TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone

Psychoneuroendocrinology (2008) 33, 1183—1197

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone Albert Eugene Pekary a,c,f,*, Albert Sattin a,b,d,e, James Blood a, Stephanie Furst a a

Research Services, VA Greater Los Angeles Healthcare System, USA Psychiatry Services, VA Greater Los Angeles Healthcare System, USA c Center for Ulcer Research and Education, VA Greater Los Angeles Healthcare System, USA d Department of Psychiatry & Biobehavioral Sciences, University of California, Los Angeles, CA 90073, USA e Research Institute, University of California, Los Angeles, CA 90073, USA f Department of Medicine, University of California, Los Angeles, CA 90073, USA b

Received 9 January 2008; received in revised form 4 April 2008; accepted 5 June 2008

KEYWORDS Chronic corticosterone; Thyrotropin-releasing hormone; Limbic system; Depression

Summary Sustained abnormalities of glucocorticoid levels have been associated with neuropsychiatric illnesses such as major depression, posttraumatic stress disorder (PTSD), panic disorder, and obsessive compulsive disorder. The pathophysiological effects of glucocorticoids may depend not only on the amount of glucocorticoid exposure but also on its temporal pattern, since it is well established that hormone receptors are down-regulated by continuously elevated cognate hormones. We have previously reported that TRH (pGlu-His-Pro-NH2) and TRH-like peptides (pGlu-X-Pro-NH2) have endogenous antidepressant-like properties and mediate or modulate the acute effects of a single i.p. injection of high dose corticosterone (CORT) in rats. For these reasons, two accepted methods for inducing chronic hyperglucocorticoidemia have been compared for their effects on brain and peripheral tissue levels of TRH and TRH-like peptides in male, 250 g, Sprague-Dawley rats: (1) the dosing effect of CORT hemisuccinate in drinking water, and (2) s.c. slow-release pellets. Overall, there were 93% more significant changes in TRH and TRH-like peptide levels in brain and 111% more in peripheral tissues of those rats ingesting various doses of CORT in drinking water compared to those with 1—3 s.c. pellets. We conclude that providing rats with CORTin drinking water is a convenient model for the pathophysiological effects of hyperglucocorticoidemia in rodents. Published by Elsevier Ltd.

1. Introduction * Corresponding author at: VA Greater Los Angeles Healthcare System, Building 114, Room 229, 11301 Wilshire Boulevard, Los Angeles, CA 90073, United States. Tel.: +1 310 268 4430; fax: +1 310 441 1702. E-mail address: [email protected] (A.E. Pekary). 0306-4530/$ — see front matter. Published by Elsevier Ltd. doi:10.1016/j.psyneuen.2008.06.001

We have previously reported that i.p. injection of 4.0 mg of corticosterone (CORT) into young adult male rats results in rapid changes in the levels of TRH (pGlu-His-Pro-NH2) and TRH-like peptides (pGlu-X-Pro- NH2) where ‘‘X’’ can be any amino acid residue (Pekary et al., 2005, 2006a,b,c,d) in

1184 multiple regions of rat brain and peripheral tissues. These endogenous neuropeptides have antidepressant-like, analeptic and neuroprotective effects which may contribute to, or moderate, neuropsychiatric disorders (Pekary et al., 2005, 2006a,b,c,d; Zeng et al., 2007). An important characteristic of major depression and post-traumatic stress disorder (PTSD) is chronic elevation, or suppression, respectively, of serum CORT levels (Yehuda, 2006). Ingestion of 400 mg/L CORT in 2.5% ethanol for 28 days suppressed CORT levels below that in rats consuming either water or 2.5% ethanol despite a reduction in plasma clearance time for CORT by day 28 (Pung et al., 2003). Consumption of 25 mg/L CORT in drinking water for 20 days resulted in a sustained depressogenic effect 2 weeks after exposure while higher doses did not (Gourley et al., 2006, 2008). Evidently, prolonged ingestion of high doses of CORT induce alterations in its metabolism that are complex and potentially informative of the pathophysiological responses to repeated stress that can lead to major depression, or PTSD, in humans (Yehuda, 2006). Subcutaneous implantation of a single slow (21-day) release pellet containing 100 mg CORT into adrenally intact rats has the effect, after 7 days, of flattening the endogenous rhythm of CORT, reducing body weight gain and decreasing adrenal weights compared to control rats implanted with a cholesterol pellet (Bush et al., 2003). Removal of the pellet 7 days later resulted in a persistent decrease in body weight, adrenal weight, cortical 5-HT1A receptor binding and alteration in the diurnal variation of 5-HT turnover in the frontal cortex (Bush et al., 2003). Flattening of glucocorticoid rhythm decreases hippocampal expression of mRNAs coding essential structural and functional proteins and desensitizes the 5HT1A autoreceptors in a regionally selective manner. These changes may be relevant to the cognitive deficits of aging and depression (Gartside et al., 2003; Leitch et al., 2003). The therapeutic effects of antidepressants may depend on neurogenesis within the hippocampus which requires, in turn, rhythmic changes in glucocorticoid levels (Huang and Herbert, 2006). Decreased 5-HT1A receptor binding in the dentate gyrus and increased 5-HT2A receptor binding in the parietal cortex following implantation of a single 100 mg CORT pellet for 1 week was associated with increased depression-like behavior but not anxiety (Fernandes et al., 1997) as a result of enhanced occupation of mineralocorticoid receptors (Meijer et al., 1997). Mild hypothyroidism also induces depression-like behavior in rats (Solberg et al., 2001) and has rapid and profound effects on the levels of TRH-like peptides in limbic regions of male Sprague-Dawley rats (Pekary et al., 2006a). For these reasons we have determined the chronic dosing effect, in male rats, of CORT in drinking water, or slowrelease pellets implanted subcutaneously, on the levels of TRH and TRH-like peptides in brain and peripheral tissues. In a separate experiment blood CORT levels were monitored daily in rats drinking low, intermediate or high levels of CORT. Suppression of daytime CORT levels in the serum of adrenally intact rats after 15 days of ingesting a high dose of CORT was confirmed. This process includes episodic water ingestion, adrenal involution, slowing of CORT metabolism, and flattening of the diurnal CORT rhythm, resulting in a very complex temporal pattern.

A.E. Pekary et al.

2. Materials and methods 2.1. Animals Male Sprague-Dawley (SD) rats (Harlan, Indianapolis, IN) were used for all experiments. These animals were group housed (4 animals per cage), maintained with standard Purina rodent chow #5001 and water ad libitum during a standard one 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 receipt and on the morning of each experiment. Initial 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. All efforts have been made to minimize the number of animals used and their suffering. All animals were transferred from the Veterinary Medical Unit to the laboratory 12 h before the start of experiments to minimize the stress of a novel environment.

2.2. Experiment 1: Effect of chronic ingestion of CORT hemisuccinate in drinking water on levels of TRH and TRH-like peptides in brain and peripheral tissues Male Sprague-Dawley rats, weighing 232  13 g upon receipt, were divided into 4 groups (n = 4/group). The control (CON) group was provided tap water for drinking. The next group (LOW) received 25 mg/L CORT in tap water for 20 days that was replaced every third day by diluting a stock solution of CORT in DMSO with tap water at pH 12 and then neutralizing with HCl to pH 7.5 as previously described (Gourley et al., 2006, 2008). The third group (HIGH) received 400 mg/L CORT in drinking water for 20 days. The final group (WD, withdrawal) was also given 400 mg/L CORT for 18 days but was then returned to regular tap water for 48 h prior to decapitation. More than 80% of the initial CORT immunoreactivity (25, 100 and 400 mg/L distilled water) was measured after 3 days in the glass water bottles used for chronic CORT ingestion experiments.

