Pentylenetetrazole-induced convulsions affect cellular and molecular parameters of the mechanism of action of triiodothyronine in adult rat brain

Neuropharmacology 48 (2005) 894e902 www.elsevier.com/locate/neuropharm

Pentylenetetrazole-induced convulsions affect cellular and molecular parameters of the mechanism of action of triiodothyronine in adult rat brain Stamatis Bolarisa,1, Caterina Constantinoub,1, Theony Valcanab, Marigoula Margarityb,* a Department of Human Reproduction, ‘‘Helena Venizelou’’ Hospital, 2 Venizelou Sqr., 11521 Athens, Greece Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26500 Patra, Greece

b

Received 19 December 2003; received in revised form 16 August 2004; accepted 28 October 2004

Abstract The aim of the current study was to elucidate whether the response of the adult rat brain to thyroid hormones is affected by the intensity of neuronal activity. For this purpose, the kinetic characteristics of nuclear T3 binding, the relative expression of thyroid hormone receptor (TR) isoforms and the synaptosomal content of thyroid hormones in adult rat brain were examined after administration of a single convulsion dose of pentylenetetrazole (PTZ). Experiments in adult Wistar rats revealed an increase (33%) of the density of specific T3 nuclear receptors in cerebral hemispheres 4 h after PTZ-induced seizures while no changes were observed in the dissociation constant. The relative expression of the T3binding isoforms of TRs was not affected, while there was a gradual decrease of the relative expression of the TRa2 variant (non-T3 binding isoform). The above changes were coupled with an increase of the synaptosomal T3 levels during the epileptic seizures. Our study revealed inversely proportional changes between the nuclear T3 binding sites and the TRa2 mRNA levels 4 h after PTZ-induced seizures, suggesting that the regulation of the expression of the non-T3 binding variant of TRs determines the nuclear T3 binding sites in adult rat brain, while the synaptosomal T3 levels could play a novel functional role in the signaling from the synapse to the nucleus. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Pentylenetetrazole; Adult brain; Nuclei; Triiodothyronine; Thyroid hormone receptors (TRs); Synaptosomal T3

1. Introduction Triiodothyronine (T3), the active form of thyroid hormones, exerts its physiological role in several target tissues through binding to specific nuclear receptors, affecting thus important metabolic processes both in developmental and adult stages (Eberhardt et al., 1976, * Corresponding author. Tel.: C30 2610 997430; fax: C30 2610 969273. E-mail address: [email protected] (M. Margarity). 1 Both authors equally contributed to this paper. 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.10.020

1978). There are three main isoforms of thyroid hormone receptors (TRs): TRa1, TRb1 and TRb2, which not only bind to T3 with high affinity, but also bind on thyroid hormone response elements (TREs) on DNA, regulating thus transcriptional processes in several target tissues, including adult rat brain (Yen, 2001). Binding of unliganded TRs on positive TREs inhibits the T3-dependent gene expression (Brent et al., 1991), a process easily reversed by binding of T3 (Yen et al., 1992, 1993). TRa1 and TRb1 isoforms are widely expressed in the adult rat brain (Strait et al., 1990), while TRb2’s expression is pituitary specific (Hodin et al.,

