Transcriptional regulation of hippocampal 5-HT1a receptors by corticosteroid hormones

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular

Brain Research 29 (1995) 23-34

Research

Transcriptional

report

regulation of hippocampal SHT,, by corticosteroid hormones Pingyu Zhong

*, Roland

receptors

D. Ciaranello

Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Stanford Unioersity Medical Center, Stanford, CA 94305-5485, USA

Accepted 27 September 1994

Abstract S-HT,, receptors in the hippocampus play a critical role in modulating limbic system output. The activity and level of SHT,, receptors are modulated by glucocorticoid levels. The present study was undertaken to test the hypothesis that glucocorticoids attenuate the transcriptional activity of the SHT,, receptor gene. Using in situ hybridization and RNase protection assays, we

observed a substantial

increase in 5-HT,,

mRNA expression

after adrenalectomy

in the same hippocampal

regions in which

SHT,, binding sites are increased. This increase in SHT,, mRNA expression occurs as early as 1 h after adrenalectomy and precedes the increase in receptor binding sites. Further in situ hybridization analysis showed that SHT,, mRNA is increased within individual hippocampal cells after adrenalectomy. Administration of dexamethasone completely prevents the adrenalectomy-induced elevation in hippocampal SHT,, receptor mRNA. Nuclear run-on assays showed that the rate of transcription of SHT,. mRNA after adrenalectomy increased 70% above the rate from control preparations and could be reduced to basal levels by the administration of dexamethasone. Adrenalectomy did not cause an increase in functional coupling of 5-HT,, receptors to adenylyl cyclase or phospholipase C. These results suggest that transcription of hippocampal 5-HT,, receptor mRNA is under negative regulation by corticosteroid hormones. Keywords: Serotonin Transcription

5-HT,,

receptor;

Hippocampus;

Corticosteroid

1. Introduction

Serotonin (5hydroxytryptamine, 5-HT) has been implicated as a neurotransmitter in a number of human and animal behaviors. Some of these behaviors, such as mood, the response to stress, depression, anxiety and neuroendocrine responses show striking similarity to the biological effects of corticosteroids on the brain. In animal models, both 5-HT and corticosteroids modulate certain behaviors in common, including exploratory activity and aggression. Several lines of evidence have indicated a regulatory role for corticosteroids at various points in serotonergic neurotransmis-

Abbreviations: 5-HT, 5-hydroxytryptamine; CRH, corticotropin releasing hormone; ACTH, corticotropin; 8-OH-DPAT, 8-hydroxy-2(di-n-propylamino)tetralin. * Corresponding author. Present address: Neurex Inc., 3760 Haven Avenue,

Menlo

Park, CA 94025-1012,

0169-328X/95/$09.50 0 1995 Elsevier SSDI 0169-328X(94)00225-8

USA. Fax: (1) (415) 853-1538. Science

B.V. AI1 rights reserved

hormone;

Corticosteroid

receptor;

Receptor

regulation;

sion in the CNS. Corticosteroids have been reported to influence the activity of tryptophan hydroxylase, the initial enzyme in serotonin synthesis; serotonin uptake and turnover at nerve terminals [7,18,361. Corticosteroids also appear to modulate the level of [3H]5-HT binding in certain brain regions. Removal of [ 3H]5-HT corticosteroids by adrenalectomy increases binding in hippocampus; this effect is reversed by glucocorticoid administration [18]. Although [3H]5-HT binds to at least fourteen different serotonin receptor subtypes, the changes in [3H]5-HT binding induced by adrenalectomy and corticosteroids appear to reflect principally changes in the 5-HT,, receptor subtype [13,34]. Several groups have shown that 5-HT,, receptor binding sites in rat hippocampus are elevated following adrenalectomy [10,12,13,18,32,34,35]. This effect, monitored through quantitative receptor autoradiography, occurs as early as 1 h after removal of the adrenal glands and is reversed by corticosteroid administration [18]. Adrenalectomy also increases the level of 5-HT,, mRNA (using in situ hybridization and North-

