Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain

Peptides 25 (2004) 647–658

Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain A. Eugene Pekary a,b,∗ , Albert Sattin a,c,d,e,f , James L. Meyerhoff g , Mark Chilingar d,e a

g

Research Services, West Los Angeles VA Medical Center, Bldg. 114, Rm. 229, 11301 Wilshire Blvd., Los Angeles, CA 90073, USA b Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA c Psychiatry Services, VA Greater Los Angeles Health Care System, Los Angeles, CA, USA d Neuropsychiatric Institute, UCLA School of Medicine, Los Angeles, CA, USA e Psychiatry and Biobehavioral Sciences, UCLA School of Medicine, Los Angeles, CA, USA f Brain Research Institute, UCLA School of Medicine, Los Angeles, CA, USA Department of Neurochemistry, Division of Neuroscience, Walter Reed, Army Institute for Research, USA Received 22 October 2003; accepted 27 January 2004

Abstract We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH2 , His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior. © 2004 Elsevier Inc. All rights reserved. Keywords: Valproate; TRH-like peptides; TRH receptors; Depression; Cortex; Limbic system

1. Introduction Severe depression, including bipolar disorder, represents a major, worldwide, public health issue. While a variety of treatment modalities and strategies is available, each has its limitations in terms of cost, latency or duration of response, side effects and percentage of responders [44]. Depression and other related neuropsychiatric disorders are products of complex combinations of genetic and environmental risk factors. This undoubtedly contributes not only to the variability of treatment response, but also to uncertainty as to the optimum therapeutic approach [17]. Augmentation by valproate (Valp), lithium (Li) or thyroid hormone has been an important strategy for inducing remis∗

Corresponding author. Tel.: +1-310-268-4430; fax: +1-310-268-4982. E-mail address: [email protected] (A.E. Pekary).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.01.016

sion in patients who are resistant to standard antidepressants [7]. On the other hand, withdrawal of antidepressants or of Li or Valp as mood stabilizers increases risk of relapse of bipolar disorder [2]. The current experiments explore the chronic effects of low dose Valp and Li treatment on: (a) TRH receptor levels and (b) the responses of TRH and other TRH-like peptide expression and secretion to acute, chronic and withdrawal treatment with high dose Valp. Chronic Valp treatment resulted in the induction of liver enzymes that rapidly cleared this drug from the circulation. A high dose of Valp (20 g/kg diet) produced a rapid rise in serum levels into the therapeutic range at days 9 and 10 followed by a precipitous decline. Maintaining steady, therapeutically relevant levels of Valp in rats for several days, was not achieved in this study. Nevertheless, we observed profound effects of Valp treatment on the levels of TRH receptors and TRH and TRH-like

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peptides with the general structure pGlu-X-Pro-NH2 , where “X” can be Glu, Val, Tyr, Leu or Phe. The neurotropic effects of these peptides are consistent with their involvement in the therapeutic functions of Valp. For example, the anti-epileptic, anticonvulsant and neuroprotective effects of TRH may be due, in part, to its ability to reduce AMPA-stimulated calcium influx by almost 50% in neuronal cultures of rat embryonic forebrain [20]. TRH reduces ataxia in mutant mice by normalizing glucose metabolism in the cerebellum and the ventral tegmental area via NMDA receptor activation [19]. TRH alleviates symptoms in 50% of severely depressed patients within a few hours of its intrathecal administration [26]. EEP (pGlu-Glu-Pro-NH2 , Glu-TRH); Phe-TRH [23] and Tyr-TRH [33] also have antidepressant-like activities in the rat forced swim test. TRH reduces anxiety-like behavior and learning impairment in senescence-accelerated mice [27]. TRH and some TRH-like peptides, e.g. EEP, Val2 -TRH and Leu2 -TRH, have potent analeptic properties while others, e.g. Phe2 -TRH and Tyr2 -TRH, have none [15]. Unlike TRH, TRH-like peptides are not readily metabolized by the serum enzyme pyroglutamate aminopeptidase II [18] and therefore have therapeutic potential for the treatment of psychiatric illnesses. We have recently shown that chronic thyroxine alters the levels of TRH and TRH-like neuropeptides in rat brain [35]. We now report that chronic Li and Valp treatment significantly increase the levels of TRH receptors in nucleus accumbens, amygdala, frontal cortex, posterior cortex and cerebellum and that acute, chronic and withdrawal treatment with Valp profoundly alters levels of TRH and TRH-like peptide levels throughout the brain of WKY male rats.