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1989). However there is another TR isoform, the TRa2 variant, which is abundantly expressed in adult rat brain (Strait et al., 1990) and it cannot bind T3 (Mitsuhashi et al., 1988) since it lacks part of its ligand-binding domain (Katz et al., 1992). The physiological relevance of the TRa2 remains unclear. Among T3 target tissues, adult brain is of particular interest. Despite the presence of a large number of specific TRs which are affected under altered thyroidal states (Valcana and Timiras, 1978), adult brain is also under tight metabolic control regarding its responsiveness to thyroid hormones. It has been suggested that local conversion of T4 to T3 is responsible, to some extent, for the purported unresponsiveness of adult brain to thyroid hormones (Yiannakouris and Valcana, 1994). T3 is concentrated in the nerve ending fractions of adult rat brain (Dratman et al., 1976) deriving presumably from local deiodination of T4 (Dratman et al., 1976; Dratman and Crutchfield, 1978). The physiological significance of this local distribution of T3 is rather unclear; in vitro experiments have showed the existence of specific T3 binding sites on the synaptosomal membranes (Mashio et al., 1982) and that endogenous T3, but not T4, is released from depolarized synaptosomes primarily by a CaC2-dependent process (Mason et al., 1993). However, several data in the literature suggest that a large number of genes, which are expressed in adult rat brain, are specific targets of T3 (Yen, 2001). In addition, studies in our laboratory revealed that T3 receptors are functional in adult rat brain, affecting cellular parameters of the brain’s metabolism including the total and ribosomal RNA levels, the rate of rRNA synthesis and the translation of specific proteins (Yiannakouris and Valcana, 1994). Pentylenetetrazole (PTZ), an epileptogenic drug, causes absent-type seizures when given at a daily dose of 25 mg/kg of body weight (B.W.), whereas an acute administration of 50 mg PTZ/kg B.W. results to convulsions (Purpura et al., 1972; Fisher, 1989). PTZinduced convulsions are coupled with abnormal electrical recordings of brain activity (Prince, 1978), which resemble those induced by the administration of high doses of thyroxine (T4) (status epilepticus) (Saundaram et al., 1985). The induction of convulsions by PTZ is attributed to repression of gamma aminobutyric acid type A (GABAA) receptor Clÿ channel (Corda et al., 1990), which attenuates, thus, the GABA-dependent inhibition. Furthermore, PTZ administration may provoke alterations in the expression of several genes including c-fos and c-jun (Belle et al., 1994), the cholecystokinin gene (Takeda et al., 1997) and the thyrotropin-releasing hormone gene (Jaworska-Feil et al., 1999). Aim of this study was to investigate whether PTZinduced alterations in adult brain activity affect the mechanism of action of thyroid hormones at the

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molecular level, and thus the response of this tissue to thyroid hormones. In particular, the kinetic characteristics of nuclear T3 binding and the relative expression of all TR isoforms by Northern analysis were examined in cerebral hemispheres of adult rat brain after a single convulsant dose of PTZ. The synaptosomal levels of thyroid hormones were also examined in order to delineate whether the synaptic events that follow the PTZ-induced convulsions can influence the thyroid hormone availability and/or metabolism in the adult brain.

2. Methods 2.1. Animals and treatment Adult Wistar rats (40 days) of both sexes, bred in our Laboratory, were housed 4 per cage and had ad libitum access to laboratory chow and water. Animals were treated according to the standards of the international statues on animal handling. They were exposed to a regular lightedark cycle (light period: 7 am to 7 pm; dark period: 7 pm to 7 am; at 22 G 1  C) for at least 1e 2 weeks prior to PTZ treatment and until sacrifice. Convulsions were induced by an intraperitoneal injection of PTZ (50 mg/kg B.W.) between 9:00 and 10:00 am. After the injection, all the experimental animals were kept in individual cages and were continually observed in order to establish behavioral changes that characterize toniceclonic convulsions according to the criteria of Ito et al. (1977a). Only the animals showing characteristic toniceclonic convulsions (duration of convulsions: 60e70 s) within 3e5 min after PTZ injection were included in this study. Ineffective PTZinduced convulsions were evaluated to be 0e5% in the total number of animals in each experiment. Control animals received intraperitoneal injection of physiological saline (0.9% NaCl). PTZ-treated and control animals were sacrificed by rapid decapitation at several intervals after the appearance of convulsions (2 h and 4 h for the nuclei isolation - during convulsions, 15 min, 1 h, 2 h, 4 h and 24 h for the mRNA and synaptosomes’ isolation). Brains were rapidly removed into a sterile cooled glass plate and cerebral hemispheres were isolated and weighed. For the binding studies and the determination of synaptosomal thyroid hormone levels, fresh cerebral hemisphere tissue was used. For the mRNA isolation, tissue samples were frozen in liquid N2 until assayed by Northern analysis within two weeks. In order to elucidate whether PTZ-induced seizures affect the free fraction of thyroid hormone levels in blood circulation, serum T3 and T4 concentrations were determined in all experimental and control animals by radioimmunoassay (T3-RIA and T4-RIA kits, Hellenic Center of Natural Research, ‘‘Demokritos’’).