24

P. Zhong, R.D. Ciaranello /Molecular

ern analysis) in the same regions 112,131, but the mechanism underlying this change is undetermined. The goal of the present study was to examine the mechanism underlying corticosteroid modulation of 5HT,, receptor activity in the hippocampus. The results described here demonstrate that both adrenalectomy and hypophysectomy significantly increase the B,,, of 5-HT,, receptor binding sites in the hippocampus. Administration of ACTH reverses increase in 5-HT,, binding after hypophysectomy. Hippocampal 5-HT,, mRNA increases by 70% following adrenalectomy, while 5-HT,, radioligand binding increases more than 40%. The increase in hippocampal 5-HT,, mRNA occurs as early as 1 h after adrenalectomy and precedes the increase in 5-HT,, binding. The increase in 5-HT,, mRNA is observed in discrete hippocampal neurons, and does not appear to involve recruitment of cells which previously did not express 5-HT,, receptors. Administration of dexamethasone to adrenalectomized rats reverses the increase in 5-HT,, mRNA, although this treatment does not reduce 5-HT,, mRNA below basal levels, nor does dexamethasone administration to sham-operated animals reduce 5-HT,, mRNA levels. The transcription rate of hippocampal 5-HT,, mRNA is increased by 70% after adrenalectomy and declines to basal levels after dexamethasone administration. Functional coupling of 5-HT,, receptors to adenylyl cyclase and phosphoinositol hydrolysis do not increase after adrenalectomy. These results suggest that corticosteroids normally exert a negative regulatory effect on hippocampal 5-HT,, receptor expression by attenuating transcription of the receptor gene.

2. Materials 2. I. Animals

and methods

and tissue collection

Male Sprague-Dawley rats weighing from 200-230 g (Simonsen Labs, Gilroy, CA) were used for most experiments. Animals were housed 3 per cage one week before surgery under standard light (on from 06.00 to 20.00 h) and temperature (23°C) conditions. Food and water were given ad libitum. Before surgery, animals were anesthesized by halothane. The adrenals were removed bilaterally via a dorsal approach. Sham-operated control animals were subject to the same procedure except the adrenals were not removed. Those animals receiving glucocorticoid replacement treatment were administered a single dose of dexamethasone subcutaneously, as designated. Control animals were given an equal volume of saline. Animals were sacrificed by decapitation 1 h. 1, 2, 3, 5 and 7 days after surgery. In some experiments, hypophysectomized rats (male Sprague-Dawley) were also purchased from Simonsen Labs. These animals were treated with ACTH or vehicle (i.p.1 and decapitated 3 weeks after hypophysectomy. The entire brain was rapidly removed from the skull, frozen on dry-ice, and stored at -80°C for preparation of slide-mounted coronal brain sections. The hippocampus and other brain regions were dissected, frozen on dry-ice and stored at -80°C for preparation of total RNA or nuclei.

Brain Research 29 (199.7) 23-34 2.2. Receptor binding assay Frozen hippocampi were homogenized in 10 mM Tris-HCI buffer (pH 7.6) and washed twice by pelleting at 35,000X g, 4°C for 10 min each. Final pellets were resuspended in incubation buffer (50 mM Tris-HCI, pH 7.6, 0.1% ascorbic acid, 4 mM CaClz and 10 /IM pargyline). Aliquots of the membranes were incubated with [‘H]8OH-DPAT in the presence and in the absence of 10 PM 5-HT. The incubation was carried out at 25°C for 40 min and stopped by filtration through glass filters using a Brandel Cell Harvester (M-48R) followed by washing three times in ice-cold 50 mM Tris-HCI buffer (pH 7.6). The filters were then dried and counted by liquid scintillation spectroscopy. 2.3. Quantitati1.e in vitro receptor autoradiography Twenty pm slide-mounted brain sections were preincubated in 0.17 M Tris-HCI, pH 7.6, containing 4 mM CaClz and 0.1% ascorbic acid for 30 min at room temperature. The sections were incubated with 2 nM [3H]8-OH-DPAT in the same buffer for 60 min at room temperature. They were then washed in 2 changes of the same buffer at 4°C and air dried. Non-specific binding to adjacent tissue sections was determined in the presence of 2 PM 5-HT. The dried sections were exposed to [‘HI-sensitive Hyperfilm (Amersham) at room temperature. Autoradiograms were scanned using the computerized image analysis system that was pre-calibrated for optical density measurement. The amount of specific binding was obtained as O.D. values and averaged from 5-6 coronal brain sections. 2.4. Preparation

of total RNA

Brain tissues were homogenized separately in 1 ml of ice-cold 4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.2% sodium N-laurosylsarcosine, 0.2 M P-mercaptoethanol. Total RNA was purified on a discontinuous CsCl, gradient and quantitated by measuring the optical density at 260 nm. 2.5. Preparation