ceipt and on the morning of each experiment. Initial and final body weights did not differ between experimental groups. Research was approved by the VA Greater Los Angeles Healthcare System Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act and the federal statutes and regulations related to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and use of Laboratory Animals, NRC Publication, 1996 edition. 2.3. Dissection of rat brains All rats were sacrificed by decapitation. Nucleus accumbens (NA), amygdala (AY), frontal cortex (FCX), posterior cortex (PCX), cerebellum (CBL), medulla (MED), anterior cingulate (ACNG), posterior cingulate (PCNG), septum (SE), striatum (STR), pyriform cortex (PYR), hypothalamus (HY), hippocampus (HC), entorhinal cortex (ENT), and bed nucleus of the stria terminalis (BNST) were hand dissected on an ice-chilled glass plate, weighed rapidly, and then extracted as described below. 2.4. Experiment 1: low dose valproate and lithium treatment for TRH receptor assay Rats (6 animals/group) were fed standard rat chow with 0 or 2 g sodium Valp/kg or 1.7 g LiCl/kg rodent diet for 1 week until sacrifice. Lithium-fed rats also had a supplemental bottle containing 1.45 M NaCl to compensate for the effects of LiCl on kidney function. 2.5. Experiment 2: time-dependence of TRH receptor and serum valproate responses to high dose valproate

2. Materials and methods 2.1. Valproate, lithium and thyroid hormone assays Valproate levels were measured by immunoturbidimetry using the Beckman Coulter Synchron LX Systems Analyzer (Fullerton, CA). Serum lithium concentrations were measured by an ion-selective electrode method (AVL 9181 Electrolyte Analyzer, AVL Scientific Corp., Roswell, GA). Rat TSH was measured using reagents obtained from the National Pituitary Agency. Serum total T3 and total T4 were measured by the Coat-A-Count method (Diagnostic Products Corp., Los Angeles, CA). 2.2. Animals Male WKY rats (Charles River Laboratories, MA) were used for all experiments. These animals were maintained with standard Purina rodent chow #5001 and water ad libitum during a standard 1 week initial quarantine in a controlled temperature and humidity environment; lights on 6 a.m. to 6 p.m. All animals were weighed on the day of re-

Because serum Valp was not detectable in Experiment 1, the Valp treatment was repeated at 0 g Valp/kg (n = 6) and 20 g Valp/kg (high dose) diet (n = 6). Rats were sacrificed at 7–15 days after the start of continuous Valp treatment, (n = 1) for each time point. 2.6. TRH receptor assay The effect of low dose Valp and Li treatment on the total TRH receptor binding capacity in each of the dissected brain regions was measured using a TRH receptor binding assay [4]. Rats were decapitated between 10 a.m. and 1 p.m. Tissues were homogenized in 20 vol. ice-cold 20 mM sodium phosphate buffer, pH 7.4 (Buffer A) using a Polytron (Brinkmann Instruments, Westbury, NY). These homogenates were centrifuged in the cold for 20 min at 3000 × g. All pellets (except for MED) were resuspended with the Polytron in 10 vol. (relative to the original wet weight) Buffer A containing 10% glycerol and frozen at −70 ◦ C. Immediately before the TRH receptor assay, tissues were thawed and centrifuged at 3000 × g at 4 ◦ C,