2.3. Experiment 2: Effect of subcutaneous implantation of CORT-containing pellets on levels of TRH and TRH-like peptides in brain and peripheral tissues Male Sprague-Dawley rats weighing 261  15 g upon receipt were divided into 4 groups (n = 4/group). All animals were anesthetized with Nembutal (35 mg/kg body weight). Body temperature was monitored with a digital thermometer while animals lay prone on a hot pad set at ‘‘low’’ temperature. The skin between the scapulae was shaved and cleaned with povidone-iodine solution (Aplicare, Branford, CT). Ethanol was not used to maintain the integrity of the slow release pellets to be implanted. A subcutaneous tunnel adequate for

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone insertion of up to three 21-day release pellets containing 100 mg CORT/pellet (Steraloids, Newport, RI) was made by blunt dissection with round-tipped scissors that had been sterilized in a high-temperature bead bath. The control (CON) group received 0, the second (ONE) group received one pellet, the third (TWO) group received two pellets and the forth group (THREE) had three pellets inserted before closing the wound with two autoclips. Animals were observed continuously until fully ambulatory and given the antibiotic Baytril (2.27% enarofloxacin, 11 ml/500 ml in drinking water) for 3 days.

2.4. Experiment 3: Effect of chronic ingestion of CORT in drinking water on serum CORT versus time Male Sprague-Dawley rats weighing 208  19 g upon receipt were divided into 3 groups (n = 4/group). All animals received CORT in tap water for 15—19 days. The LOW group received 25 mg/L, the MED group was given 100 mg/L for 17 days, and the HIGH group drank 400 mg/L for 15 days. Each rat was bled between 8 a.m. and 10 a.m. once every 4th day as follows. The base of the tail was swabbed with 70% ethanol then the rat was placed inside a bag which was closed and tied with draw strings leaving the tail protruding. A vein near the base of the tail was punctured with a 20 gauge syringe

1185

needle and 20 ml of blood was drawn up into a heparinized capillary pipet. This procedure is readily performed by one person. This is an adaptation of a previously described method that requires 2 people (Fluttert et al., 2000). This blood was then immediately rinsed into a vial containing 2.0 ml of diluent for a CORT RIA kit (MP Biomedical, Irvine CA) and stored at 20 8C until all samples had been collected for measurement within the same assay.

2.5. Experiment 4: Effect of acute ingestion of CORT in Hawaiian Punch on serum CORT versus time The consistent observation of a large single spike in serum levels of CORT, within the first week of providing CORT in drinking water (Fig. 1), may have been due to the occasional ingestion of a large amount of CORT just before tail vein bleeding between 8 a.m. and 10 a.m. Rats are most active during the dark phase (6 p.m. to 6 a.m.) and do most of their eating and drinking during this time. An acute experiment was devised in which four Sprague-Dawley male rats, 200— 220 g on receipt, housed in the same cage, were first adapted to a free-choice of tap water and Hawaiian Punch (York, 1981). Hawaiian Punch containing 400 mg/L CORT hemisuccinate was then prepared as above and made available for 60 min with direct observation of active drinking by all rats.

Figure 1 (A) Serum CORT versus time in male SD rats drinking 400 mg/L of CORTad libitum for 15 consecutive days. Note not only the extraordinary, 25,000 ng/ml, peak of serum CORT at day 4 but also the significant rebound in CORT levels between days 7 and 13 compared to days 6 and 15 ( p < 0.05 by one tailed t-test). (B and C) Corresponding profiles for rats drinking 100 mg/L and 25 mg/L of CORT, respectively. (D) Serum CORT versus time in SD rats drinking 400 mg/L of CORT in Hawaiian Punch for 1 h.

1186 One rat was bled from the tail vein, as described above, before, and at 0, 1, 2, 4, 6 and 18 h after removal of the CORT-containing bottle.

2.6. Dissection of rat brain, pancreas and reproductive organs All rats were decapitated between 9 a.m. and 11 a.m. Nucleus accumbens (NA), amygdala (AY), frontal cortex (FCX), cerebellum (CBL), medulla oblongata (MED), anterior cingulate (ACNG), posterior cingulate (PCNG), striatum (STR), pyriform cortex (PYR), hippocampus (HC), entorhinal cortex (ENT), pancreas, prostate, epididymis, and testes were hand dissected, weighed rapidly, and then extracted as previously described (Pekary et al., 2005, 2006a,b,c,d).

2.7. Serum hormone assays Serum CORT, free T4 and total T3 were measured with the following commercial RIA kits: CORT (MP Biomedicals, Aurora, OH), free T4 and total T3 (DPC Coat-A-Count, Los Angeles, CA). Serum glucose was measured with the Beckman LX-20 Pro Chemistry Analyzer (Fullerton, CA).

2.8. HPLC and RIA procedures, HPLC peak identification and quantitation HPLC and RIA procedures, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis of multiple antibodies to TRH and TRH-like peptides, mass spectrometry and resolution of overlapping peaks by least squares fitting of a 2Gaussian statistical model have been previously reported in detail (Pekary et al., 2007, 2005, 2002; Pekary and Sattin, 2001). Briefly, after boiling, tissues were dried, re-extracted with methanol, dried and defatted by water—ethyl ether partitioning. Dried samples were dissolved in 0.1% trifluroacetic acid (TFA), and loaded onto reverse phase C18 Sep-Pak cartridges (Water, Milford, MA). TRH and TRH-like peptides were eluted with 30% methanol. Dried peptides were again dissolved in TFA, filtered and then fractionated by HPLC using a 4.6 mm  150 mm Econosphere, 3 mm C18 reverse phase column (Alltech Associates, Deerfield, IL) and a 0.2%/min gradient of acetonitrile. The 0.5 ml fractions collected were dried completely and reconstituted with 0.15 ml of 0.02% NaN3 just before RIA. The antiserum used (8B9) cross-reacts with TRH and eight TRH-like peptides with a relative potency of displacement ranging from 2.31 (Lys-TRH) to 0.288 (Ser-TRH) relative to Tyr-TRH (Table 2, Pekary et al., 2004). Two of the regularly observed peaks (2a and 2b) consist of a mixture of unidentified TRH-like peptides. Of the seven observed peptides three have so far been confirmed by mass spectrometry: TRH, Glu-TRH and Tyr-TRH (Pekary et al., 2005) and mass spectrometry of the remaining four is planned. Tissue samples from the 4 rats within each treatment group were pooled prior to HPLC to provide the minimum amount of immunoreactivity needed for reliable RIA measurements.

A.E. Pekary et al. The mean recovery of TRH and TRH-like peptide immunoreactivity from all tissues studied was 84  15% (mean  SD). The within-assay and between-assay coefficient of variation for measuring 333 pg/ml TRH was 4.8% and 16.9%, respectively. All HPLC fractions obtained from a given brain region or peripheral tissue were analyzed in the same RIA. The minimum detectable dose for TRH was 5 pg/ ml. The specific binding of [1215I]TRH (Bo/T) was 25%.

2.9. 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 multi-group comparisons were carried out by one way analysis of variance using post hoc Scheffe contrasts with the control group. The mean within-group coefficient of Variation (CV) (SD/mean, CV-within group) for each tissue and TRH/ TRH-like peptide combination, across four photoperiod intervals, has been previously reported (circadian rhythm experiment) for untreated Sprague-Dawley rats (Pekary et al., 2006c). Mean within-group CVs in brain ranged from 6.3% for TRH levels in AY to 48% for Leu-TRH in ACNG, and from 3.1% for Val-TRH in testis to 21% for Tyr-TRH in pancreas. These CVs were then used to estimate the level of significance, by one-way ANOVA, of changes in the pooled mean values of TRH and TRH-like peptide levels following chronic ingestion of various doses of CORT or chronic s.c. implantation of 0-3 CORT-containing pellets. The SD for measurements of TRH or TRH-like peptide (XTRH) following CORT treatment in a given tissue extract (T), SD (X-TRH,T), was equal to CV (X-TRH,T; cycle experiment) multiplied by the mean for the corresponding X-TRH from Tables 1—4.

3. Results 3.1. Experiment 1: Effect of chronic ingestion of CORT in drinking water on body and tissue weights The body weights for the CON, LOW, HIGH and WD groups after 20 days were: 310  21; 323  31 (n.s.); 229  13 ( p < 0.02), and 255  28 g ( p < 0.05), respectively. The only tissue weight that was significantly affected by CORT in drinking water was that of the prostate. Prostate weights, in the above treatment sequence, were: 0.59  0.07; 0.68  0.20 (n.s.); 0.40  0.07 ( p < 0.05), and 0.35  0.04 g ( p < 0.05), respectively.