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2.2. Nuclei isolation The cerebral hemispheres were homogenized (20% w/ v) in ice-cold 0.32 M sucrose by 10 up and down strokes in a glass homogenizer with a loose fitting motor driven Teflon pestle (1000 rpm). The homogenates were then centrifuged at 1000 ! g for 10 min (4  C). The crude nuclear pellets were resuspended in 0.32 M sucrose according to the initial dilution and recentrifuged as above. In order to obtain pure nuclei the pellets were suspended in 1.7 M sucrose and centrifuged at 53,000 ! g for 90 min (4  C) in a Beckman SW 27 ultracentrifuge rotor. The final nuclear pellets were suspended in potassium phosphate buffer (Kp buffer: 25 mM KH2PO4, 1 mM MgCl2, 20 mM b-mercaptoethanol, pH 7.5) which contained 0.5% Triton X-100 and were centrifuged at 10,000 ! g for 10 min (4  C). The pellets were then resuspended in Kp buffer without Triton X-100 and were washed at 10,000 g for 10 min (4  C). The final, pure, nuclear preparations were sampled and DNA content was estimated in each one with the method of Burton (1956). 2.3. In vitro triiodothyronine binding assay For the determination of T3 binding constants [maximal binding capacity (Bmax) and equilibrium dissociation constant (Kd)], nuclear samples (40e60 mg of DNA) were incubated at 37  C for 15 min in 1 ml of Kp buffer (without Triton) containing various concentrations (0.5e10 ! 10ÿ1 M) of 125I-T3 (spec. activ. 1200 mCi/mg, New England Nuclear). At all concentrations of radioactive T3 examined, identical incubations were carried out with 100 M excess of unlabeled T3. Duplicate assays were performed at each concentration employed. The incubation was terminated by placing the samples on ice for 2 min. Samples were then centrifuged at 10,000 ! g for 10 min (4  C). The pellets were subsequently washed once in Kp buffer containing 0.5% Triton X-100 and were recentrifuged as above. The final pellets were then counted in a Beckman gcounter (5500 model) and the specific binding was determined by subtracting the radioactivity of nonspecific bound T3 (i.e. the radioactivity which could not be competitively displaced by incubating the nuclei in 100-fold molar excess of unlabeled T3) from the total bound radioactivity. The results were expressed as moles T3 bound per mg DNA. The DNA content in each sample was determined by the method of Burton (1956) and the protein content by the method of Bradford (1976). The binding constants were estimated by Scatchard analysis (1949) of the binding data. The in vitro total 125I-T3 binding in cerebral hemispheres from control and PTZ-treated animals was estimated by incubating, in duplicate, the nuclei at 37  C for 15 min in 1 ml Kp buffer (without Triton) containing