of SHT,,

and p-actin

[‘5S]riboprobes

A 0.9 kb BamHI/PstI fragment of the rat genomic 5-HT,, clone in pGEM-blue was obtained from Dr. 0. Civelli (Vellum Institute, Portland, OR). This fragment contains the 5-HT,, coding sequence from the second transmembrane domain to the middle of the third intracellular loop of the receptor. A 0.45 kb subclone of /3-actin cDNA (pActDS1 was obtained from Dr. D.L. Wong of Stanford University. Antisense riboprobes for each cDNA were generated by in vitro transcription using T, RNA polymerase in the presence of [“SIUTP (1200 Ci/mMol; NEN) after linearizing the 5-HT,,, plasmid DNA with Hind111 and the p-actin plasmid DNA with Asp 718. The transcription procedure was based on the manufacturer’s (Stratagene, La Jolla, CA) instructions. 2.6. In sttu hybridization Ten mm slide-mounted brain sections were fixed in 4% formaldehyde (v/v) for 60 min at room temperature. They were then rinsed in isotonic phosphate buffered saline (2x5 min) and treated with proteinase K (I pg/ml in 100 mM Tris-HCI, pH 8.01 for 10 min at 37°C. The sections were successively rinsed in water (1 min), 0.1 M triethanolamine (pH 8.01, 0.25% acetic anhydride for 10 min and 2xSSC (0.3 M NaCl. 0.03 M NaCitrate, pH 7.2) for 5 min. They were then dehydrated through graded alcohols and air dried. The fixed sections were hybridized with a [“S]UTP labeled 5-HT,, riboprobe (1 X lo6 dpm) in a volume of approximately 50 ~1 hybridization buffer (75% formamide, 10% dextran sulfate. 3 X SSC. 50

P. Zhong, R.D. Ciaranello /Molecular mM Na phosphate buffer, pH 7.0, 1 X Denhardt’s solution, 0.1 mg/ml yeast tRNA, 0.1 mg/ml sheared salmon sperm DNA and 10 mM dithiothreitol). After overnight hybridization at SYC, the sections were washed in 2xSSC for 5 min and treated with RNAse A (200 fig/ml in 10 mM Tris-HCI, pH 8, 0.5 M NaCl) for 30 min at 37°C. They were then washed in 2XSSC for 10 min, 1 XSSC for 10 min, 0.5 XSSC for 60 min at 55°C and 0.5 X SSC for 10 min at room temperature. Finally, the sections were dehydrated in graded alcohols and air dried. To detect 5-HT,, mRNA hybridization, brain sections were placed on Kodak XAR-5 X-ray film and exposed for 3 days at room temperature. For microscopic examination of 5-HT,, mRNA density over individual neurons, the brain sections were then dipped in Kodak NTB-2 emulsion and stored dessicated in light tight boxes at 4°C for 10 days before development. They were then processed for Nissl-staining.

2.7. Solution hybridization /RNase protection assay For the hybridization reaction, purified total RNA was ethanol co-precipitated with [‘5S]5-HT,,, or [35S]/3-actin riboprobes plus 25 pg yeast tRNA, and then resuspended in 20 ml hybridization buffer (40 mM MOPS, pH 6.7, 400 mM NaCI, 2 mM EDTA, 30 mM DTT, 80% deionized formamide). After heat denaturation at 85°C for 5 min, hybridization was carried out overnight in a 58°C waterbath. The mixture was then treated with 10 pg/ml RNase A (Pharmacia, Piscataway, NJ) and 2 U/ml RNase Tl (Boehringer Mannheim, Indianapolis, IN) in 300 yl RNase buffer (10 mM Tris-HCI, pH 7.5, 300 mM NaCl, 2 mM EDTA) at 25°C for 1 h. The reaction was stopped by adding 20 ~1 10% SDS and 10 ~1 10 mg/ml proteinase K (BRL, Bethesda, MD) followed by incubation at 37°C for 30 min. The mix was extracted in phenol-chloroform and RNA hybrids were precipitated in ethanol with 15-20 pg yeast tRNA. The recovered RNA was dissolved in 1.5 ~1 loading dye (100 mM Tris-HCI, 100 mM boric acid, pH 8.0, 80% deionized formamide, 0.1% Bromophenol blue, 0.1% xylene cyanol, 0.1% SDS) and fractionated on a 4% denaturing polyacrylamide gel (7 M urea, 0.4 mm thick) in running buffer (100 mM Tris/HCI, 100 mM boric acid, pH 8.0) at 1500 V for 3 h. The gel was vacuum-dried on Whatman 3 MM paper and exposed to Kodak X-ray film for 2 days at room temperature. The protected fragments of 5-HT,, and p-actin mRNA bands were quantitated by computerized densitometry. Readings of 5-HT,, mRNA were normalized to those of p-actin mRNA. Positive (sense RNA instead of tissue RNA) and negative (no RNA) controls were included in each experiment. The linearity range of the autoradiograms was calibrated by hybridization of 5-HT,, sense RNA (0.1 pg-100 ng) and p-actin sense RNA (0.01-l ng) to their corresponding [35S]riboprobes.

