, Urbana, Illinois 61802
Address all correspondence and requests for reprints to: Duncan Ferguson, Department of Veterinary Biosciences, University of Illinois Urbana-Champaign, Urbana, Illinois 61801. E-mail: dcf{at}uiuc.edu.
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Abstract |
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Introduction |
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With respect to measures of impact on brain function, it is well established that disruption in the level of thyroid hormone during development can alter synaptic transmission in regions such as the hippocampal formation. Significant effects on long-term potentiation (LTP) have also been reported (18, 19, 20, 21, 22, 23, 24, 25, 26, 27). LTP has been denoted as a cellular model for learning and memory processes; therefore, we and others have theorized that changes in LTP could provide a functional link between thyroid hormone deficits with deficiencies in cognitive function (28). In addition, prior studies have also demonstrated that inhibitory and excitatory pathways may be differentially impacted by thyroid insufficiency (29, 30). Such complexities may contribute to the wide range of outcomes reported concerning the assessment of thyroid hormone deficiency on electrophysiological parameters measured in the hippocampus (15, 19, 24).
Whereas significant animal research has been performed on thyroid toxicants, including studies of neurodevelopmental toxicity, thyroid status is most commonly determined by serum hormone concentrations such as total T4 and TSH (31, 32, 33, 34, 35). Many factors influence the relationship between serum and tissue levels of free and bound thyroid hormones, and the relationships may be tissue and region specific (36, 37, 38). Propylthiouracil (PTU) will have both dam-mediated and direct effects on the gestating pups because it will cross the placental barrier (39). Whereas PTU has been shown to have effects on the liver and immune systems, many of these effects have been demonstrated at doses well beyond those used in this study. PTU has been routinely used to induce developmental hypothyroidism to study neurological impact.
The hypothalamic-pituitary-thyroid axis has a variety of mechanisms that would allow potential adaptation to a toxicological insult, including alteration of pituitary TSH secretion, alteration of intrathyroidal sodium-iodide symporter and thyroglobulin synthesis, change in deiodinase enzyme activity, and altered thyroid receptor number or affinity. However, little is known about the threshold for triggering these changes nor about their compensatory limit. These issues become particularly critical to understand in subpopulations at greater risk from thyroid disruption, the developing fetus and neonate. The primary goal of the present study was to use gestational and lactational exposure to the mechanistically well-described antithyroid drug, PTU, in the rat to study serum, thyroid, and brain hormone markers of thyroid status in both offspring and dams. These parameters were then correlated with electrophysiology results measured in the CA1 region of hippocampal slices obtained from the offspring. This study identifies the biomarkers most tightly linking gestational and neonatal thyroid insufficiency with the altered neurophysiological outcomes measured in the hippocampus.
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Materials and Methods |
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Animals were weighed every other day and water intake recorded. Water bottles were refilled every 2–4 d, depending on the amount remaining in the bottle. New PTU solutions were made at least every 2 wk. Average water intake during gestation was approximately 200 ml/kg·d for the dams. Pups in the control and 3-ppm groups began to supplement their diet with food and water before weaning at varying times after postnatal d (PND) 14, but pups in the 10-ppm group were unable to do so due to reduced size and coordination. Pups were killed beginning on PND21, with two males per litter killed over the next 11 d. Data from these animals are presented as PND25 pups. Additionally, all female pups were culled at PND23. Remaining males were weaned at the end of this period when the animals were killed (on PND32) and followed up to PND90–110 referred to hereafter as adults. In this study, with this weaning protocol, the weaned 10-ppm pups were so physically and neurodevelopmentally delayed that it influenced their ability to independently seek adequate food and water. Despite attempted supportive measures, it was not possible to consistently maintain these animals for study in the PND90–110 adult grouping.
Animal studies were approved by the University of Georgia Institutional Animal Care and Use Committee and were in accordance with procedures outlined in the National Institutes of Health Guidelines for Care and Use of Laboratory Animals.