3.2. Experiment 2: Effect of implantation of CORT-containing pellets on body and tissue weights The body weights for rats 20 days after implantation of 0, 1, 2 or 3 CORT-containing pellets were: 308  9; 224  36 ( p < 0.02); 182  21 ( p < 0.001), and 149  29 g ( p < 0.001), respectively. The weights of prostate, epididymis and testis were all significantly decreased 20 days after implantation of CORT pellets. The prostate weights, in the

1187

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone above treatment sequence, were: 0.52  0.08; 0.25  0.06 ( p < 0.01); 0.13  0.06 ( p < 0.001), and 0.12  0.04 ( p < 0.001), respectively. The combined weight for the two epididymides was: 1.15  0.08; 1.04  0.16 (n.s.);

0.63  0.09 ( p < 0.01), and 0.47  0.09 ( p < 0.01), respectively. Combined weight for both testes were: 3.44  0.11; 3.15  0.35 (n.s.); 2.16  0.23 ( p < 0.05), and 1.71  0.82 ( p < 0.02), respectively.

Table 1 Effects of ingesting LOW (25), or HIGH (400 mg/L) corticosterone hemisuccinate (CORT) for 20 days in drinking water or high CORT for 18 days followed by 2 days of normal drinking water only (WD) on the levels of TRH and TRH-like peptides in rat brain relative to the corresponding levels in rats ingesting CON (control, 0 mg/L) CORT Treatment

Glu-TRH

Peak ‘‘2’’

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

Nucleus accumbens LOW #52.7 * HIGH #34.2 * WD #26.9 *

#66.0 * #36.9 * #58.1 *

191.1 125.8 122.7

63.7 #21.0 * 72.1

76.8 69.8 72.8

109.9 55.0 68.8

#39.8 * #31.0 * #35.0 *

94.0 86.7 81.9

Striatum LOW HIGH WD

#33.5 * #26.5 * #47.0 *

137.6 99.6 80.9

#33.1 * #11.3 * 66.3

43.2 97.8 77.7

75.9 63.5 62.4

#23.4 *** #19.6 *** #33.9 **

197.2 146.5 131.0

Anterior cingulate LOW 82.9 HIGH 88.4 WD 135.5

71.3 #35.5 * 75.3

81.4 49.8 103.7

88.2 41.0 83.2

122.5 35.0 73.0

102.5 57.6 129.7

87.0 19.8 80.7

60.3 48.2 71.3

Posterior cingulate LOW 101.0 HIGH 52.8 WD 121.0

58.0 41.6 46.7

#43.5 * #25.2 * #55.6 *

35.4 #18.2 * 47.8

63.8 #18.6 * #37.1 *

30.0 26.5 31.5

#40.0 * 51.5 #19.6 *

125.9 63.4 56.9

47.7 50.5 60.4

#19.4 * #28.9 * 57.5

#22.2 ** 107.5 "130.5 *

#12.6 ** #24.8 ** #67.0 *

#20.0 * 44.6 50.0

32.8 26.6 46.6

#24.3 * #23.3 * #36.6 *

41.8 161.3 35.2

Entorhinal cortex LOW 129.4 HIGH 144.3 WD 80.4

84.5 #34.4 * #47.6 *

141.2 85.3 85.3

120.1 #28.3 * 62.3

107.6 47.4 110.8

125.6 57.4 73.3

85.4 20.2 35.9

100.9 51.9 63.2

Hippocampus LOW HIGH WD

49.4 #14.7 * #31.6 *

83.1 45.4 70.8

71.3 #12.7 ** 64.2

67.5 17.9 49.8

59.3 16.5 41.7

64.0 #13.7 * #44.3 *

78.8 #35.4 * #50.0 *

Pyriform cortex LOW 109.7 HIGH 36.5 WD 95.5

55.3 #17.2 * #38.1 *

95.5 49.7 42.6

68.4 #17.4 * 60.0

81.6 27.3 66.4

56.2 #10.6 * 29.2

46.4 #10.0 * #27.0 *

82.5 55.8 77.8

Frontal cortex LOW HIGH WD

#42.0 * #52.5 * #33.4 *

31.3 #15.3 * 35.0

44.3 #12.0 * 104.8

143.3 #21.2 * 28.3

#13.4 *** #14.2 *** #40.6 **

#39.4 * #14.2 * #44.6 *

#40.9 * #24.6 * 88.4

#23.3 * #7.4 ** #47.2 **

Medulla oblongata LOW #34.2 * HIGH #6.6 * WD #20.1 *

#13.6 ** #3.2 ** #20.1 **

57.3 #9.2 * 95.6

#11.4 * 71.1 132.0

#9.6 * #5.0 * #13.1 *

#13.3 * #4.6 * 26.8

#14.9 * #11.4 * #18.4 *

#10.2 * #1.7 * #6.9 *

Cerebellum LOW HIGH WD

#23.8 *** #8.4 *** #11.3 ***

#21.5 * #4.5 ** #41.2 *

70.0 #7.1 ** #13.5 **

#15.3 * #11.4 ** #10.0 **

27.2 9.1 20.9

#23.6 * #7.8 ** #16.3 **

34.8 #2.5 * 15.9

Amygdala LOW HIGH WD

*

91.2 153.0 "561.1 **

68.5 34.4 #30.9 *

#32.0 ** #21.4 ** #17.6 ***

p < 0.05; **p < 0.025, and ***p < 0.005 by one way ANOVA using post hoc Scheffe contrasts versus the control group as previously described (Pekary et al., 2006c).

1188

A.E. Pekary et al.

3.3. Experiment 1: Effect of chronic ingestion of CORT in drinking water on serum CORT, T3, free T4 and glucose levels

3.5. Experiment 3: Effect of chronic ingestion of CORT in drinking water on serum CORT levels versus time

The serum CORT levels for rats drinking 0 (CON), 25 (LOW), 400 (HIGH) mg/L of CORT for 20 days or 400 mg/L CORT followed by 2 days of regular tap water (WD) were: 110  69, 96  82 (n.s.); 45  30 (n.s.), and 10  4 ( p < 0.05) ng/ml, respectively. Serum T3 levels were: 63  3; 62  10 (n.s.); 74  14 (n.s.) and 78  12 (n.s.) ng/dl, respectively. Serum free T4 levels were: 1.26  0.11; 1.24  0.30 (n.s.); 1.26  0.30 (n.s.); 2.01  0.14 ( p < 0.05), respectively. Serum glucose levels were: 127  7; 129  7 (n.s.); 115  5 ( p < 0.05); 130  8 (n.s.) mg/dl, respectively.

The serum CORT levels versus time in male Sprague-Dawley rats drinking 400 mg/L of CORT ad libitum for 15—19 consecutive days are displayed in Fig. 1. An extraordinary 25,000 ng/ml peak of serum CORT at day 4 was observed as well as a significant rebound to 450 ng/ml at day 12 (CORT levels between days 7 and 13 compared to days 6 and 15, p < 0.05 by one tailed t-test). Ad libitum ingestion of a low (25 mg/L) or medium (100 mg/L) CORT in drinking water gave similar results with a peak of serum CORT occurring at day 7 (310 ng/ml) and day 6 (1200 ng/ml), respectively (Fig. 1).

3.4. Experiment 2: Effect of implantation of CORT-containing pellets on serum CORT, T3, free T4, and glucose levels

3.6. Experiment 4: Effect of acute ingestion of CORT in drinking water on serum CORT levels versus time

The serum CORT levels for rats with 0, 1, 2, or 3 slow-release CORT-containing pellets implanted s.c. were: 36  16, 126  63 (n.s.), 156  60 (n.s.) and 233  130 ( p < 0.05) ng/ml, respectively. Serum T3 levels were: 60  6; 83  8 ( p < 0.01); 77  5 ( p < 0.05); 76  5 ( p < 0.05) ng/dl, respectively. Serum free T4 was: 2.17  0.26; 2.56  0.61 (n.s.); 1.87  0.37 (n.s.); 2.29  0.25 (n.s.) ng/dl, respectively. Serum glucose: 102  8; 110  10 (n.s.); 79  21 ( p < 0.05 vs. 1 pellet); 105  17 (n.s.) mg/dl, respectively. These serum hormone values, combined with normal appearance and behavior, are consistent with full recovery of all animals from the initial stress of s.c. implantation of CORTcontaining pellets 20 days prior to decapitation.