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and resuspended in Buffer A. MED pellets were resuspended in Buffer A and assayed immediately after dissection as more than 90% of the TRH receptor binding was lost upon freezing and thawing, even in 20% glycerolBuffer A. Resuspended tissue (100 ␮l) was added to 50 ␮l of 40 nM (3-methyl-His2 ) [l-His-4-3 H,l-pro-3,4-3 H]TRH (98 Ci/mmol, Perkin-Elmer Corp., Boston, MA) and 25 ␮l of 0 or 1.0 mg/ml [3-Me-His2 ]TRH in triplicate. Tubes were incubated for 2 h on ice. Bound radioactivity was separated by filtration through 2.4 cm Whatman GF/B glass fiber filters (Whatman, Inc., Clifton, NH) under vacuum with four rapid rinses of 3 ml ice-cold 0.9% sodium chloride. Filters were dried and counted by liquid scintillation spectrometry at an efficiency of 60%. 2.7. Production of antibodies to TRH-like peptides Lys-TRH (pGlu-Lys-Pro-NH2 ) was conjugated to keyhole limpet hemocyanin (Pierce Chemical Co.) using glutaraldehyde [33]. Two pathogen-free New Zealand white rabbits (Harlan, Indianapolis, IN) were immunized subcutaneously (s.c.) with a stable emulsion prepared from equal volumes of Lys-TRH-KLH conjugate (1 mg/ml sterile saline) and Freund’s complete adjuvant. After shaving a 10 cm × 10 cm area of the flank, rabbits received 10–12 s.c. injections, equaling 1.0 ml of emulsion. At 6-week intervals, all rabbits received similar injections of emulsion prepared from equal volumes of Lys-TRH-KLH and Freund’s incomplete adjuvant. The rabbits were bled at 3-week intervals from the ear artery. 2.8. Evaluation of titer and binding specificity of rabbit antisera TRH was labeled with Na[125 I] and [125 I]TRH binding was measured as previously described in the EEP radioimmunoassay (RIA) [36,41] using serially diluted sera from rabbits immunized with the Lys-TRH-KLH conjugate. The specificity of binding was determined by adding serial dilutions of TRH and TRH-like peptides to the RIA in which the antibody dilution was adjusted to give 20% specific binding of [125 I]TRH. The relative potency of displacement by TRH and TRH-like peptides was determined as previously described [32]. 2.9. Experiment 3: acute, chronic and withdrawal treatment of rats with high dose valproate for HPLC analysis of TRH and TRH-like peptides Rats weighing 200 g were divided into four groups, 5 rats/group. Animals received either normal rat chow (CON group), were injected i.p. with 100 mg Valp/rat in 0.5 ml sterile saline 2 h prior to sacrifice (AC group), fed 20 g (high dose) Valp/kg diet for 10 days (CHR) group or fed

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high dose Valp for 10 days and then given normal diet for 2 days (WD group). 2.10. HPLC Brain tissues, after dissection and rapid weighing (see above), were boiled in 1.0 M acetic acid at 95 ◦ C for 15 min in glass test tubes [36,41]. After Polytron homogenization (Brinkman Instruments, Westbury, NY), the samples were dried completely, redispersed in 2.0 ml methanol, centrifuged, and the supernatant transferred to glass test tubes and dried completely. Dried samples from a given experiment were reconstituted with 1.0 ml 0.02% NaN3 and stored at −70 ◦ C. Aliquots of tissue extracts were pooled within each treatment group, defatted by water–ethyl ether partitioning, centrifuged, and the aqueous phase was lyophilized. After dissolving the residue in 0.1% trifluoroacetic acid (TFA), each pooled extract was loaded onto a C18 Sep-Pak cartridge (Waters, Milford, MA) that had been previously activated by washing with methanol and water. The cartridges were eluted with 3 ml of methanol and the combined sample pass-through and methanol eluates were dried completely by lyophilization and redissolved in 0.1% TFA. Particulates were removed with an HPLC-certified Acro LC3A filter (Pall Gelman Laboratories, Ann Arbor, MI). These pooled extracts were then injected into a programmable HPLC system (Shimadzu Corp., Kyoto) equipped with a 4.6 mm × 150 mm Econosphere, 3 ␮m C18 reverse phase column (Alltech Associates, Deerfield, IL) previously equilibrated with 0.1% TFA. At the time of injection, a 0.33%/min gradient of acetonitrile at a flow rate of 1 ml/min was started. At 30 min the gradient was increased to 1%/min and at 70 min the gradient was further increased to 5%/min. The 0.5 ml fractions collected were dried completely and reconstituted with 1 ml of 0.02% NaN3 just before RIA. 2.11. Experiment 4: effect of valproate on degradation of TRH by fresh human plasma Equal volumes of fresh human plasma from two healthy male volunteers were pooled and 1.0 ml aliquots were placed in two glass test tubes. Sodium valproate (1.0 ml, 0 or 2 mg/ml PBS, pH 7.5) was added to each tube followed by 0.5 ml of 5 ␮g TRH/ml PBS. Zero, 1, 2, 5, 10, 15, 20, 25, 30 and 40 min after mixing, 0.1 ml aliquots were taken from each tube and diluted in 1.0 ml of cold methanol. Extracts were centrifuged, the supernatants dried completely and then diluted in 0.02% NaN3 just prior to TRH RIA. 2.12. Experiment 5: effect of valproate on intra-vesicular procession of TRH and TRH-like peptides by rat brain and testicular homogenates Total brain cortex, 577 mg, and both decapsulated testes from a 400 g Sprague–Dawley rat, total weight 1.9 g, were each homogenized in 10 vol. of ice-cold PBS, pH 7.5, with