Euthanasia protocol
Male pups were weighed, anesthetized with halothane, and killed via decapitation. Blood, thyroid, and liver were collected. The brain was dissected for tissue collection and slices prepared for electrophysiology experiments (see below). In all pup and adult samples, the majority of the hippocampus was used for slice preparation. Cortex samples were taken from areas of the brain not impacted by dissection of the hippocampus, generally including prelimbic, motor, sensory, and cingulate regions, and brain tissue samples were divided equally between deiodinase and hormone assays. Liver and cortex samples intended for deiodinase assays were homogenized on the day the animals were killed in a buffer of 250 mM sucrose, 20 mM KH2PO4, 1 mM EDTA, 20 mM dithiothreitol (DTT) and then stored in aliquots at –80 C until assayed. Blood was allowed to clot and serum collected after centrifugation. All other tissues were flash frozen and stored at –80 C until analysis.
Biochemical assays
Serum thyroid hormone and TSH RIAs.
T3 and T4 RIAs were validated for rat serum as previously described, with coefficients of variation of approximately 12 and 5%, respectively (40). TSH was measured in dams with a commercial immunoassay kit (MP Biomedicals, Solon, OH). Free T4 concentrations were measured by use of a two-step direct dialysis commercial RIA kit (Nichols Institute, San Juan Capistrano, CA) with a coefficient of variation of approximately 5%. All samples for serum hormone quantification were run in a single assay for each hormone.
Thyroid RNA extraction and analysis.
Thyroglobulin (Tg) and the sodium iodide symporter (NIS) were selected for mRNA analysis because they are, at least partly, TSH dependent. Thyroids were extracted by use of the Absolutely RNA kit (Stratagene, La Jolla, CA). After extraction, real-time RT-PCR was performed for NIS and Tg at the UGA Functional Genomics Resource Facility using Taq-Man primers and probes (see Table 1
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Cerebrocortical type II 5'-deiodinase activity
This protocol was based on a modification of that of Leonard et al. (41). Cerebrocortical type II 5'-deiodinase activity was assayed using 2 nM reverse T3 (rT3), including 1.9 MBq [125-I]-T3 in 100 mM KH2PO4, 1 mM EDTA, 20 mM DTT (pH 7) with and without 1 mM PTU. Triplicate tubes were incubated at 37 C for 1 h with 50 µg protein for pups, or 200 µg for dams and adults, in a total volume of 320 µl along with tissue-free blanks. The reaction was stopped by addition of 150 µl ice-cold 10% BSA. Tubes were then incubated 30 min at 4 C before addition of 500 µl of 20% trichloroacetic acid. The tubes are spun at 2600 x g at 4 C for 45 min. Then 400 µl of supernatant are added to a 1.5-ml column of 50WX2–200 resin (Sigma) and eluted with 2 ml 70% acetic acid. Tubes were then counted on a 
-counter (Wizard 1470; Wallac, now part of PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA) along with tubes for total counts. The percentage of free iodide was calculated by comparison of the elution fractions to the total counts. The radioactivity in the eluted fractions minus that of the blanks was used to calculate the total deiodinase activity. The percent free iodine generated in the samples containing PTU was multiplied by 2 to account for the equal possibility of liberating a radioactive or nonradioactive iodide and then divided by the time and protein amount and then multiplied by the total rT3 concentration in the assay to arrive at the final activity measurements. The type 2 deiodinase (D2) activity was shown to be inhibited by iopanoic acid and excess of rT3 (data not shown).
Hepatic type I 5'-deiodinase (D1) activity
Liver D1 assays were carried out using approximately 2 nM rT3 including 1.9 MBq [125-I]-rT3 in 200 mM KH2PO4, 1 mM EDTA, 2 mM DTT (pH 7) with and without 1 mM PTU. Triplicate tubes were incubated at 37 C for 2 and 12 or 62 min (PTU-treated animals) with 2–3 µg protein in a total volume of 120 µl. The reaction was stopped by addition of 500 µl ice-cold 10% calf serum and then incubated for 30 min on ice. Then 500 µl of ice-cold 10% trichloroacetic acid was added to precipitate the reaction tubes. The tubes were centrifuged at 2590 x g 4 C for 10 min. Five hundred microliters of supernatant were placed into a separate tube and counted alongside the pellet. The percent free iodine was calculated by multiplying by a factor of 2 to account for liberation of nonradioactive iodine, dividing the supernatant activity by that of the pellet, and multiplying by 2.24 to account for volume. The percentage of free iodine above that of the 2-min point was used to calculate the total deiodinase activity (40).