Serum CORT versus time in SD rats drinking an average of 5 ml of 400 mg/L of CORT in Hawaiian Punch ad libitum for 1 h is displayed in Fig. 1. The peak CORT level occurred 2 h after removal of the CORT-Hawaiian Punch mixture. CORT levels declined continuously to very low levels by 18 h due to the suppression of the HPA axis.

3.7. Experiment 1: Effect of chronic ingestion of CORT in drinking water on brain levels of TRH and TRH-like peptides Ingestion of low or high levels of CORT in drinking water for 20 days or high levels of CORT for 18 days followed by plain

Table 2 Effects of ingesting LOW (25), or HIGH (400 mg/L) corticosterone hemisuccinate (CORT) for 20 days in drinking water or high CORT for 18 days followed by 2 days of normal drinking water only (WD) on the levels of TRH and TRH-like peptides in rat brain relative to the corresponding levels in rats ingesting CON (control, 0 mg/L) CORT.

Prostate LOW HIGH WD

Glu-TRH

Peak ‘2’

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

130.1 131.5 #10.8 **

147.4 86.4 #17.1 **

151.7 "381.6 * #36.8*

"239.7 * "399.0 * 199.0

#44.2 * 179.0 #40.0 *

50.4 85.8 137.7

#36.7 * 125.0 #26.6 *

87.3 "293.2 * 114.8

#49.1 * "261.4 * 54.0

#25.6 * #45.4 * #25.7 *

#27.8 * 70.1 #41.8 *

#25.4 * 146.4 #44.2 *

"231.5 * "525.0 ** "289.8 *

#14.2 ** #30.7 * #9.6 ***

#11.4 *** #27.1 * #8.4 ***

#14.4 ** #27.0 * #14.9 **

a

Epididymis LOW HIGH WD Pancreas LOW HIGH WD

#29.1 * #13.8 ** #14.7 **

#13.9 ** #3.0 *** #1.4 ***

#1.6 *** #0.9 *** #0.8 ***

#22.4 ** #5.5 *** #4.1 ***

#9.5 *** #2.1 *** #6.2 ***

#21.1 ** #0.6 *** #0.5 ***

#13.9 ** #0.2 *** #0.3 ***

#27.8 * #2.5 *** #2.3 ***

Testis LOW HIGH WD

53.2 #6.1 *** #15.5 **

#13.5 ** #1.2 *** #1.2 ***

#10.3 *** #8.3 *** #6.1 ***

#13.0 ** #1.4 *** #1.8 ***

#6.2 *** #3.3 *** #1.7 ***

#1.7 *** #0.2 *** #0.1 ***

72.6 #0.8 *** #0.3 ***

#2.6 *** #6.4 *** #1.2 ***

*

p < 0.05; **p < 0.025, and ***p < 0.005 by one way ANOVA using post hoc Scheffe contrasts versus the control group as previously described (Pekary et al., 2006c). a Peptide levels divided by tissue weights prior to normalization with corresponding control values to correct for significant decrease in tissue weight with HIGH and WD treatment.

1189

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone Table 3

Effect of s.c. pellet implantation on TRH and TRH-like peptide levels in peripheral tissues

Treatment

Glu-TRH

Peak ‘‘2’’

TRH

Val-TRH

Tyr-TRH

Phe-TRH

Trp-TRH

"347.1 ** 181.7 183.7

"389.8 * 110.7 140.7

145.9 #9.1 * #35.2 *

"246.4 * 197.2 64.8

68.6 87.1 #8.4 *

118.0 73.7 #11.6 *

157.3 106.9 65.2

68.9 #38.9 * 95.6

47.8 67.5 94.3

68.3 #38.9 * #60.9 *

78.2 56.4 74.1

98.1 104.7 26.1

#22.2 ** #49.3 * #30.7 *

104.9 122.4 100.5

65.6 #43.6 * #54.1 *

Anterior cingulate 1 pellet #31.9 * 2 pellets #28.1 ** 3 pellets #48.8 *

93.5 55.9 58.5

89.7 30.9 26.0

51.4 98.1 33.3

#36.9 * #38.9 * #40.0 *

29.1 41.5 13.0

44.8 89.6 31.9

129.3 79.9 244.0

Posterior cingulate 1 pellet 77.3 2 pellets 51.6 3 pellets 183.1

69.8 43.9 #5.3 *

#28.4 ** #29.9 ** #10.0 **

74.8 29.5 #8.4 *

54.5 #21.6 * #13.5 *

15.2 17.9 #2.4 *

#19.4 * #27.2 * 55.9

75.8 #15.1 * 48.8

138.3 93.6 158.8

#76.2 * 77.3 117.1

149.1 58.1 #41.2 *

146.2 143.6 225.6

76.0 82.7 69.3

140.0 120 200.0

175.3 66.0 210.3

64.2 "198.3 * 82.0

60.2 182.4 91.6

102.3 235.6 "285.3 *

61.5 101.6 155.9

39.3 155.5 114.0

37.7 143.6 79.7

88.8 82.9 96.5

132.4 98.0 207.9

111.3 86.9 89.3

114.7 90.7 81.7

71.2 68.7 76.8

113.9 225.9 "322.3 *

"430.6 ** 115.6 144.3

Pyriform cortex 1 pellet "5742.4 *** 2 pellets 26.7 3 pellets 263.5

110.2 80.0 142.4

175.9 75.1 203.5

194.4 174.8 "617.7 *

116.0 161.3 316.0

63.0 128.5 84.0

53.8 307.7 208.9

444.3 545.7 400.0

Frontal cortex 1 pellet 2 pellets 3 pellets

171.0 305.3 101.1

218.6 286.8 91.5

#17.3 * #48.0 * #35.5 *

"291.3 * "533.3 *** "342.1 *

117.5 94.7 58.5

84.8 104.7 65.9

135.4 157.9 83.7

#56.2 * 70.3 100.3

50.8 53.2 111.2

130.5 52.5 62.4

104.9 107.3 130.1

42.1 77.2 105.2

108.9 60.8 84.4

68.1 39.9 49.9

69.1 "151.5 * 100.7

114.0 "224.5 * 156.7

"187.5 * 83.3 66.7

111.7 77.7 82.3

50.0 57.7 31.1

58.8 98.0 #43.2 *

175.0 104.4 117.3

Nucleus accumbens 1 pellet 135.7 2 pellets "274.3 * 3 pellets 106.7 Striatum 1 pellet 2 pellets 3 pellets

Amygdala 1 pellet 2 pellets 3 pellets

269.2 358.9 "5948.7 ***

Entorhinal cortex 1 pellet 69.5 2 pellets 230.9 3 pellets 146.4 Hippocampus 1 pellet 2 pellets 3 pellets

140.8 52.7 68.4

110.9 "258.0 * 101.5

Medulla oblongata 1 pellet 59.3 2 pellets 28.0 3 pellets 75.2 Cerebellum 1 pellet 2 pellets 3 pellets

136.8 "186.0 * "203.5 *

Leu-TRH

75.9 "501.2 * 355.6

*

p < 0.05; **p < 0.025, and ***p < 0.005 by one way ANOVA using post hoc Scheffe contrasts versus the control group as previously described (Pekary et al., 2006c).

water for 2 days (WD group) resulted in either no change or a significant decrease in the levels of TRH and TRH-like peptides in all brain regions examined (Table 1; Figs. 2 and 3). A significant increase was observed in the TRH level of AY following a 2 day withdrawal of CORT. The overall number of significant changes in TRH and TRH-like levels throughout

the brain in response to LOW, HIGH, and WD CORT was 110. Brain regions, ranked according to the number of significant reductions in TRH and TRH-like peptide levels, given in parentheses, were: MED (19); CBL (18); FCX (17); NA (10); AY (10); STR (8); PCNG (9); HC (8); PYR (6); ENT (3) and ACNG (1). The ranking of frequency of decreases in specific

1190 Table 4

A.E. Pekary et al. Effect of s.c. pellet implantation on TRH and TRH-like peptide levels in peripheral tissues Glu-TRH

Peak ‘2’