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10 strokes of a Dounce homogenizer. The two homogenates were then subdivided into four equal aliquots in capped plastic test tubes. An equal volume of either 0 or 2 mg sodium valproate/ml PBS was then added to two test tubes from each set of homogenates and after mixing, pairs of tubes, with and without added valproate, were incubated at either 4 or 24 ◦ C. Single 200 ␮l aliquots were removed at 0, 0.16, 0.33, 0.67, 1, 1.5, 2, 4, 6 and 8 h and boiled in 1 M acetic acid at 95 ◦ C for 15 min. Extracts were dried completely and reconstituted with 1.0 ml of 0.02% NaN3 just before RIA of combined TRH and TRH-like peptide levels. 2.13. RIA procedure, HPLC peak identification and quantitation RIA procedure, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis with multiple antibodies to TRH and TRH-like peptides, mass spectrometry and resolution of overlapping peaks by least squares fitting of a 2-Gaussian statistical model have been previously reported in detail [32,33,36,41]. 2.14. Statistical analysis Statistical comparisons were made with the aid of Statview (Abacus Concepts, Inc., Berkeley, CA), a statistical software package for the Macintosh computer. All multigroup comparisons were carried out by one-way ANOVA using post hoc Scheffe contrasts with the control group.

3. Results 3.1. Serum assay results The serum TSH, total T3 and total T4 values did not differ significantly between groups in Experiments 1–3 (results not shown). All rats consumed about 135 g rat chow/kg body weight per day. Serum Valp levels in Experiment 1 were undetectable with low dose Valp (2 g/kg diet). Rats continuously fed a high dose Valp diet (20 g/kg) in Experiment 2 had rapidly increasing serum Valp levels that peaked at 10 days of treatment and then declined rapidly, as seen in Fig. 1. The mean serum Valp levels in the acute (AC), chronic (CHR) and withdrawal (WD) groups of Experiment 3 were 281±83 ␮g/ml (AC), 41±12 ␮g/ml (CHR) and <0.1 ␮g/ml (WD). The mean serum lithium value in Experiment 1 was 0.5 ± 0.1 mM. 3.2. TRH receptor binding Table 1 summarizes the effects of 1 week of low dose Valp and Li treatment on [3 H]Me-TRH binding to the NA, AY, FCX, PCX and CBL. Low dose Valp significantly increased TRH receptor levels in FCX and NA. Lithium treatment produced a significant increase in the TRH receptor level in AY, FCX and CBL. TRH receptor binding in other brain areas did not change significantly in response to low dose Valp or Li (results not shown). High dose Valp did not result in significant changes in TRH receptor levels when TRH receptor binding results were pooled, most likely due to the large and rapid rise and fall in serum (and tissue?).

Fig. 1. Time-dependence of serum valproate levels in individual rats treated continuously with high dose (20 g/kg diet) valproate (n = 1 for each data point).

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Table 1 Effect of lithium and valproate on TRH binding to various rat brain regions

Control Lithium Valproate

NA

AY

FCX

PCX

CBL

16.1 ± 5.6 20.6 ± 3.4 29.7 ± 1.5∗

19.2 ± 5.1 27.2 ± 4.7∗ 21.5 ± 7.1

22.7 ± 6.1 30.3 ± 5.1 37.9 ± 16.5∗

15.1 ± 3.4 19.0 ± 1.9∗ 14.6 ± 4.4

8.0 ± 5.2 2.1 ± 3.3∗ 19.3 ± 12.0

Results are mean ± S.D., fmol 3 H-3Me-TRH bound/mg protein. Nucleus accumbens (NA), amygdala (AY), frontal cortex (FCX), posterior cortex (PCX) and cerebellum (CBL). ∗ P < 0.05 by one-way ANOVA using post hoc Scheffe contrasts vs. the control group.