2.8. Nuclear run-on assays 2.8.1. Nuclei preparation Nuclei from frozen hippocampi were prepared as described previously [33]. Briefly, hippocampi were put into buffer I (0.32 M sucrose, 3 mM CaCl,, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol, 0.25% Triton X-100 and 10 mM Tris, pH 8.0) and gently homogenized on ice following mixing with an equal volume of buffer II (2.2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol and 10 mM Tris, pH 8.0). The isolated nuclei were then fractionated through a cushion of buffer II by ultracentrifugation in a Beckman Model u-70 ultracentrifuge (SW 28 rotor, 25,000 rpm, 4°C for 1 h). The nuclear pellets were then washed once with storage buffer (25% glycerol, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM dithiothreitol and 50 mM Tris, pH 8.0) and resuspended in storage buffer to a final DNA concentration of approximately 1 mg/ml). The integrity of the nuclei was confirmed by

25

Brain Research 29 (1995) 23-34 inspection under light microscopy at 25 xmagnification. ml each) of nuclei were stored at -80°C until use.

Aliquots

(50

2.8.2. Transcription run-on assays Each reaction mixture contained: 0.5 mM ATP, GTP and CTP, 0.6 PM cold UTP, 100 PCi [32P]UTP (3000 Ci/mmol), 0.12 M KCI, 2.5 mM magnesium acetate. Transcription was initiated by adding a 50 ~1 aliquot of nuclei to the reaction mix and incubating at 35°C for a designated period. Labeled RNA was then treated with proteinase K and RNase-free DNase I twice and extracted in phenol-chlorform. 2.8.3. Slot blot hybridization Pellets of labeled RNA were dissolved in hybridization buffer solution, 50 mM (50% deionized formamide, 5 x SSC, 5 x Denhardt’s sodium phosphate, pH 6.8, 0.05% SDS, 100 pg/ml of yeast tRNA and salmon sperm DNA) and hybridized with 10 pg of 5-HT,, cDNA, p-actin cDNA or the corresponding vector plasmid DNAs previously immobilized on a Duralon-VU membrane slot blot (52°C for 40 h). The slot blots were then washed three times in 1 x SSC, 0.5% SDS at 65°C and once in 0.1 X SSC, 0.5% SDS at 60°C (20 min each). They were then air-dried before exposure to X-ray film. 5-HT,, mRNA hybridization was quantitated by determining the O.D. value of each band and normalized relative to the optical density of the p-actin control band. 2.9. Data analysis Autoradiograms of 5-HT,, binding sites, 5-HT,, mRNA protected bands and slot blot hybridization were analyzed through computer-assisted densitometry (Image 1.52, National Institutes of Health) using a Sony (CCD Video Camera Module) video camera connected to a Macintosh IIfx computer. Values of optical density (O.D.) represented the relative levels of _5-HT,, binding sites and mRNA. The signals of 5-HT,, mRNA detected from the RNase protection or nuclear run-on assay were always normalized to those of p-actin mRNA. Final data are expressed as the mean of O.D. values from 5-8 animals. The level of statistical significance in all studies was determined by ANOVA followed by a Scheffer’s post-hoc analysis. 2.10. Materials [“H&OH-DPAT (150 Ci/mmol), [35S]aUTP (1200 Ci/mmol), [32P]aUTP (3000 Ci/mmol) and [“*P]ATP (800 Ci/mmol) were purchased from New England Nuclear Co. (Boston, MA). Myo[3H]inositol was purchased from American Radiolabeled Chemicals, Inc. (St Louis, MO). Dexamethasone was purchased from Merck (Rahway, NJ). All restriction enzymes, T, and T, RNA polymerases and Duralon-UV membrane were purchased from Stratagene (La Jolla, CA). RNase A and proteinase K were purchased from Boehringer Mannheim (Indianapolis, IN). RNase T, and ribonucleotides were purchased from BRL (Gaithersburg, MD).

3. Results 3.1. SHT,, receptor binding after adrenalectomy hypophysectomy

and

To confirm the increase in SHT,, receptor binding sites in response to adrenalectomy reported previously, we examined the binding of [3H]8-OH-DPAT to hip-