saturating levels (5 ! 10ÿ10 M) of 125I-T3, while identical incubations were performed in the presence of 100 M excess of cold T3. The experiments were repeated several times in order to determine the significance of difference in the data obtained from experimental and control groups. In a separate experimental procedure we examined whether PTZ exerts any direct in vitro effect on T3 binding. In this case the nuclear samples were incubated at 37  C for 15 min in 1 ml Kp buffer in the presence of 10ÿ4 M PTZ, a concentration similar to that achieved in the brain after the administration of a convulsant dose of PTZ (Yonekawa et al., 1980). This performance was done in subsaturating levels of 125I-T3 (3 ! 10ÿ10 M). 2.4. Extraction of polyACRNA PolyACRNA was extracted from 1 g of cerebral hemispheres from PTZ-treated and control animals using the Poly(A)PureÔ kit (Ambion), which is based on the guanidine thiocyanate method. The final RNA pellets were dissolved in RNase free water and quantified in duplicate by determining the optical density at 260 nm (O.D.260). In each experiment three different polyACRNA preparations from PTZ-treated and control animals were analyzed by Northern blot hybridization twice in duplicate for each thyroid hormone receptor isoform mRNA. 2.5. Northern blot hybridization The relative expression of the mRNA of each thyroid hormone receptor isoform was determined by Northern blot hybridization according to the method of Sambrook et al. (1989). Briefly, aliquots of polyACRNA, appropriate for each TR isoform, were subjected to electrophoresis in a 1% agarose/formaldehyde gel (20 mM 3-[n-morpholino]-2-hydroxy-propanesulfonic acid (MOPS), 1 mM ethylene diamine tetraacetic acid (EDTA), 5 mM sodium acetate, pH 7.0, and 2.2 M formaldehyde). The mRNA was transferred overnight to positively charged nylon membranes (New England Nuclear) and subsequently cross-linked by baking the membranes at 80  C for 2 h. The blots were prehybridized at 68  C in UltraHyb hybridization solution (Ambion) for 2 h. Hybridization was done overnight at 62  C for TRa1 isoform, 68  C for TRa2 isoform and 65  C for TRb1 and TRb2 isoforms with 3 ! 106 cpm/ ml of random labeled probes arising by digestion of the TRa1, TRa2 and TRb2 full-length cDNAs with restriction enzymes (a1 specific probe: EcoRI and XbaI in a1 cDNA, a2 specific probe: EcoRI and XhoI in a2 cDNA, b1/b2 common probe and b2 specific probe: XbaI in b2 cDNA). Blots were washed twice in 2! Standard Saline Citrate bufferdSSC (30 mM sodium citrate, 300 mM sodium chloride, pH, 7)/0.1% sodium

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dodecyl sulphate (SDS) at 62  C for 20 min, twice in 0.1 ! SSC/0.1% SDS at 65  C for 10 min and finally in 0.1 ! SSC at room temperature for 1e2 min. The washed blots were exposed immediately to the Phosphor imager cassette (Molecular Dynamics) for 24 h and quantified by computer-assisted densitometry. The size of each thyroid hormone receptor isoform mRNA was determined by comparison with RNA molecular weight markers (Promega). PolyACRNA recovery was determined by rehybridization of membranes with the glyceraldhehyde-3-phosphate-dehydrogenase (GAPDH) probe and all values were normalized to the GAPDH signal. 2.6. Isolation of synaptosomes The preparation of synaptosomes was performed according to the method of Sarkar and Ray (1992) with slight modifications. Briefly, the cerebral hemispheres (y800 mg) were homogenized (10% w/v) in ice-cold 0.32 M sucrose (pH 7.4) by eight up and down strokes in a glass homogenizer with a loose fitting motor driven Teflon pestle (1000 rpm). The homogenates were then centrifuged at 1000 ! g for 10 min and the supernatant was layered slowly on top of a sucrose gradient composed of 8 ml of 1.2 M and 8 ml of 0.32 M sucrose (4  C, pH 7.4) and centrifuged at 34,000 ! g for 50 min, plus 8 min acceleration time, using a Beckman SW27 ultracentrifuge rotor. The crude synaptosomal fraction was banded between the 0.32 M and the 1.2 M sucrose layers and was carefully removed by suction at 4  C and the sucrose concentration was adjusted to 0.32 M by adding slowly ice-cold bi-distilled water (y1:1.8) and then layered slowly on the top of a sucrose gradient composed of 8 ml of 0.85 M and 8 ml of 0.32 M sucrose (4  C, pH 7.4) with a glass Pasteur pipette and centrifuged at 34,000 ! g for 30 min, plus 8 min acceleration time using a Beckman SW27 ultracentrifuge rotor as above. The bottom pellet thus obtained was the synaptosomal fraction which was further purified by washing once with 5 ml 0.32 M sucrose at 4  C and repelleted at 20,000 ! g for 20 min to finally get pure synaptosomes. The synaptosomes were then ruptured hypo-osmotically in 1.5 ml of 5.5 mM imidazoleeHCl buffer (pH 7.4, 4  C). The ruptured synaptosomal suspension was used immediately for T4 and T3 radioimmunoassay (RIA). 2.7. Determination of thyroid hormone synaptosomal levels T4 and T3 synaptosomal concentrations were measured using radioimmunoassay kits from the Hellenic Center of Natural Research, Demokritos, (Athens, Greece). Prior to analyses, in each synaptosomal suspension sample (100 ml) 10 ml of 0.1 N NaOH was added in order to obtain an appropriate alkaline