Valp levels during the broad treatment interval used prior to sacrifice. The inability to detect serum Valp after 7 days of low dose treatment and the rapid rise and fall of serum Valp levels during a 7–15 days high dose treatment is consistent with a rapid induction of Valp-metabolizing enzymes in rat liver. Dosage increase to maintain a therapeutic response to Valp has been reported in the clinical literature [47]. 3.3. Antibody production Immunization of two rabbits with the Lys-TRH-KLH conjugate resulted, initially, in the production of antisera specific for Tyr-TRH and Phe-TRH. Results for rabbit #8 are shown in Table 2. With continuing immunization, the affinity of binding for other TRH-like peptides including TRH itself, EEP, Val-TRH, Leu-TRH, Lys-TRH and Ser-TRH, increased in antisera from both rabbits. All of the RIA measurements for the HPLC studies were obtained using antibody 8B9 because it has nearly equivalent relative potency of binding to each of the 9 TRH and TRH-like peptides evaluated (0.247 for Gln-TRH to 2.31 for Lys-TRH).

crease in Leu-TRH. In NA, on the other hand, only modest changes were detected, most of these not achieving statistical significance. In CBL, no significant changes were observed (Table 3). 3.5. Chronic effects of valproate There were no grossly observable changes in the behavior or response to handling in this and all following experiments. Chronic feeding of Valp significantly decreased TRH in ACNG, PCNG and HC (Table 3). In contrast, large increases in Tyr-TRH were noted (Table 3). The fold increases for Tyr-TRH (in parenthesis) were: STR (8) (Fig. 4), NA (7), PYR (7) (Fig. 2), ACNG (6), ENT (5) (Fig. 3), PCNG (4), MED (3) and HC (2). The levels of TRH and other TRH-like peptides were relatively unchanged or in some cases substantially decreased. For example, in PCNG (Table 3), the following decreases were detected: Phe-TRH (94%), Leu-TRH (89%), TRH (85%), Val-TRH (80%) and EEP (58%). 3.6. Effect of valproate withdrawal

3.4. Acute effects of valproate The acute response to an i.p. injection of a high dose of Valp was, in general, to increase the levels of TRH and TRH-like peptides (Table 3 and Figs. 2–4). In many tissues, these increases were extraordinary (Fig. 2). For example, the increases ranged from 58-fold (Val-TRH) to 2.7-fold (Leu-TRH). In ENT (Fig. 3), the range was 23-fold (TRH) to 2.4-fold (Phe-TRH). In other tissues, increases and decreases were observed. In PCNG, an 18-fold increase in TRH and a 7.5-fold increase in Tyr-TRH occurred simultaneously with a 67% decrease in Phe-TRH and a 64% de-

Huge increases in the levels of TRH and TRH-like peptides were measured in the FCX following Valp withdrawal ranging from 121-fold for TRH (FCX) to 10-fold for Leu-TRH (FCX) as seen in Table 3. In other tissues, large increases and decreases in the levels of TRH and TRH-like peptides accompanied a 2 days withdrawal from chronic Valp diet. In addition to FCX, large TRH increases were observed in other tissues except MED. The fold increases were: CBL (38), HC (12.5), NA (12.4), AY (9), ACNG (8.5), ENT (7) (Fig. 3), PYR (6) (Fig. 2), STR (6) (Fig. 4), and PCNG (5). With the exception of NA, decreases were

Table 2 Optimization of a radioimmunoassay used for detection of TRH and TRH-like peptides in HPLC-fractionated brain extracts Ab1

Tracer

Tyr2 -TRH

Phe2 -TRH

Leu-TRH

Val-TRH

TRH

Gln-TRH

EEP

Lys-TRH

Ser-TRH

8B1 8B9

[125 I]TRH

1.00 1.00

0.87 1.02

0.099 0.620

0.092 0.591

0.061 1.14

0.003 0.247

0.007 0.990

0.021 2.31

0.004 0.288

[125 I]TRH

Potencies for displacement of tracer were calculated relative to Tyr-TRH, potency = 1.00, as determined with the aid of a previously described computer program [32]. Ab: antibody. Repeated immunization and bleeding of rabbit #8 resulted in nearly equivalent binding of all TRH-like peptides. Compare results for bleeding #9 (8B9) with those for the initial bleeding #1 (8B1) that was specific for Tyr-TRH and Phe-TRH binding.