P. Zhong, R.D. Ciaranello /Molecular

26

pocampal 5-HT,, binding sites. As an independent verification of these results, we also studied the effects of hypophysectomy on S-HT,, binding. Scatchard analysis showed that both surgical procedures significantly increased the BKZX of 5-HT!, binding (Fig. 1) by approximately the same magnitude. Neither treatment altered the K, of [3H]8-OH-DPAT for 5-HT,, sites, however. Daily administration of ACTH for 5 days reverses the increase in receptor binding seen after hypophysectomy. 3.2. Localization and hippocampal 5-HT,, adrenalectomy

time course of the increase in receptors and mRNA after

We next determined the relationship between the up-regulation of 5-HT,, binding sites and mRNA by in vitro receptor autoradiography and in situ hybridization. In dorsal hippocampus, total 5-HT,, receptor binding sites in both dentate gyrus and CA fieid regions increase by approximately 60% one day after adrenalectomy (Table 1). In contrast to previous reports 1181, however, no significant change in receptor binding was seen in these regions 1 h after adrenalectomy. We speculate that these different results could derive from: (a) animals might experience different level of stress due to different handling procedure rirlrino the EIITOPI?I 2nd YllV”LllV”lU anectheria hptw,vn n,,r nrn,,n ..U”“b .llV YU’ bu’J UllU ““C..~VII VUI b’ “LLF

loo0 1 800

-l

*

1

f

**

***

-

l600 -

400 -

200 -

0 1

r

HYPOX

HYPOX/ACTH

u

TREATMENT

Effects of adrenalectomy (ADX) and hypophysectomy (HYPOX) on B,,, of hippocampal 5-HT,, receptor sites. Data were summarized from Scatchard analysis of [ ‘H]X-OH-DPAT binding to membranes prepared from hippocampi taken from the following groups of rats: control, 1 day after adrenalectomy, 3 weeks after hypophysectomy and hypophysectomy plus ACTH (4 IU daily for 5 days, i.p.). None of the treatments significantly affected the K, of [ ‘HIS-OH-DPAT. Data are expressed as mean + S.E.M. (fmol/mg protein) with II = 6-8. (‘, * *: P < 0.01 and 0.05 compared to control, * * * : P < 0.01 compared to hypophysectomy).

Brain Research 29 (1995) 23-34

and the other group; (b) different strain rats were used for adrenalectomy so that their responses to adrenalectomy might be different. The adrenalectomy-induced up-regulation of 5-HT,, sites appears to be regionally specific because we found no change in [“H&OHDPAT binding in the cerebral cortex (data not shown). We next took brain sections immediately adjacent to those used to determine 5-HT,, binding, and examined them for corresponding changes in 5-HT,, mRNA by in situ hybridization. The density of the signal corresponding to 5-HT,, mRNA within the dorsal hippocampus increases dramatically after adrenalectomy compared to sham-operated controls (Fig. 2). The increase in 5-HT,, mRNA is localized primarily within the dentate gyrus and the CA field regions, which is anatomically coincident with the increase in the receptor binding sites. However, 5-HT,, mRNA in these regions is up-regulated as early as 1 h after adrenalectomy, whereas no significant changes at the receptor ievei was detected at the same time point. This suggests that the increase in 5-HT,, receptor binding follows the increase in receptor mRNA. The increase in receptor mRNA and binding persists for at least 7 days after adrenalectomy. 3.3. Determination of distribution within hippocampal subregions

SHT,,

receptor mRiVA

The adrenalectomy-induced increase in 5-HT,, mRNA could occur as the consequence of increased synthesis within a population of hippocampal neurons already expressing S-HT,, receptors, or by recruitment of previously non-expressing cells. To test these alternatives, 10 pm sections of hippocampi from rats taken 1 day after adrenalectomy or sham surgery were subjected to in situ hybridization and concurrent Nissl staining. Examination of 5-HT,, mRNA in dentate gyrus and CA3 fields shows that receptor mRNA substantially increases 1 day after adrenalectomy (Fig. 2). The distribution of 5-HT,, mRNA was also examined by localizing silver grains over Nissl-stained hippocampal neurons. Hippcampal sections from adrenalectomized animals appear to show a higher number of labeled neurons. To examine this further, we counted the number of cells with silver grains over them under bright-field microscopy, limiting the count to only those cells with definable borders. When this was done the number of positive cells was not different between adrenalectomized and sham-operated animals, although a significantly higher density of silver grains was seen in hippocampal sections from adrenalectomized animals (Fig. 3). We next counted the distribution of silver grains over the labeled neurons; the results are shown as frequency distribution histograms in Fig. 4. In the dentate gyrus from sham-operated animals, 50% of the

P. Zhong, R. D. Ciaranello /Molecular

Brain Research 29 (I 995) 23-34

B

27

.

Fig. 2. Expression of hippocampal SHT,, receptor mRNA. In situ hybridization was performed on brain coronal sections using a “S-labeled antisense 5-HT,, riboprobe prepared as described in Materials and methods. A: sham-operated; B, C, D and E: 1 h, 1, 2 and 7 days after adrenalectomy, respectively. (Changes of SHT,, mRNA expression in subregions of the CA fields (CA1,2,3) and dentate gyrus (DC) in hippocampus are indicated.).