Cerebrocortical T3 extraction
Frozen cerebrocortical tissue was homogenized in cold 100% methanol containing 1 mM PTU with approximately 171 kBq [125I]3,5,3'-T3 tracer. The homogenate was centrifuged for 15 min at 2000 x g, and the supernatant fluid was collected into another tube and kept on ice. The pellet was resuspended in methanol and centrifuged again. The supernatants were pooled and passed over an AG 1-X2 column (Bio-Rad). The columns were washed and then eluted with 3 ml of 70% acetic acid (23). The elution fractions were lyophilized and resuspended in 50 mM KH2PO4 and 0.25% BSA before counting in a -counter to determine recovery. Average recovery for this procedure was 57%. T3 concentrations in the extracts were determined by specific RIA as with the serum T3 assay. The determined concentration was divided by the product of the extraction efficiency, the proportion of the extract tested, and the initial mass of the tissue to give the final tissue concentration.
Electrophysiology
In general, the electrophysiological procedures and analysis were conducted as previously reported (42), except as noted.
Hippocampal slice preparation
Freshly prepared hippocampal slices (500 µm) were obtained after anesthesia and decapitation. Horizontally cut slices were dissected in ice-cold, oxygenated (95% O2-5% CO2) dissection artificial cerebrospinal fluid (aCSF) containing (in millimoles) NaCl (120), KCl (3), MgCl2 (4), NaH2PO4 (1), NaHCO3 (26), and glucose (10). Slices recovered for 1 h in an oxygenated interface holding chamber with standard aCSF containing (in millimoles) NaCl (120), KCl (3), MgCl2 (1.5), NaH2PO4 (1), CaCl2 (2.5), NaHCO3 (26), and glucose (10). Slices were then transferred to a submerged recording chamber and recovered for an additional hour at 30 C with continuously perfused standard aCSF saturated with 95% O2-5% CO2 at approximately 1 ml/min before experiments were begun.
Extracellular recording
Extracellular recording electrodes were placed in the stratum radiatum of CA1. Field excitatory postsynaptic potential (fEPSP) responses were evoked with a bipolar stimulating electrode placed on either the CA3 or subicular side of the recording electrode in the stratum radiatum. Stimulus pulses consisted of a single square wave of 270 µsec duration delivered at 60–160 µA.
Data acquisition and analysis
Data were digitized at 10 kHz, low pass filtered at 1 kHz, and analyzed with pCLAMP9.2 software (Molecular Devices, Sunnyvale, CA). The initial slope of the population fEPSP was measured by fitting a straight line to the first millisecond of the fEPSP immediately after the fiber volley. Stimulus response curves were performed at the beginning of each experiment. Baseline stimulation pulses of an intensity that gave 40–60% of the maximum response were given at a frequency of 0.05 Hz for the entire length of the experiment. Synaptic responses were normalized by dividing all slopes by the average of the 15 fEPSP slopes 5 min before tetanus.
Long-term potentiation induction
The LTP high-frequency stimulation protocol consisted of a single 100-Hz train of 1 sec duration.
Statistics
All statistical analysis was performed using SAS (SAS Group, Cary, NC). All group comparisons were performed using ANOVA, followed by Duncan’s multiple range test. P < 0.05 was taken as the level of significance.
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Results |
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PND100 adults.
In 3-ppm dosed pups allowed to recover for approximately 2 months after weaning, all serum concentrations of T3 and T4 returned to control levels.