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Table 3 Effects of valproate on HPLC peak areas corresponding to TRH and related peptides EEP Entorhinal AC CHR WD Posterior AC CHR WD Striatum AC CHR WD Hippocampus AC CHR WD Amygdala AC CHR WD Pyriform AC CHR WD ACNG AC CHR WD Nucleus AC CHR WD Cerebellum AC CHR WD Medulla AC CHR WD Frontal cortex AC CHR WD

Val-TRH

Tyr-TRH

2250∗∗∗ 49∗ 670∗∗∗

830∗∗∗ 51∗ 345∗∗

420∗∗ 510∗∗ 196

1766∗∗∗ 15∗∗ 534∗∗∗

202∗ 20∗∗ 129

748∗∗∗ 402∗∗ 215∗

36∗ 11∗∗ 21∗∗

304∗∗ 162 60

1698∗∗∗ 132 592∗∗∗

599∗∗ 68 32∗

1175∗∗ 820∗∗∗ 28∗

260∗ 38∗ 19∗∗

454∗∗ 37∗ 86

173 108 162

1120∗∗∗ 15∗∗ 1247∗∗∗

500∗∗∗ 21∗∗ 443∗∗

927∗∗∗ 210∗ 200∗

372∗∗ 27∗ 51∗

583∗∗∗ 140 180

110 63 58

992∗∗∗ 125 902∗∗∗

418 81 151

156 78 140

134 156 26∗

100 32∗ 66

403∗∗ 173 96

856∗∗∗ 83 619∗∗∗

5781∗∗∗ 61 616∗∗∗

2546∗∗∗ 664∗∗∗ 89

465∗∗ 41∗ 848∗∗∗

191 72 240∗

168 596∗∗∗ 203∗

139 77 55

237∗ 163 417∗∗

195 106 1242∗∗∗

149 110 395∗∗

139 742∗∗∗ 709∗∗∗

166 421∗∗ 242∗

75 227∗ 188

150 75 228

157 76 3781∗∗∗

113 45∗ 731∗∗∗

96 95 824∗∗∗

85 35∗ 70

115 132 476∗∗

147 73 64

311∗∗ 83 113

287∗∗ 62 31∗

294∗∗ 54 27∗

136 25∗∗ 80

139 16∗∗ 21∗

265∗∗ –a 2939∗∗∗

117 –a 3437∗∗∗

343∗∗ –a 1050∗∗∗

176 –a 3595∗∗∗

Cortex 173 159 244∗ Cingulate 241∗ 42∗ 50∗

105 48∗ 328∗∗ Accumbens 232∗ 180 500∗∗

585∗∗∗ –a 1376∗∗∗

TRH

617∗∗∗ –a 12122∗∗∗

Leu-TRH 290∗ 118 80

266∗ 11∗∗ 4∗∗∗

Phe-TRH 260∗ 129 153 33∗ 6∗∗∗ 23∗∗

5400∗∗∗ 63 165

Results are % of corresponding peak area in sham-treated controls. Acute (AC), chronic (CHR) and withdrawal (WD) groups. a Test tube failed during water–ethyl ether extraction. ∗ P < 0.05 by one-way ANOVA using post hoc Scheffe contrasts vs. the control group. ∗∗ P < 0.01 by one-way ANOVA using post hoc Scheffe contrasts vs. the control group. ∗∗∗ P < 0.001 by one-way ANOVA using post hoc Scheffe contrasts vs. the control group.

observed in Leu-TRH levels as follows: PYR (96%) (Fig. 2), STR (81%) (Fig. 4), PCNG (79%), AC (74%), HC (49%), ACNG (45%).

final concentration) in fresh pooled human plasma diluted to 40% in PBS and incubated at 24 ◦ C (results not shown).