environment for the dissolution of thyroid hormones from the membrane fraction of synaptosomes. All samples were analyzed in duplicate. Protein concentration was evaluated by the method of Bradford (1976), using bovine serum albumin as standard. 2.8. Statistics Data are expressed as the mean G S.E. of separate preparations in each experimental procedure. The statistical analysis of the data was estimated by performing One-Way Analysis of Variance with the p ! 0.05 as the significance cutoff point. One-Way Analysis of Variance with a Bonferroni correction for multiple comparisons was performed for the effect of PTZ-induced convulsions on the relative expression of all TR isoforms and on T3 synaptosomal levels.

3. Results 3.1. Effects of PTZ-induced convulsions on the in vitro I-T3 nuclear binding to adult rat cerebral hemispheres

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The effect of a single convulsant dose of PTZ (50 mg/ kg B.W.) on the in vitro specific 125I-T3 binding to cerebral nuclei from adult rats 2 and 4 h after PTZinduced convulsions is presented in Table 1. These data show that the specific 125I-T3 in vitro binding to nuclei from adult rat cerebral hemispheres was increased (25%) 4 h after the PTZ-induced convulsions, while no statistically significant changes were observed 2 h after the convulsions. Notably, in all PTZ-treated animals, no significant changes at the serum T3 and T4 levels with respect to the control levels (T3: 0.829 G 0.2 ng/ml; T4: 36 G2 ng/ml) were observed. In order to delineate whether the increase of the in vitro 125I-T3 specific nuclear binding was due to alterations in the maximal number of binding sites and/or in the equilibrium dissociation constant, we

Table 1 The effect of PTZ-induced convulsions on the in vitro specific binding of 125I-T3 to nuclei isolated from adult rat cerebral hemispheres Time after PTZ-induced convulsions

2h 4h

Specific 125I-T3 binding (10ÿ17 moles/ mg DNA) Control

PTZ treated

32.7 G 0.7 (n Z 4) 43.8 G 2.1 (n Z 6)

34.7 G 3.9 (n Z 4) 54.8 G 2.5* (n Z 6) ([25%)

Pentylenetetrazole (50 mg/kg B.W.) was injected intraperitoneally to adult rats. Control animals received equal volume of 0.9% NaCl. The numbers represent means G S.E. of n nuclear preparations (five animals per preparation), which were analyzed in duplicate. *Statistically different from control, p ! 0.05 (Analysis of Variance).

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performed equilibrium competitive binding assays of nuclei prepared from control and PTZ-treated animals 4 h after the convulsions. Scatchard analysis of the binding data is presented in Fig. 1. These results show that the increase in T3 binding to cerebral nuclei was attributable only to an increase (33%) in the maximal binding capacity; while no significant differences were observed in the dissociation constant (Table 2). The increase in the T3Bmax was not due to direct pharmacological effect of PTZ on the in vitro 125I-T3 binding, since the presence of PTZ (10ÿ4 M) in the incubation medium of nuclei T3 binding procedure did not cause any significant differences in the specific, nuclear T3 binding (Fig. 2). Moreover, in this study we observed that PTZ-treated animals which did not develop convulsions did not also show any changes in the in vitro nuclear 125I-T3 binding to cerebral nuclei in comparison to the control animals (data not shown).

3.2. Effects of PTZ-induced convulsions on the relative expression of all thyroid hormone receptor isoforms in adult rat cerebral hemispheres Northern blot hybridization was employed for the evaluation of the relative expression of all TR isoforms in cerebral hemispheres from control and PTZ-treated animals at 15 min, 2 h, 4 h and 24 h after the appearance of convulsions, in order to delineate whether the changes in the molecular basis of the observed increase in T3Bmax 4 h after PTZ-induced convulsions, are firstly estimated and which is their duration. Our results (Table 3) show that no significant differences were observed in any of the T3-binding isoforms of TRs (TRa1 and TRb1), in any interval after PTZ-induced convulsions. Moreover, in agreement with the literature (Hodin et al., 1989), we also observed that the TRb2 isoform could not be