Body weight (Fig. 1)
Almost from birth, the mean body weights of the 10-ppm pups were significantly reduced in comparison with the undosed pups. In addition to the low body weights, a delay in eye opening and decrease in overall physical coordination were observed (data not shown). At weaning, the 10-ppm pups were unable to reach food and water in standard rat cages, so special housing arrangements were provided. Whereas the 3-ppm pups were not as affected as the 10-ppm pups, they also weighed less than controls, with the difference becoming significant around PND18 and expanding as the pups aged until weaning.
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Cerebrocortical thyroid parameters (Fig. 4)
D2 activity.
In the pups, cerebrocortical D2 activity was significantly increased at both 3 and 10 ppm, increasing to about 6-fold that of control values (P < 0.0001) with no significant difference between doses. In the dams, a slightly different pattern was observed. There was a tripling in D2 activity at 3 ppm and a further increase to 10 times control values at 10 ppm (P < 0.0001). However, the observed baseline and maximal activity in the dams was about 10% of the activity in the pups. D2 activity was normalized in 3-ppm adults.
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Baseline synaptic transmission is impaired in hypothyroid pups (Fig. 5)
Extracellular recordings of the fEPSP response were evoked from the stratum radiatum layer of the CA1 region of the hippocampus. The magnitude of the baseline synaptic transmission was significantly reduced (P = 0.0002) in hippocampal slices obtained from pups (Fig. 5, A and D). The maximal slope of the fEPSP was reduced by approximately 50% in both the 3- and 10-ppm dose groups, with no significant difference between these two conditions.
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). Paired-pulse analysis of the fEPSP indicated that there was no effect of PTU exposure on presynaptic function because the paired-pulse facilitation ratios were not affected. The combined results from Fig. 5, A and B, suggest that whereas the absolute magnitude of the synaptic response was decreased by PTU treatment, the sensitivity of this response was not impacted, nor was the function of the presynaptic nerve terminals (Fig. 5C).
Long-term potentiation is impaired in recovered adults (Fig. 6)
Unlike the results observed in PND21–32 animals, hippocampal slices prepared from littermates allowed to mature in the absence of PTU from weaning until PND90–110 did not exhibit any persisting change in baseline synaptic transmission (Fig. 6A). However, a significant impairment in the magnitude of long-term potentiation was observed in the 3-ppm adult group (Fig. 6B), suggesting that there may be some consequences of PTU exposure during development that persist after recovery to the euthyroid state.
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Discussion |
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It is noteworthy that cerebrocortical D2 activity increased in PND25 pups to maximal levels (
6-fold), even at the 3-ppm dose, a point at which cT3 concentrations had fallen by 50%. No further increase was seen in D2 at the 10-ppm dose, yet cT3 concentration fell further to 12% of control levels. In the dams, a tripling of cerebrocortical D2 appeared to allow maintenance of cT3 concentrations at the 3-ppm dose, but despite a 10-fold increase in D2 at the 10-ppm dose, the cT3 concentrations fell to 20% of control. Therefore, based on a maximal stimulation of D2 activity at 3 ppm PTU and reduction of cerebrocortical T3 at this dose, the brains of pups were more severely impacted than those of the dams, which were not impacted until the 10-ppm dose. Recordings of the fEPSP response in the CA1 region of hippocampal slices revealed significantly decreased basal synaptic transmission in both the 3- and 10-ppm pups, indicating the presence of a functional impairment in neuronal signaling as compared with 0-ppm control animals. Although we cannot point to a specific deficit based on extracellular recording results alone, the lack of effect on either the sensitivity of the fEPSP or paired-pulse facilitation suggests that neither postsynaptic responsiveness nor presynaptic release was a primary underlying factor. Because the fEPSP recorded in the stratum radiatum reflects a population synaptic response, one remaining possibility is that the synaptic density was decreased in these PND25 pups. A prior report consistent with this speculation describes a decrease in CA1 pyramidal cell density in hypothyroid rats (44).
Cerebrocortical D2 activity was found to be a sensitive respondent to reductions in serum T4 concentrations. Whereas this study focused on total T4, showing parallel changes in free T4 concentration in pooled samples from pups, the true correlation is undoubtedly to the unbound free T4 concentration. Both the dams and pups had a strong exponential correlation between declining T4 and increasing cerebrocortical D2 activity (Fig. 7). D2 is known to be regulated both pre- and posttranslationally by T3 and T4, respectively. One recognized method of posttranslational regulation by T4 is control of the rate of ubiquitination and thus degradation of D2 (45).