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

#16.9 ** 76.7 #37.2 *

#16.2 ** #41.5 * #18.0 **

53.9 #6.9 *** #1.7 *** 122.3 #12.7 ** #23.3 **

Prostate a 1 pellet 2 pellets 3 pellets

92.8 #11.2 *** #2.8 ***

144.8 53.1 #29.7 *

124.6 #31.3 * #12.0 ***

#28.9 * 148.7 74.9

#13.3 ** #35.1 * #31.5 *

Epididymis a 1 pellet 2 pellets 3 pellets

92.8 #7.6 *** #2.8 ***

#35.4 * #31.5 * 54.1

"781.4 ** #46.7 * 74.4

#38.8 * #21.6 ** 74.5

"3852.4 *** "700.4 ** "294.0 *

160.0 54.8 65.1

100.9 #18.9 ** #18.8 ** 77.0 129.7 "220.9 *

Pancreas 1 pellet 2 pellets 3 pellets

63.9 128.5 "248.1 *

#43.1 * 126.3 164.0

#22.4 ** 86.9 53.0

70.1 155.3 142.7

#20.1 ** 73.0 119.1

60.5 113.4 120.1

Testis a 1 pellet 2 pellets 3 pellets

91.5 "222.7 * 99.2

67.5 87.6 175.0

75.5 157.1 185.0

161.4 197.0 251.9

88.2 105.3 143.2

71.3 137.2 90.5

73.0 81.2 80.9

135.0 109.5 125.7 82.8 64.5 106.1

*

p < 0.05; **p < 0.025, and ***p < 0.005 by one way ANOVA using post hoc Scheffe contrasts versus the control group as previously described (Pekary et al., 2006c). a Peptide levels divided by tissue weights prior to normalization with corresponding control values to correct for significant decrease in tissue weight with increasing number of implanted pellets.

peptides, across all brain regions studied were: Phe-TRH (23); peak ‘2’ (22); Val-TRH (14); Tyr-TRH (12); Glu-TRH (14; TRH (9); Trp-TRH (9); Leu-TRH (6).

3.8. Experiment 1: Effect of chronic ingestion of CORT in drinking water on peripheral tissue levels of TRH and TRH-like peptides LOW, HIGH and WD CORT suppressed levels of TRH and all TRH-like peptides in pancreatic tissue. Suppression of TRH and TRH-like peptide to very low levels by these same treatments was also observed in testis with two exceptions. Glu-TRH and Phe-TRH were not significantly decreased by low dose CORT. Most CORT treatments also decreased TRH and TRH-like peptides in prostate and epididymis. Val-TRH was increased by low and high CORT and TRH, Val-TRH, and TrpTRH increased with high CORT while withdrawal of high dose CORT suppressed Glu-TRH, Peak 2, TRH, Tyr-TRH, and PheTRH in prostate. Glu-TRH rose with high CORT in epididymis. Interestingly, Tyr-TRH was increased in epididymis by LOW, HIGH and WD CORT (Table 2). The overall total of significant peptide level changes was 78.

3.9. Experiment 2: Effect of s.c. CORT pellets on brain levels of TRH and TRH-like peptides Sustained elevation of serum CORT levels using 1, 2 or 3 s.c. CORT pellets resulted in both significant reductions as well as elevations in TRH and TRH-like peptide levels in various brain regions as seen in Table 3. The overall total of these changes throughout the brain was 64. Ranking brain regions in order of total number of significant changes (either direction) we find that: PCNG had 11 decreases (11 #); STR (8 #); NA (5 #, 3 "); FCX (3 #, 4 "); ACNG (6 #); CBL (5 ", 1 #); AY (2#, 1 "); ENT

(3 "); HC (2 "), PYR (2 "); MED (1 #). Ranking peptides across brain regions according to number of significant changes gave: Glu-TRH (5 #, 5 "); TRH (6 #, 2 "); Val-TRH (5 #, 3"); Tyr-TRH (4 #, 4 "); Leu-TRH (4 #, 1 "), Phe-TRH (4 #, 1 "), TrpTRH (3 #, 1 "), and peak ‘2’ (1 #, 3 ") (Fig. 4).

3.10. Experiment 2: Effect of s.c. CORT pellets on peripheral tissue levels of TRH and TRH-like peptides As for TRH and TRH-like peptide levels in brain, both decreases (30) and increases (7) were observed in peripheral tissues with sustained elevation of serum CORT for a total of 39 significant changes compared to the corresponding control peptide level. The ranking of peptides according to the overall number of significant changes was: Tyr-TRH (4 #, 3 "); Phe-TRH (5 #, 1 "); Glu-TRH (4 #, 2 "); TRH (4 #, 1 "); TrpTRH (4 #); Peak ‘2’ (4 #); Val-TRH (3 #); Leu-TRH (2 #) (Fig. 5).

4. Discussion This is the first report of a comparison of the effects of episodic versus continuous elevation of CORT on the levels of TRH and TRH-like peptides in rat brain. Episodic elevation of serum CORT produced by ingestion of CORT in drinking water produced 93% more significant changes in TRH and TRH-like peptide levels in brain than did a sustained increase produced by s.c. implantation of slow-release CORT pellets. The corresponding increase in peripheral tissues was 111%. This reduction in response sensitivity following pellet implantation is familiar to endocrinologists and can be attributed to down-regulation of the MR and GR receptors that mediate the negative feedback inhibition of prepro-TRH (and TRH-like peptide precursors) gene expression (Pekary et al., 2005).

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone

1191

Figure 2 Effect on levels of TRH and TRH-like peptides in hippocampus of control (0 mg/L), low (25 mg/L), high (400 mg/L) dose CORT or withdrawal (WD) for 2 days after 20 days of high dose CORT in drinking water.

This suggests that using CORT in drinking water as a model for studying the depressogenic effects of this glucocorticoid is not only more convenient but more physiologic than s.c. implantation of slow-release CORT-containing pellets. While not identical, it more closely simulates normal endogenous release than the continuous release paradigm. Genes coding for prepro-TRH, the TRH receptor, and enzymes involved in the biosynthesis of TRH and TRH-like peptides all contain glucocorticoid response elements (Bruhn et al., 1998; Fragner et al., 2001; Hovring et al., 1999; Mains and Eipper, 1984). Glucocorticoids are responsible for maintaining the availability of energy for critical functions such as brain glucose levels and shutting off functions such as reproduction that are not required during an emergency (Gip et al., 2004; Laugero et al., 2002). This shift in metabolic priorities is well-illustrated in previous work, and the present studies, especially the almost quantitative disappearance of all TRH and TRH-like peptides from testis within 4 h of CORT injection (Pekary et al., 2006a,b,c,d) and after 20 days of ingesting CORT in water (Table 2). This rapid clearance may be attributable to accelerated release of these peptides, which has been verified by in vitro studies (Pekary et al., 2007). CORT-responsive enzymes such as glycogen synthase kinase-3b (GSK-3b) regulate microtu-

bule assembly and transport of large dense core vesicles containing various neuropeptides including TRH and TRH-like peptides (Pekary et al., 2007). A contribution of reduced synthesis or increased intracellular degradation to the decline in TRH and TRH-like peptides in epididymis and pancreas after chronic ingestion of CORT, and prostate, following pellet implantation, must be considered since these reductions were not observed following acute i.p. injection of a high dose of CORT (Pekary et al., 2006b). Glucocorticoids stimulate insulin release and carbohydrate feeding (Laugero et al., 2002). Secretion of TRH and insulin by fetal pancreas in organ culture is stimulated by glucose, arginine and depolarization by high potassium. TRH itself stimulates insulin release in perifused rat islets and insulin-secreting clonal b-cell lines (Kulkarni et al., 1995) and this effect of TRH is attenuated by Phe-TRH, the most abundant TRH-like pancreatic peptide (Pekary et al., 2006d). Dexamethasone stimulates TRH gene promoter activity in both primary and neoplastic islet cells (Fragner et al., 2001). The acute fight/flight stress response of Selye is clearly adaptive and required for survival of any organism in a competitive environment. When the stress becomes unremitting, other coping strategies may be more sustainable, but

1192

A.E. Pekary et al.