Table 2 Effect of PTZ-induced convulsions on T3 binding characteristics to nuclei isolated from adult rat cerebral hemispheres

Control PTZ-treated (4 h)

Bmax (10ÿ17 moles/mg DNA)

Kd (10ÿ10 M)

78 G 1.9 104 G 9.5* ([33%)

2.7 G 0.4 2.8 G 0.4

Numbers represent means G S.E. from six nuclear preparations (five animals per preparation) assayed in duplicate. *Statistically different from control, p ! 0.05 (Analysis of Variance).

detected by Northern analysis in cerebral hemispheres of control or experimental animals. However, a gradual decrease in the TRa2 variant (non-T3-binding isoform) mRNA levels was observed within 2 h (11%), reached its peak (27%) at 4 h and it was conserved for at least 24 h after PTZ-induced convulsions in cerebral hemispheres (Table 3). Additionally, we observed that the reduction in TRa2 mRNA levels in cerebral hemispheres 4 h after the PTZ-induced convulsions was inversely proportional to the observed increase in the maximal binding capacity of the 125I-T3 nuclear binding in the same tissue and under the same experimental conditions. 3.3. Effects of PTZ-induced convulsions on the thyroid hormone synaptosomal levels The determination of the T3 synaptosomal levels in cerebral hemispheres from control and experimental animals is presented in Table 4. Our results reveal that during the PTZ-induced convulsions the T3 levels in cerebral synaptosomes were increased by almost 50% in comparison to the control levels and this change was returned to normal within 15 min after PTZ-induced convulsions. In agreement with the literature (Sarkar and Ray, 1994) the T4 synaptosomal levels remained undetectable

Bound T3/Free T3 (10-6 lt / µg DNA)

4

3

2

1

0

0

20

40

60

80

100

Bound T3 (moles × 10-17 / µg DNA) Fig. 1. Scatchard analysis of the specific 125I-T3 binding to brain nuclei from control ( d ) and PTZ-treated animals (B d B), which were decapitated 4 h after PTZ-induced convulsions. Numbers represent means G S.E. ( p ! 0.05, Analysis of Variance) from six different nuclear preparations (five animals per preparation) assayed in duplicate.

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Specific bound 125I-T3 (10-17 moles / µg DNA)

60

Without PTZ 10-4 M PTZ

40

Control PTZ-treated PTZ-treated PTZ-treated PTZ-treated PTZ-treated

20

0

Fig. 2. In vitro effect of pentylenetetrazole on the specific T3 binding to brain nuclei. Nuclei were incubated at 37  C for 15 min in 1 ml phosphate buffer containing 3 ! 10ÿ10 M 125I-T3 in the presence or absence of 10ÿ4 M PTZ in the incubation medium. The bars represent means G S.E. ( p O 0.05, Analysis of Variance) from five different preparations (three animals per preparation) analyzed in triplicate.

by RIA in all control and experimental conditions examined in this study.

4. Discussion It is well established that although the adult brain contains a high density of specific nuclear T3 receptors, in comparison to other T3-target tissues (Valcana and Timiras, 1978), it is also under tight metabolic control, leading thus to the long known hypothesis that this tissue is not responsive to thyroid hormones, at least, in adulthood. However, several reports in the literature have suggested that these hormones affect aspects of the neurotransmission in adult brain (Ito et al., 1977b; Codola and Garsia, 1985). Our present data provide evidence that perturbed neural activity affects many aspects of the molecular mechanism of action of thyroid hormones. Specifically, we observed a significant increase (25%) of the in vitro specific T3 nuclear binding in adult Table 3 Effect of PTZ-induced convulsions on the relative expression of TR isoforms in adult rat cerebral hemispheres TRb1 (AU/mg polyACRNA)

TRa1 (AU/mg polyACRNA)

TRa2 (AU/mg polyACRNA)