3.7. In vitro effect of valproate on TRH degradation by fresh human plasma

3.8. In vitro effect of valproate on the kinetics of intra-vesicular processing of TRH precursor peptides in rat brain and testis

Valproate at 0.8 mg/ml final concentration had no significant effect on the rate of degradation of TRH (1.0 ␮g/ml

The post-translational processing of the prepro-TRH precursor protein to tripeptide TRH, in brain and extra-CNS

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653

Fig. 2. Effect of valproate treatment on HPLC profile of TRH and TRH-like peptide immunoreactivity (IR) in pyriform cortex. Note differences in scale of ordinates in this and successive figures. Each chromatogram was obtained from a pool of extracts from all five animals in each treatment group.

tissues, occurs within secretory vesicles by means of a multi-enzyme cascade [30]. TRH occurs within a variety of neuronal cell types that have a multitude of functions including neuroendocrine control of hypothalamic secretion of TSH and prolactin as well as intercellular communication within the CNS itself [30]. No time-resolvable, valproate-induced, changes in the in vitro processing of endogenous TRH and TRH-like precursors by Dounce homogenates of rat brain cortex were observed at 4 or 24 ◦ C (results not shown). Testicular TRH and TRH-like peptides, on the other hand, are biosynthesized only in the Leydig cells [34]. Combined

TRH and TRH-like peptide immunoreactivity (IR) rose significantly following Dounce homogenization in testicular homogenates and then declined to low levels by 8 h. The addition of Valp at 1 mg/ml final concentration had the effect of accelerating this rise and fall in testicular TRH-IR levels. At 4 ◦ C, the total TRH-IR maximum that occurred at 4 h in the control homogenate was advanced to 2 h by Valp (results not shown). At 24 ◦ C, the control peak occurred at 1.5 h while the TRH-IR for the homogenate containing Valp peaked at 1.0 h. Interestingly, the TRH-IR level in the Valp-treated homogenate fell to nearly baseline

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Fig. 3. Effect of valproate treatment on HPLC profile of TRH and TRH-like peptide immunoreactivity (IR) in entorhinal cortex.

levels at 1.5 h when the control TRH-IR level reached its maximum (Fig. 5). 3.9. Valproate-induced acceleration of the post-translational processing of TRH and TRH-like peptides depends on the integrity of secretory vesicles Dounce homogenization in hypotonic buffer or vigorous Polytron homogenization completely disrupts secretory vesicles and abolishes the incubation-induced rise in TRH and TRH-like peptide levels in all tissues examined (results not shown). The Valp and temperature-induced changes in the levels of TRH-Gly, a final prepro-TRH precursor peptide of TRH [30] in the testis homogenates, followed a similar time-dependent profile to that for TRH (results not shown).

4. Discussion This is the first report that Valp modulates TRH receptor binding and TRH and TRH-like peptide levels in rat brain. These effects of Valp on the expression of TRH receptors are not likely due to a Valp-induced central “hypothyroidism,” which may not be detectable by changes in peripheral blood thyroid hormone levels [8]. Hypothyroidism increases the level of pituitary TRH receptors [21] while leaving TRH levels in the CNS unchanged [3]. We have, on the other hand, observed a highly significant increase in TRH receptor levels during chronic Valp and Li treatment in important limbic regions but no change in pituitary TRH receptor levels [41] and no change in circulating thyroid indices. A recently described TRH re-

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655

Fig. 4. Effect of valproate treatment on HPLC profile of TRH and TRH-like peptide immunoreactivity (IR) in striatum.