28

P. Zhong, R.D. Ciarunello /Molecular

Brain Research 29 (1995) 23-34

cells have less than 10 silver grains overlying them, and 80% have less than 15. Adrenalectomy induces a shift to the right in this distribution, so that nearly 50% of the cells have more than 20 silver grains (Fig. 4A). In the CA3 region, about 50% of cells from the sham-operated animals have less than 5 silver grains, and 90% have less than 10. Adrenalectomy again induces a right-shift in the distribution of silver grains, with 60% of the cells having more than 10 (Fig. 4B). Among the cell populations that highly express 5-HT,, mRNA after adrenalectomy, 20% of cells in dentate gyrus or CA3 contain extremely high levels of silver grains (> 25 in the dentate gyrus and > 15 in the CA,), whereas no cell in the same regions of a control animal expresses that level. These results are consistent with the hypothesis that the increase in hippocampal 5-HT,, mRNA is

caused by increased receptor mRNA in individual neurons and not by an increase in the total number of expressing cells. 3.4. Quantitation mRNA

of the increase in hippocampal

SHT,,

after adrenalectomy

We next sought to quantitate the elevation in 5-HT,, mRNA after adrenalectomy. To accomplish this, we modified the standard solution hybridization/RNase protection technique to improve its sensitivity. We observed a significant increase in 5-HT,, mRNA as early as 1 h after adrenalectomy, which is consistent with the result shown by in situ hybridization (Fig. 5). Maximal increase in the expression, which is observed 1 day after adrenalectomy, is approximately 70% (Fig. 5).

c

’

a.,..: ,._ .

-

.-

..

8

r

r

f .

*

1

.

-.

.

.

.

.

)* .

.

.

.

I: ; ‘&

.

I

.‘!

Fig. 3. Bright-field photomicrographs of 5-HT,, mRNA in hippocampus after adrenalectomy. Hippocampal sections were assayed by in situ hybridization with a %labeled antisense 5-HT,, riboprobe, followed by emulsion dipping and Nissl staining. Labeled cells in corresponding regions were examined using the computerized image analysis system described in Materials and methods. A: sham-operated, dentate gyrus; B: 1 day post-adrenalectomy, dentate gyrus; C: sham-operated, CA3 region: D: 1 day post-adrenalectomy, CA3 region. The number of labeled cells within a computerized image was counted and compared: dentate gyrus (A vs B): 105 f 3 and 100 f 4; CA3 region (C and D): 28 * 2 and 29 f 2. These were not significantly different using a paired f-test. Arrow 1 and arrow 2 indicate that silver grains were counted (associated with a definable cell) and not counted (not associated with a definable cell), respectively. Size bar: 20 pm.

P. Zhong, R.D. Ciaranello /Molecular Table 1 Effect of adrenalectomy oocamous Treatment Sham-operated Adrenalectomy, Adrenalectomy, Adrenalectomy, Adrenalectomy,

1 1 2 7

hour day days days

on 5-HT,,

receptor

Dentate

gyrus

lOO.O+ 105.6+ 166.3t 179.8+ 189.5 +

2.7 3.4 4.1 * 11.2 * 13.2 *

binding

sites

Brain Research 29 (1995) 23-34

29

in hip-

CA fields 100.0 + 2.3 109.6*3.5 157.7+4.7 * 169.2+ 8.7 * 183.3 k 9.3 *

Quantitative autoradiography or mppocampai 5-HT,, binding sites. Adrenalectomized animals were sacrificed at the designated time points. Control animals (sham-operated) underwent the same anesthetic and surgical procedures as the adrenalectomized rats except the adrenals were not removed. 5-HT,, receptor sites within the dentate gyrus and the CA field region (CA1,2,3) were determined by quantitative in vitro receptor autoradiography of the brain sections using 2 nM [ 3H]8-OH-DPAT. Optical density of the specific binding was determined by pre-calibrated computerized densitometry from film autoradiograms. Data were expressed as the means of percent of sham Q.D. vaiues* SEM. with n = 6 for each treatmeni groups. ( * P < 0.01, compared to the corresponding sham control).