6426 G 156

17,254 G 76

159,988 G 322

6289 G 168 6329 G 98

17,193 G 83 17,322 G 88

4h

6398 G 105

17,265 G 94

24 h

6487 G 146

17,186 G 101

160,102 G 292 142,389 G 285* (Y11.2%)Y 116,471 G 266* (Y27.2%) 119,511 G 302* (Y25.3%)

Control PTZ-treated 15 min 2h

Table 4 Effects of PTZ-induced convulsions on the T3 synaptosomal levels

All values are expressed as arbitrary units (AU) per mg polyACRNA and represent means G S.E. of five different experiments as described under Sections 2.4 and 2.5. *Statistically different from control, p ! 0.05 (One-Way Analysis of Variance, Bonferroni test).

(during convulsions) (15 min) (2 h) (4 h) (24 h)

n

T3 (ng/mg protein)

12 15 14 14 14 16

0.4525 G 0.002 0.6650 G 0.005* ([47%) 0.4550 G 0.08 0.4530 G 0.01 0.4420 G 0.009 0.4250 G 0.01*

All values represent means G S.E. from n different synaptosomal preparations assayed in triplicate. * Statistically different from control, p ! 0.05 (One-Way Analysis of Variance, Bonferroni test).

cerebral hemispheres (Table 1) 4 h after the seizures induced by a single convulsant dose of PTZ (50 mg/kg B.W.) while there were no changes in thyroid hormone availability in blood circulation. The above effect was due to the increase (33%) in the Bmax of the active form of thyroid hormones and not due to changes in the Kd (Fig. 1, Table 2). The increase of the maximal number of nuclear T3 binding sites is not a direct effect of PTZ on the in vitro nuclear T3 binding, since addition of the drug in the incubation medium did not alter the total nuclear T3 binding (Fig. 2). Moreover, the observation that experimental animals, which did not develop convulsions after the PTZ treatment, did not also show any changes in the in vitro T3 nuclear binding, relative to the control animals, strongly suggest that the above increase in the T3Bmax is a result of the perturbed neural activity of adult brain, as it is caused by the PTZinduced convulsions. Previous reports that other drugs (i.e. LiCl) which affect neural activity could also affect the kinetic characteristics of the in vitro nuclear 125I-T3 binding capacity (Bolaris et al., 1995) support our present data. In order to delineate which is the molecular basis of the increase in the nuclear T3 binding sites in adult rat cerebral hemispheres, we studied the relative expression of all TR isoforms in the same tissue. Although PTZinduced convulsions did not cause significant alterations in the relative expression of the T3-binding isoforms of TRs (TRa1 and TRb1), they, however, induced a gradual decrease in the relative expression of the TRa2 variant, a non-T3-binding isoform, in adult rat cerebral hemispheres. This gradual decrease was firstly recorded 2 h after PTZ-induced convulsions (11%), increased 4 h after convulsions (27%) and kept constant for the next 24 h, although a trend towards returning to normal levels was observed at this time point of treatment (Table 3). In addition, we observed that the percent reduction in TRa2 mRNA levels was inversely proportional to the observed increase in T3Bmax 4 h after the PTZ-induced convulsions. The above observation provides strong evidence that the regulation of the TRa2 mRNA levels could be responsible for the alterations observed in the T3Bmax under increased neural activity