Figure 3 Effect on levels of TRH and TRH-like peptides in pyiform cortex of control (0 mg/L), low (25 mg/L), high (400 mg/L) dose CORT or withdrawal (WD) for 2 days after 20 days of high dose CORT in drinking water.

often require changes in behavioral, neuroendocrine, biochemical and immune systems. These alterations may become irreversible or damaging to long-term survival and reproductive success even after the threat has passed. A number of human neuropsychiatric disorders, including major depression, bipolar disorder, PTSD, panic disorder, schizophrenia, and obsessive compulsive disorder, have been associated with, and may be precipitated by, a genetic vulnerability to stress (Muller et al., 2002, 2004; Raison et al., 2002). Many aspects of the transition from acute to chronic response to stress and its pathophysiological implications are poorly understood. Clearly, the normal CNS is central to an acute behavioral response, but cognitive, behavioral, hormonal and immune performance decline when the fight/ flight response is unsuccessful. Sustained or repeated elevation of glucocorticoid levels can be damaging to the CNS, particularly the HC which can shrink in size and is associated with the cognitive deficits of prolonged stress. The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are responsible for mediating the acute fight/flight and chronic coping responses, respectively (deKloet and Derijk, 2004), while the GR is responsible for the chromatin remo-

deling and adaptive behavioral response to forced swimming (Chandramohan et al., 2007). These receptors are concentrated in limbic regions including the HC, AY and PVN, CRH neurons, the inhibitory GABA network of the PYR and FCX, and pituitary corticotrophs. The MR has a high affinity for glucocorticoids while the GR has low affinity and is occupied only during severe stress. Chronic alterations of the ratio of MR/GR activity in response to repeated or prolonged stress can have profound and sustained effects, even after removal of the stressful environment. Analyzing the responses to temporal and intensity variations of MR/GR activation requires animal models. Rat strains with high or low vulnerability to long-term effects of stress have been identified (Cohen et al., 2006) and have been utilized for studies of Exaggerated Behavioral Response (EBR) as a model for PTSD. Rats ingesting 25 mg/L CORT hemisuccinate in drinking water for 20 days have been reported to exhibit depressive behavior two weeks after being returned to normal drinking water (Gourley et al., 2006, 2008). Chronic ingestion of 400 mg/L corticosterone produces atrophy of apical dendrites in HC CA3 pyramidal neurons (Magarinos et al., 1998). The importance of the present observations is the implication that glucocorticoid levels, either episodic or

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone

1193

Figure 4 Effect of implanting 0—3 slow-release (21-day) pellets containing 100 mg CORT on TRH and TRH-like peptide levels in nucleus accumbens.

sustained, may alter neuroendocrine-immune system communication and/or metabolism permanently (Yehuda, 2006). We have recently reported that levels of TRH and TRH-like peptides in rat brain and peripheral tissues, particularly the testes, are rapidly altered by a single i.p. injection of 4.0 mg CORT (Pekary et al., 2006b). Because these peptides are neuroprotective, and have antidepressant-like, analeptic and anticonvulsant properties (Pekary et al., 2005), we hypothesized that they could mediate, or modulate not only acute but also chronic central and peripheral tissue responses to altered glucocorticoid regulation or metabolism. We initially replicated, with modification, the original study of Gourley et al. by providing CORT in water for 20 days and withdrawal for 2 days in some high dose-treated rats. The rats receiving low or high dose CORT did not show any elevation of daytime serum CORT levels compared to controls. The serum CORT in the WD group fell significantly, consistent with atrophic adrenal glands taking more than 2 days to regenerate after sustained hyperglucocorticoidemia (Hodges and Sadow, 1969; Hodges and Mitchley, 1970; Loose et al., 1980). For this reason we carried out Experiment 2, involving the s.c. implantation of slow-release CORT-containing pellets which guaranteed a sustained (day and night) elevation of serum CORT within the 20-day release period. A withdrawal experiment was also planned but proved impossible because

the implanted pellets disintegrated in vivo and could not be removed quantitatively without an unacceptable amount of trauma. A trauma-free withdrawal experiment that would allow a study of the in vivo effects of a transition from high sustained serum CORT to low levels on TRH and TRH-like peptides suggested itself. Why not follow the serum CORT levels in rats fed high levels of CORT in water on a daily basis? Once a high steady-state level of serum CORT had been achieved, time the decapitations to just before and after the withdrawal-induced fall-off in serum CORT. As seen in Fig. 1, this experiment was not possible because high sustained levels of serum CORT were not observed. This experiment was repeated at medium (100 mg/L) and low (25 mg/L) doses but, again, sustained elevation of CORT in serum was not observed during daytime sampling. The early peak of serum CORT (Fig. 1A—C) is unlikely to be due to a transition from low to high liver clearance of CORT since a similar experiment has previously shown that CORT clearance actually slows in animals given CORT in drinking water for 20 days (Pung et al., 2003). This early peak of serum CORT may be due to ingestion of CORT-containing drinking water about 2 h before tail vein bleeding (Fig. 1D). The rise in serum CORT from day 6 to day 13 may be due to the reported slowing of CORT clearance with repeated ingestion (Pung et al., 2003). Other possibilities include a reduction in nega-

1194

A.E. Pekary et al.

Figure 5 Effect of implanting 0—3 slow-release (21-day) pellets containing 100 mg CORT on TRH and TRH-like peptide levels in epididymis.

tive feedback inhibition of CRH release due to involution of the HC (McEwen, 2005). Glucocorticoids prime the innate immune response, suppress cellular (Th1) immunity and promote humoral (Th2) immunity (Franchimont, 2004). Glucocorticoids are elevated in most cases of severe depression, bipolar disorder and other neuropsychiatric illnesses (McEwen, 2005). This can desensitize glucocorticoid receptors, elevate proinflammatory cytokines and increase prostaglandins (Lenard, 2001; Beilin et al., 2006; Uz et al., 1999; Zakar et al., 1995). Hypersecretion of glucocorticoids and proinflammatory cytokines result in malfunction of noradrenergic and serotoneric neurotransmission in brain (Berendsen et al., 1996; Wei et al., 2004). On the other hand, antidepressants, including TRH, moderate proinflamatory cytokine release, inhibit cyclooxygenases and modulate release of IL-10, an IL-1 receptor antagonist (Kubera et al., 2000; Lenard, 2001). These observations suggest that chronic ingestion of CORT by rats may offer a useful model for studies on depression, at low CORT doses (and PTSD at high doses), given the greater effect of intermittent ingestion of CORT on TRH and TRH-like peptide levels in brain compared to sustained serum CORT levels following s.c. pellet implantation. Finally, it should be mentioned that TRH is present throughout the CNS as a cotransmitter within glutamatergic neurons (Review, Sattin, 1999). Release, action and catabo-

lism of TRH is analogous to that for acetylcholine, i.e., receptor activation followed by metabolism by a specific membrane bound enzyme (Heuer et al., 1998, 2000). However, to date no TRH receptor antagonists have been found. Of the TRH-like peptides, only the endogenous pGlu-Glu-ProNH2 has a documented receptor, and only on the sperm (Adeoya-Osiguwa et al., 1998, and see Hinkle et al., 2002 for negative results). The molecular precursors of the TRHlike peptides have not yet been determined (their sequence is not found in prepro-TRH; work in progress). Their CNS functions are unknown, although they do increase TRH levels in vivo by inhibiting the major TRH-degrading enzyme, pyroglutamate aminopeptidase II (Scalabrino et al., 2007) and may play a role in the modulation of TRH receptor activity (Engel et al., 2006; Monden et al., 1995). The localization of the TRH-like peptides in glutamatergic neurons is likely to be the same as TRH itself. When the initial histochemistry was done (Hokfelt et al., 1975; Low et al., 1989; Kubek et al., 1989) TRH-like peptides were not known to occur in brain. However, we now know (Pekary et al., 2005) that most TRH antisera cross-react with other TRH-like peptides as well. In summary, 20 days of ingestion of low (25 mg/L) or high (400 mg/L) CORT in drinking water reduced (and sometimes increased) TRH and TRH-like peptide levels in brain and peripheral tissues of male SD rats more consistently than s.c. implantation of 1—3 pellets containing 100 mg of slow