In addition to a correlation with serum T4 concentrations, D2 activity was also found to correlate with an important functional endpoint. A strong correlation between cerebrocortical D2 activity and the maximal synaptic response in pups was observed (Fig. 8). Synaptic response also correlated positively with serum T4 and cT3 concentrations, although neither of these relationships was as strong as the correlation with D2 activity (Fig. 8). Whether representative of a mechanistic link or reflective of a codependence, the correlation between D2 activity and synaptic potential is not fully understood at this time, but D2 and cT3 concentrations did recover in 3-ppm pups as they returned to euthyroidism at PND90–110 along with the baseline fEPSP response. Nonetheless, D2 activity did reflect the degree of availability of T4 from serum to cerebrocortical tissue at the time the animals were killed (37).
Analysis of thyroid mRNA levels for Tg and NIS, chosen to be markers of serum TSH bioactivity, further demonstrated the greater sensitivity to PTU in pups, compared with their dams. The up-regulation of these genes reflect an attempt to compensate for diminished hormone production. The lack of significant up-regulation in the dams, compared with the pronounced up-regulation in pups at both doses, further reinforces the hypothesis that the developing rat is more sensitive to thyroid disruption than is the adult rat. If this observation is paralleled in other mammalian systems, it reinforces the need to consider juveniles as a hypersensitive subpopulation with regard to thyroid disruption.
Although the serum and tissue thyroid hormone concentrations returned to normal after weaning, the LTP data obtained from littermates approximately 60 d after weaning demonstrated that some of the effects of treatment could not be reversed by the removal of PTU at weaning. This observation supports the conclusion that persisting changes can occur in the brain after a developmental insult, possibly involving alterations in myelination and migration patterns of neurons (14, 23, 25, 26, 27). An interesting aspect of our results was that the deficit in baseline synaptic transmission recovered concomitantly with the hormone levels, as expected from the relationships illustrated in Fig. 8. Despite this, our LTP data suggest that the PTU-treated rats retained a neuronal deficiency from which they did not recover over the time frame assessed in our studies. Thus, in contrast to LTP of the population spike response recorded from the dentate gyrus, an impaired capacity for synaptic plasticity of the fEPSP in the CA1 region may be predictive of the well-established cognitive impairments that persist after recovery to the euthyroid state (15).
Whereas PTU exposure is not a major environmental concern, we have attempted to use this drug as a model goitrogenic compound. PTU targets thyroid secretion and tissue type I 5'-deiodination, with mechanistic effects common to dietary and environmental goitrogens, including apigenin and luteolin, and mimics the T4-lowering effects of some polychlorinated biphenyls, polybrominated diphenyl ethers, and atrazine (46). Therefore, the results from this study should be applicable to other compounds that disrupt the thyroid axis. Data gathered from this study provide a correlative if not causative link between serum (free) T4, cerebrocortical D2 activity, and a neurophysiological consequence in hypothyroid rats. Such relationships could become the basis for a predictive model of developmental thyroid disruption with regard to neurological outcomes.
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Footnotes |
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This work was presented in part at the 2007 Annual Meeting of the Society of Toxicology, Charlotte, NC (Abstract 140).
This research does not represent official EPA policy.
Disclosure Statement: The authors of this paper have nothing to declare.
First Published Online March 27, 2008
Abbreviations: aCSF, Artificial cerebrospinal fluid; cT3, cerebrocortical T3; D1, type I 5'-deiodinase; D2, type 2 deiodinase; DTT, dithiothreitol; fEPSP, field excitatory postsynaptic potential; GD, gestational day; LTP, long-term potentiation; NIS, sodium iodide symporter; PND, postnatal day; PTU, propylthiouracil; Tg, thyroglobulin.
Received January 4, 2008.
Accepted for publication March 17, 2008.
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