ceptor 2 (TRHR2) is highly expressed in rat brain regions involved in the regulation of attention, learning, arousal, sleep, central motor control and processing of sensory information [14]. TRHR1 predominates in limbic regions such as the perirhinal cortex, entorhinal cortex and shell of the accumbens, areas associated with mood regulation [14]. TRH-like peptides do not cross-react with Type I and Type II TRH receptors [15]. TRH can down-regulate the TRH receptor [15]. On the other hand, TRH-like peptides may alter TRH receptor levels indirectly by competitive inhibition of TRH-degrading enzymes [18] leading to increased levels of tripeptide TRH [37] or by allosteric mechanisms [28]. Valp inhibits protein kinase C (PKC) epsilon expression [22,25]. Since this PKC isozyme increases TRH receptor

mRNA degradation [10], in vivo treatment with Valp should increase TRH receptor levels, as we have observed. Acute i.p. injection of Valp, and 2 days withdrawal after chronic treatment with Valp, resulted in remarkable changes in the levels of TRH and TRH-like peptides, particularly in the FCX, PYR, ENT, HC, CBL, ACNG, PCNG and NA. Because 2 h is too short a time to significantly alter protein synthesis [43], changes in peptide levels in response to acute Valp treatment may result from alterations in any or all of the following: intra-vesicular processing of peptide precursors, vesicular stability, intracellular degradation of TRH and TRH-like peptides by pyroglutamate amino peptidase I (PAPI), release, or extracellular degradation by PAPII [5]. Extracellular degradation of TRH and TRH-like peptides is due primarily to membrane-bound, TRH recep-

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Fig. 5. In vitro effect of valproate on the kinetics of intra-vesicular processing of TRH and TRH-like precursor peptides in a Dounce homogenate of Sprague–Dawley testes. ∗ P < 0.05 by one-tailed nonpaired Student’s t test.

tor associated-PAPII [14], or thyrolibrinase, which is the free-form of PAPII found in blood [42]. The rate of in vitro degradation of TRH observed in fresh human plasma was Valp insensitive, suggesting that the extracellular clearance of TRH and most TRH-like peptides, following release in brain, is also Valp insensitive. Intrathecal TRH has been clinically shown to reverse treatment-resistant depression within hours [26]. It has been suggested that these acute (though short-lasting) remissions result from the sudden flooding of TRH receptor sites on the overactive glutamatergic neurons [39,40]. Our observations that chronic, low dose, Valp and Li treatment substantially increases the number of TRH receptors in various brain regions complement this idea and suggest that enhanced sensitivity to endogenous TRH within limbic regions may contribute to mood stabilization. The nucleus accumbens serves as a critical interface between the limbic and motor systems linking motivation with action [9]. The amygdala mediates processes that invest sensory experience with emotional significance [1]. Both the accumbens and the amygdala receive direct glutamatergic projections from medial prefrontal cortical regions [16]. Our findings are consistent with the newly recognized contribution of glutamatergic dysfunction to neuropathology [31] and the ability of chronic Valp and Li to moderate glutamate toxicity [6,12,24]. The therapeutic potential of this endogenous tripeptide is limited, however, by the necessity for intrathecal injection to minimize its rapid degradation by TRH-degrading enzymes in the circulation [37] and to maximize its restricted transport across the blood–brain barrier by a nonsaturable, enzyme

degradation-coupled mechanism [48]. These limitations do not apply, of course, to Valp-induced intracerebral synthesis and/or release of TRH and other prepro-TRH-derived peptides that have effects which augment the antidepressant action of TRH and increase responsiveness to endogenous TRH [38]. Valp and Li are neurotrophic/neuroprotective agents that can robustly increase the expression of the cytoprotective protein bcl-2 and increase significantly the total gray matter volume in patients with manic-depressive illness [11,25]. Imaging studies have provided evidence for a role of the anterior cingulate, amygdala, and prefrontal cortex in the pathophysiology of bipolar disorder [11,25]. Long-term exposure to Valp and Li dramatically protects cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity, which involves apoptosis mediated by N-methyl-d-aspartate (NMDA) receptors [12,13,29,45]. The protection in cerebellar neurons is specific for glutamate-induced excitotoxicity and can be attributed to inhibition of NMDA receptor-mediated calcium influx. TRH (and TRH-like peptides) are colocalized in, and secreted by, glutamate-containing neurons in the hippocampus [40]. Valp is a histone deacetylase inhibitor that decreases the electrostatic interaction between the negative sugar-phosphate backbone of DNA and the lysine residues of the histones forming the core of the nucleohistone particles [46]. This results in chromatin decondensation and increased gene transcription. These effects may play a role in the dramatic Valp-induced changes in TRH and TRH-like peptide lev-

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