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in this tissue. We therefore suggest that the reduction of the TRa2 mRNA levels leads to a release of DNA binding sites (TREs) available for the T3-binding isoforms, increasing thus the maximal nuclear binding capacity of the active form of thyroid hormones. This conclusion is supported by the observation that the TRa2 isoform has an intact DNA binding domain (Mitsuhashi et al., 1988) which allows it to bind on DNA after dimerization with the 9-cis-retinoic acid receptor (RXR) (Katz and Koenig, 1994; Yang et al., 1996) and consequently act as a T3-independent transcription factor, which competes with the T3binding isoforms for site-specific binding on the same TREs in chromatin. It has been shown that these TREs have specific structure and constitute optimal DNA binding sites for the TRa2eRXR complex, as well as for the TRa1eRXR complex and the monomeric forms of TRs (Katz and Koenig, 1994; Yang et al., 1996). Additionally, it has been shown that the ability of TRa2 isoform to bind on DNA is also relative to its increased expression levels (Katz and Lazar, 1993). This observation could explain why the small reduction (11%) in TRa2 relative expression 2 h after the PTZinduced convulsions (Table 3) does not reflect into changes in the nuclear T3 binding at the same time point of animal treatment (Table 1). In addition, the possibility that the alteration (decrease 11%) in TRa2 mRNA expression presumably needs more time than 2 h in order to be translated into changes in the respective protein levels should not be excluded. It could be very interesting to investigate if the above changes in the TRa2 relative expression are due to changes in the transcriptional activity of the c-erbAa gene and/or due to alterations in the alternative splicing pattern of this gene or both, inasmuch as c-erbAa gene codes for both TRa1 and TRa2 isoforms. Furthermore, a definitive resolution of many of the issues raised in this study will depend upon the development of specific antibodies for identifying the translational products of TRa1, TRb1 and TRa2 mRNAs and the identification of the specific target genes where the competitive substitution between the T3-binding TR isoforms and the non-T3-binding variant takes place. In this case, the suggested competitive substitution of the TRa2 isoform by the T3-binding TR isoforms in adult rat cerebral hemispheres should cause induction of the transcriptional activity of their common target genes in the presence of sufficient amounts of intracellular T3. Estimation of the synaptosomal levels of T3 in adult cerebral hemispheres reveals an increase (47%) in these levels during the PTZ-induced convulsions (Table 4), suggesting thus that increased neural activity resulting from the administration of PTZ could induce the T3dependent gene expression. In support of our above suggestion, data in the literature reveal that PTZinduced convulsions provoke almost instant increase in

the expression of several immediate early genes, such as c-fos, c-jun, jun-b and zif/268 (Belle et al., 1994), which also participate in the formation of significant transcription factors (i.e. AP1), suggesting thus another molecular mechanism of induction of gene expression in adult brain under perturbed neural activity. Furthermore, we propose that the precocious increase in the T3 synaptosomal levels during the PTZinduced convulsions, which preceded the observed changes in TRa2 relative expression (firstly estimated 2 h after the convulsions) and the changes in nuclear T3 binding sites (firstly estimated 4 h after the convulsions), presumably classifies the active form of thyroid hormones to a novel, intracellular, retrograde message which could travel from the synapse to the nucleus in order to induce the T3-dependent gene expression. Another possible physiological relevance of the observed increase in T3 synaptosomal levels could be the local enhancement of the depolarization-induced CaC2 uptake (Mason et al., 1990) and/or the release of GABA (Hashimoto et al., 1991). However, the origin of the increased triiodothyronine levels in the synaptosomal fraction remains unknown, since the T4 synaptosomal levels cannot be estimated by radioimmunoassay in order to delineate whether there is an increase in the local conversion of T4 to T3 or an increase in the T3 transport ratio in brain cells under perturbed neural activity. Furthermore, we consider that the inhibition of the local deiodination of T4 to T3, simultaneously with the increase in neural activity, could be an experimental model that would contribute a lot to the understanding of the physiological relevance of synaptosomal availability on nuclear and/or synaptic events in PTZ-treated animals. In conclusion, the effects of the PTZ-induced altered neural activity on the molecular mechanism of action of thyroid hormones could consequently reflect into changes in the transcriptional and metabolic T3-dependent processes and also constitute a novel, adaptive network between nuclear and synaptic aspects of thyroid hormone action. This mechanism could be used by the adult brain in order to respond to its elevated metabolic needs and reestablish its normal neural activity.

Acknowledgments The authors gratefully acknowledge Professor W. W. Chin (Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, and Howard Hughes Medical Institute, Harvard Medical School) for his generous donation of the full-length cDNAs coding for the TRa1, TRa2 and TRb2 isoforms and Professor P. Stylianopoulou (Department of Public Health, University of Athens) for her kind gift of the GAPDH fulllength cDNA.

S. Bolaris et al. / Neuropharmacology 48 (2005) 894e902

This work was partially supported by the Hellenic Ministry of Development, General Office of Research and Technology (Grant No: PENED95ED/1611).

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