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone (21-day) release CORT. Serum levels of CORT sampled daily from the tail vein of unanesthetized rats drinking low, medium (100 mg/L), or high CORT for 15—20 days rose to a peak value of 320 ng/ml on day 6, 1200 ng/ml on day 7, and 25,000 ng/ml on day 4, respectively. This early rise in CORT levels is most likely due to sampling about 2 h after ingestion of CORT-containing water. A lower increase in serum CORT to 450 ng/ml at day 12 in rats drinking high-dose CORT may be due to a slowing of CORT metabolism, and/or reduction of negative feedback inhibition of CRH release. The particular tissues with levels of TRH/TRH-like peptide most affected by CORT in water were often different from those most affected by slow-release pellets. This contrast is most apparent in a comparison of peripheral tissues. Almost all TRH and TRH-like peptides in the pancreas and testis fell more than 90% following 20 days of HIGH and WD CORT in water. On the other hand, these peptides were more consistently suppressed in prostate and epididymis of rats with 1—3 pellets, with the following major exception: In the epididymis a marked increase in Tyr-TRH for 1—3 pellets was observed along with raised TRH with one pellet. We conclude that episodic ingestion of CORT in drinking water by rats minimizes the down regulation of glucocorticoid receptors that attends the continuous release of CORT from slow release pellets. Adding CORT to drinking water is thus a convenient as well as more realistic model for studying the pathophysiological effects of hyperglucocorticoidemia.

Role of funding source Funding for this study was provided by Department of Veterans Affairs (DVA); DVA had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Acknowledgements This work was supported by the Department of Veterans Affairs (A.E.P. and A.S.). The authors thank Fredericka Martin, Ph.D., for suggesting Experiment 4.

Conflict of interest There are no financial, personal or other relationships with other people or organizations within three (3) years of beginning the work submitted that could inappropriately influence, or be perceived to influence, this work.. AS and AEP are coauthors, along with Robert Lloyd, Associate Professor of Psychiatry, University of Minnesota, Duluth, of a patent for the use of TRH-like peptides in the treatment of neuropsychiatric disorders. Any financial benefit from this patent will belong to the Department of Veterans Affairs (DVA) since this agency supported the experimental basis for this patent.

References Adeoya-Osiguwa, S.A., Dudley, R.K., Hosseini, R., Fraser, L.R., 1998. FPP modulates mammalian sperm function via TCP-11

1195

and the adenylyl cyclase/cAMP pathway. Mol. Reprod. Dev. 51, 468—476. Beilin, B., Shavit, Y., Dekeyser, F., Itzik, A., Weidenfeld, J., 2006. The involvement of glucocorticoids and interleukin-1 in the regulation of brain prostaglandin production in response to surgical stress. Neuroimmunomodulation 13, 36—42. Berendsen, H.H., Kester, R.C., Peeters, B.W., Broekkamp, C.L., 1996. Modulation of 5-HT receptor subtype-mediated behaviors by corticosterone. Eur. J. Pharmacol. 308, 103—111. Bruhn, T.O., Huang, S.S., Vaslet, C., Nillni, E.A., 1998. Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (pro-TRH). Endocrine 9, 143—152. Bush, V.L., Middlemiss, D.N., Marsden, C.A., Fone, K.C., 2003. Implantation of a slow release corticosterone pellet induces long-term alterations in serotonergic neurochemistry in the rat brain. J. Neuroendocrinol. 15, 607—613. Chandramohan, Y., Droste, S.K., Arthur, S., Reul, J.M.H.M., 2007. The forced swimming-induced behavioral immobility response requires chromatin remodeling and transcriptional induction in dentate gyrus neurons via concurrent activation of GR and NMDA/ MAPK/ERK/MSK signaling. Society for Neuroscience, Annual Meeting, Abst. 841.4. Cohen, H., Zohar, J., Gidron, Y., Matar, M.A., Belkind, D., Loewenthal, U., Kozlovsky, N., Kaplan, Z., 2006. Blunted HPA axis response to stress influences susceptibility to posttraumatic stress response in rats. Biol. Psychiatry 59, 1208—1218. deKloet, E.R., Derijk, R., 2004. Signaling pathways in brain involved in predisposition and pathogenesis of stress-related disease: genetic and kinetic factors affecting the MR/GR balance. Ann. N.Y. Acad. Sci. 1032, 14—34. Engel, S., Neumann, S., Kaur, N., Monga, V., Jain, R., Northup, J., Gershengorn, M.C., 2006. Low affinity analogs of thyrotropinreleasing hormone are super-agonists. J. Biol. Chem. 281, 13103— 13109. Fernandes, C., McKittrick, C.R., File, S.E., McEwen, B.S., 1997. Decreased 5-HT1A and increased 5-HT2A receptor binding after chronic corticosterone associated with a behavioral indication of depression. Psychoneuroendocrinology 22, 477—491. Fluttert, M., Dalm, S., Oitzl, M.S., 2000. A refined method for sequential blood sampling by tail incision in rats. Lab. Anim. 34, 372—378. Fragner, P., Lee, S.L., Aranta de Leon, S., 2001. Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets. J. Endocrinol. 170, 91—98. Franchimont, D., 2004. Overview of the actions of glucocorticoids on the immune response: a good model to characterize new pathways of immune-suppression for new treatment strategies. Ann. N.Y. Acad. Sci. 1024, 124—137. Gartside, S.E., Leitch, M.M., McQuade, R., Swarbrick, D.J., 2003. Flattening the glucocorticoid rhythm causes changes in hippocampal expression of messenger RNAs coding structural and functional proteins: implications for aging and depression. Neuropsychopharmacology 28, 821—829. Gip, P., Hagiwara, G., Sapolsky, R.M., Cao, V.H., Heller, C., Ruby, N.F., 2004. Glucocorticoids influence brain glycogen levels during sleep deprivation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R1057—R1062. Gourley, S.L., Kiraly, D.D., Taylor, J.R., 2006. Modeling the persistent, depressive-like state in rodents: long-lasting effects of prior chronic corticosterone. Biol. Psychiatry 59, 6S (Abst.# 15). Gourley, S.L., Wu, F.J., Kiraly, D.D., Ploski, J.E., Kedves, A.T., Duman, R.S., Taylor, J.R., 2008. Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression. Biol. Psychiatry 63, 353—359. Heuer, H., Ehrchen, J., Bauer, K., Schafer, M.K., 1998. Regionspecific expression of thyrotrophin-releasing hormone-degrading ectoenzyme in the rat central nervous system and pituitary gland. Eur. J. Neurosci. 10, 1465—1478.