Elevated expression was maintained up to 7 days after surgery. We then examined whether the elevation of hippocampal 5-HT,, mRNA after adrenalectomy could be reversed by replacement of corticosteroid hormones. Animals were given a single dose (0.5-10 mg/kg body weight, subcutaneously) of dexamethasone, a synthetic glucocorticoid, immediately after adrenalectomy. The animals were sacrificed 1 day after surgery and 5-HT,, mRNA was quantitated (Fig. 6). Dexamethasone reverses the elevation of 5-HT,, .- mRNA in a dose-de-

I

11 3

PDAYS

3DAYS

7DAYS

TREATMENT

Fig. 5. Quantitation of hippocampal 5-HT,, receptor mRNA after adrenalectomy. Adrenalectomized animals were sacrificed at the designated time points. Control animals received sham surgery. Eight Kg hippocampal total RNA collected from these animals was used in each reaction for co-hybridization with ‘sS-labeled 5-HT,, receptor and /3-actin antisense riboprobes. Levels of 5-HT,, receptor mRNA were determined from the autoradiogram and normalized to p-actin mRNA. Data are expressed as means of O.D. f S.E.M. with n = 5-6 ( * : P < 0.01 compared to sham control).

pendent manner; we estimated the ED,, of this effect to be about 0.5 mg/kg. However, high-dose dexamethasone (10 mg/kg) administration to adrenalectomized animals does not suppress 5-HT,, mRNA below basal levels. Similarly, dexamethasone at doses

60

40

1 DAY

A

,

1

50 -

30

0

CONT 40 -

30 -

20

20 10 lo-

0 1

0 3-5

NUMBER

5-10

OF

10.15

SILVER

15.20

20-25

GRAINS/CELL

25-30

IN

30-35

DENTATE

35-40

GYRUS

o-5

5-10

NUMBER

OF

10-15

SILVER

20-25

15-20

GRAINS/CELL

IN

CA3

Fig. 4. Frequency distribution of 5-HT,, mRNA silver grains over individual hippocampal neurons. A: dentate gyrus; B: CA3 region. Bright field photomicrographs of 5HT,, mRNA in hippocampal sections were assayed by in situ hybridization. The number of silver grains over individual cells was counted directly (as indicated in Fig. 3 legend, only silvers grains associated with definable cells were counted. Dark-field image was also used to ensure that silver grains counted were within a cell). Hippocampal cells were sampled in corresponding regions from sham-operated and adrenaiectomized animais. The x-axis represents the range of siiver grains over individuai ceiis. +‘he y-axis represents the percentage of ceiis within each range.

P. Zhong, R. D. Ciarardo

30

as high as 10 mg/kg does not reduce levels in sham-operated animals.

SHT,,

/Molecular

Brain Research 29 (1995) 23-34

1

0.6

mRNA 2

3.5. Transcription rate of hippocampal SHT,,

mRNA

Glucocorticoids typically exert their effects by modulating gene transcription. Accordingly, we carried our nuclear run-on assays to determine whether the rise in SHT,, mRNA following adrenalectomy could result from an increase in the rate of SHT,, mRNA synthesis. We prepared hippocampal nuclei from adrenalectomized or sham-operated animals sacrificed 1 h and 1 day after surgery, and measured production of 5-HT,, mRNA 30 min after the initiation of transcription. Hippocampal nuclei from adrenalectomized rats generate significantly higher amounts of 5-HT,, mRNA (70%) compared to sham controls (Fig. 7). These increases are consistent in magnitude with those seen in the radioligand binding and mRNA studies. Single dose dexamethasone administration (5 mg/kg) to adrenalectomized rats reverses the increase in 5-HT,, transcription seen after adrenalectomy (Fig. 7). These results suggest that the upregulation of 5-HT,, receptor expression in hippocampus following adrenalectomy is mediated by increased transcription of the 5-HT,;, gene, and that glucocorticoids act normally to attenuate transcription of this gene.

F P

05-

2 ii

0.4 -

***

a E b

0.3 -

z z

0.2 -

d d

0.1 -

0.0 9 CCNI

ADX,

1 HR

ADX,

1 DAY

tEX

ADXIDEX

TREATMENT

Fig 7. Effects of adrenalectomy on the rate of transcription of hippocampal 5-HT,, mRNA. Transcription run-on assays were performed as described in Experimental Procedures. Hippocampal nuclei were prepared from sham-operated control rats, or adrenalectomized rats 1 h or I day after surgery. One group of controls (DEX) and one group of 1 day post-adrenalectomy rats (ADX/DEX) received a single dose of dexamethasone (5 mg/kg). “P-labeled RNAs were collected at 30 min after initiation of the transcription reaction, then hybridized with 5HT,, receptor cDNA, p-actin cDNA or the vector plasmid DNAs in a slot blot apparatus. 5HT,, mRNA was measured from the autoradiograms and normalized to that of p-actin. Quantitative data were expressed as means of O.D. unitskS.E.M. with =6-8 t’, **: P < 0.01 and P < 0.05 compared to sham control: * * *: P < 0.01 compared to adrenalectomy).