1196 Heuer, H., Schafer, M.K., O’Donnell, D., Walker, P., Bauer, K., 2000. Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats. J. Comp. Neurol. 428, 319— 336. Hinkle, P.M., Pekary, A.E., Senanayaki, S., Sattin, A., 2002. Role of TRH receptors as possible mediators of analeptic actions of TRHlike peptides. Brain Res. 935, 59—64. Hodges, J.R., Mitchley, S., 1970. Recovery of hypothalamo-pituitaryadrenal function in the rat after prolonged treatment with betamethasone. Br. J. Pharmacol. 40, 732—739. Hodges, J.R., Sadow, J., 1969. Hypothalamo-pituitary-adrenal function in the rat after prolonged treatment with cortisol. Br. J. Pharmacol. 36, 489—495. Hokfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S., White, N., 1975. Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry. Eur. J. Pharmacol. 34, 389—392. Hovring, P.I., Matre, V., Fjeldheim, A.K., Loseth, O.P., Gautvik, K.M., 1999. Transcription of the human thyrotropin-releasing hormone receptor gene-analysis of basal promoter elements and glucocorticoid response elements. Biochem. Biophys. Res. Commun. 257, 829—834. Huang, G.J., Herbert, J., 2006. Stimulation of neurogenesis in the hippocampus of the adult rat by fluoxetine requires rhythmic change in corticosterone. Biol. Psychiatry 59, 619—624. Kubek, M.J., Low, W.C., Sattin, A., Morzorati, S.L., Meyerhoff, J.L., Larsen, S.H., 1989. Role of TRH in seizure modulation. Ann. N.Y. Acad. Sci. 553, 286—303. Kubera, M., Kenis, G., Bosmans, E., Jaworska-Feil, L., Lason, W., Scharpe, S., Maes, M., 2000. Suppressive effect of TRH and imipramine on human interferon-gamma and interleukin-10 production in vitro. Pol. J. Pharmacol. 52, 481—486. Kulkarni, R.N., Wang, Z.L., Akinsanya, K.O., Bennet, W.M., Wang, R.M., Smith, D.M., Ghatei, M.A., Byfield, P.G., Bloom, S.R., 1995. Pyroglutamyl-phenylalanyl-proline amide attenuates thyrotropin-releasing hormone-stimulated insulin secretion in perifused rat islets and insulin-secreting clonal beta-cell lines. Endocrinology 136, 5155—5164. Laugero, K.D., Gomez, F., Manalo, S., Dallman, M.F., 2002. Corticosterone infused intracerebroventricularly inhibits energy storage and stimulates the hypothalamo-pituitary axis in adrenalectomized rats drinking sucrose. Endocrinology 143, 4552—4562. Leitch, M.M., Ingram, C.D., Young, A.H., McQuade, R., Gartside, S.E., 2003. Flattening the corticosterone rhythm attenuates 5HT1A autoreceptor function in the rat: relevance for depression. Neuropsychopharmacology 28, 119—125. Loose, D.S., Do, Y.S., Chen, T.L., Feldman, D., 1980. Demonstration of glucocorticoid receptors in the adrenal cortex: evidence for a direct desamethasone suppressive effect on the rat adrenal gland. Endocrinology 107, 137—146. Low, W.C., Farber, S.D., Hill, T.G., Sattin, A., Kubek, M.J., 1989. Evidence for extrinsic and intrinsic sources of TRH in the hippocampal formation, as determined by radioimmunoassay and Immunocytochemistry. Ann. N.Y. Acad. Sci. 553, 574—578. Magarinos, A.M., Orchinik, M., McEwen, B.S., 1998. Morphological changes n the hippocampal CA3 region induced by non-invasive glucocorticoid administration: a paradox. Brain Res. 809, 314— 318. Mains, R.E., Eipper, B.A., 1984. Secretion and regulation of two biosynthetic enzyme activities, peptidyl-glycine alpha-amidating monooxygenase and a carboxypeptidase, by mouse pituitary corticotropic tumor cells. Endocrinology 115, 1683—1690. McEwen, B.S., 2005. Glucocorticoids, depression, and mood disorders: structural remodeling in the brain. Metabolism 54 (5 Suppl 1), 20—23. Meijer, O.C., Van Oosten, R.V., De Kloet, E.R., 1997. Elevated basal trough levels of corticosterone suppress hippocampal 5-hydro-

A.E. Pekary et al. xytryptamine(1A) receptor expression in adrenally intact rats: implication for the pathogenesis of depression. Neuroscience 80, 419—426. Monden, T., Mizuma, H., Yamada, M., Murakami, M., Mori, M., 1995. A novel analog of TRH, YM14673, causes a decrease in brain TRH receptors in vitro. Endocr. Res. 21, 803—814. Muller, M.B., Keck, M.E., Steckler, T., Holsboer, F., 2002. Genetics of endocrine-behavior interactions. In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrback, S.E., Rubin, R.T. (Eds.), Hormones, Brain and Behavior, vol. 5Academic Press, pp. 263—301 (Chapter 91). Muller, M.B., Uhr, M., Holsboer, F., Keck, M.E., 2004. Hypothalamicpituitary-adrenocortical system and mood disorders: highlights from mutant mice. Neuroendocrinology 79, 1—12. Pekary, A.E., Faull, K.F., Paulson, M., Lloyd, R.L., Sattin, A., 2005. TRH-like antidepressant peptide, pyroglutamyltrosylprolineamide, occurs in rat brain. J. Mass Spectr. 40, 1232—1236. Pekary, A.E., Sattin, A., 2001. Regulation of TRH and TRH-like peptides in rat brain by thyroid and steroid hormones. Peptides 22, 1161—1173. Pekary, A.E., Sattin, A., Meyerhoff, J.L., Chilingar, M., 2004. Valproate modulates TRH receptor. TRH and TRH-like peptide levels in rat brain. Peptides 25, 647—658. Pekary, A.E., Sattin, A., Stevens, S.A., 2006a. Rapid modulation of TRH-like peptides in rat brain by thyroid hormones. Peptides 27, 1577—1588. Pekary, A.E., Senanayake, S., Sattin, A., 2002. Cocaine regulates TRH-related peptides in rat brain. Neurochem. Int. 41, 415—428. Pekary, A.E., Stevens, S.A., Sattin, A., 2006b. Rapid modulation of TRH and TRH-like peptide levels in rat brain and peripheral tissues by corticosterone. Neurochem. Int. 48, 208—271. Pekary, A.E., Stevens, S.A., Sattin, A., 2006c. Circadian rhythms of TRH-like peptides in rat brain. Brain Res. 1125, 67—76. Pekary, A.E., Stevens, S.A., Sattin, A., 2006d. Valproate and copper accelerate TRH-like peptide synthesis in male rat pancreas and reproductive tissues. Peptides 27, 2901—2911. Pekary AE, Stevens SA, Sattin A. Rapid modulation of TRH and TRHlike peptide levels in rat brain and peripheral tissues by a GSK3beta inhibitor. Program No. 98.14, 2007. Society for Neuroscience, Annual Meeting, San Diego, CA. Pung, T., Zimmerman, K., Klein, B., Ehrich, M., 2003. Corticosterone in drinking water: altered kinetics of a single oral dose of corticosterone and concentrations of plasma sodium, albumin, globulin, and total protein. Toxicol. Ind. Health 19, 171—182. Raison, C.L., Gumnick, J.F., Miller, A.H., 2002. Neuroendocrine— immune interactions: implications for health and behavior. Chapter 90 In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrback, S.E., Ru bin, R.T. (Eds.), Hormones, Brain and Behavior. Academic Press, pp. 209—261. Sattin, A., 1999. The role of TRH and related peptides in the mechanism of action of ECT. J. ECT 15, 76—92. Scalabrino, G.A., Hogan, N., O’Boyle, K.M., Slator, G.R., Gregg, D.J., Fitchett, M., Draper, S.M., Bennett, G.W., Hinkle, P.M., Bauer, K., Williams, C.H., Tipton, K.F., Kelly, J.A., 2007. Discovery of a dual action firt-in-class peptide that mimics and enhances CNSmediated actions of thyrotropin-releasing hormone. Neuropharmacology 52, 1472—1481. Solberg, L.C., Olson, S.L., Turek, F.W., Redei, E., 2001. Altered hormone levels and circadian rhythm of activity in the WKY rat, a putative animal model of depression. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 281, R786—R794. Uz, T., Dwivedi, Y., Savani, P.D., Impagnatiello, F., Pandey, G., Manev, H., 1999. Glucocorticoids stimulate inflammatory 5-lipoxygenase gene expression and protein translocation in the brain. J. Neurochem. 73, 693—699. Wei, Q., Lu, X.Y., Liu, L., Schafer, G., Shieh, K.R., Burke, S., Robinson, T.E., Watson, S.J., Seasholtz, A.F., Akil, H., 2004. Glucocorticoid receptor overexpression in forebrain: a mouse

TRH and TRH-like peptide expression in rat following episodic or continuous corticosterone model of increased emotional lability. Proc. Natl. Acad. Sci. 101, 11851—11856. Yehuda, R., 2006. Advances in understanding neuroendocrine alterations in PTSD and their therapeutic implications. Ann. N.Y. Acad. Sci. 1071, 137—166. York, J.L., 1981. Consumption of intoxicating beverages by rats and mice exhibiting high and low preferences for ethanol. Pharmacol. Biochem. Behav. 15, 207—214.

1197

Zakar, T., Hirst, J.J., Mijovic, J.E., Olson, D.M., 1995. Glucocorticoids stimulate the expression of prostaglandin endoperoxidase H synthase-2 in amnion cells. Endocrinology 136, 1610— 1619. Zeng, H., Schimpf, B.A., Rohde, A.D., Pavlova, M.N., Gragerov, A., Bergmann, J.E., 2007. Thyrotropin-releasing hormone receptor 1-deficient mice display increased depression and anxiety-like behavior. Mol. Endocrinol. 21, 2795—2804.