1 0

SHAM

3.6. Determination of functional coupling of the hippocampal SHT,, receptors after adrenalectomy

?? ADX

*

*

*

lill 5

2

TREATMENT

(mgikg

10

DEX)

Fig. 6. Effects of dexamethasone administration on the adrenalectomy-induced increase in the expression of 5-HT,, receptor mRNA. Animals were injected with either saline vehicle or the specified dose of dexamethasone (DEX) after either sham surgery (SHAM) or adrenalectomy. All animals were sacrificed 1 day after surgery. RNase protection assay was performed as described using 8 pg total hippocampal RNA in each reaction. Levels of 5HT,, receptor mRNA were determined from the autoradiogram and normalized to p-actin. Data were expressed as means of O.D. unitsfS.E.M. with n=5 t*: P < 0.01 compared to vehicle control; * * : P < 0.01 compared to adrenalectomy).

We next sought to determine whether the increase in 5-HT,, binding seen after adrenalectomy is reflected in increased functional coupling to second messenger systems. Hippocampal 5-HT,, receptors have been previously reported to inhibit adenylyl cyclase through a [45] and to inhibit [17] and stimulate Gi/,, protein phosphoinositide hydrolysis [30]. Work from our laboratory has demonstrated &OH-DPAT induced activation of phospholipase C and phospholipase A2 activities in HA7 cells stably transfected with the 5-HT,, cDNA [27]. Accordingly, we examined adenylyl cyclase activity and phosphoinositide hydrolysis to test whether functional coupling to these effecters increases after adrenalectomy. In hippocampal membranes prepared from rats 1 day after adrenalectomy, 5-HT,.-mediated inhibition of forskolin stimulated adenylyl cyclase activity is not significantly different from that of control membranes (data not shown). Similarly, 5-HT,,-mediated inhibition of carbachol-stimulated phosphoinositide hydrolysis in hippocampal slices was not significantly different between adrenalectomized and sham-operated animals, meanwhile, no significant S-OH-DPAT-

P. Zhong, R. D. Ciaranello /Molecular

stimulated phosphoinoitide turnover was observed in both adrenalectomy and sham-operated animals (data not shown). These suggest that the increase in 5-HT,, receptors after adrenalectomy is not reflected in increased coupling to the effecters studied, at least within the 24 h time period examined.

4. Discussion There is abundant evidence of a functional interaction between glucocorticoids and 5-HT,, receptors in the brain. In addition to the regulation of 5-HT,, binding sites by corticosteroids, physiological functions induced by activation of CNS 5-HT,, receptors are also modulated by corticosteroids. The well-described ‘serotonin syndrome’ consists of several limbic systemmediated behavioral responses, such as forepaw treading, head weaving, hind limb abduction, Straub tail and piloerection, suppression of exploratory behavior and decrease in body temperature [25]. This behavioral array can be brought about by 5-HT releasing agents such as fenfluramine or p-chloramphetamine, as well as by selective 5-HT,, agonists such as S-OH-DPAT [6]. In addition, agonist activation of 5-HT,, receptors increases prolactin, CRH, ACTH and corticosterone secretion into the circulation [38]. Administration of corticosterone reportedly diminishes these 5HT,, agonist-induced responses [8,211. In clinical studies, abnormal elevations of CRH, ACTH and corticosterone have been demonstrated in patients with depressive disorders [43]. Interestingly, improvement has been reported in these patients after treatment by buspirone or tandosporone, which act as agonists at 5-HT,, receptor sites [31]. Recently, a model for the pathogenesis of depression based on functional interaction between 5-HT,, receptors and corticosteroids has been proposed by Lesch and Lerer 1291 and Mendelson and McEwen 1341. These authors suggest that prolonged increase in corticosteroid secretion causes a down-regulation of hippocampal 5-HT,, receptors, resulting in a reduction of functional 5-HT,, output. This hypothesis is also supported by the observation that intracellular corticosteroid receptors are particularly rich in hippocampal granule cells and pyramidal cells [24], and both of these cell layers are major loci of 5-HT,, receptors [14,39]. Taken together, these findings suggest that corticosteroids exert a powerful suppressive influence on serotonergic neuronal activity in limbic centers. However, clinical insufficiency of corticosteroid hormones such as Addison’s disease and hypopituitarism does not usually demonstrate higher emotional status. But this is probably due to other effects of removal of corticosteroids in addtion to its regulation of 5-HT,, receptor expression. A number of studies already suggest that neuronal death in the

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31

hippocampal structure can result from prolonged adrenalectomy [26]. The results described here confirm that adrenalectomy leads to an increase in hippocampal 5-HT,, receptor binding and extend these observations in the following ways. First, we have shown that hypophysectomy, which also depletes endogenous glucocorticoids, increases 5-HT,, binding in hippocampus, while administration of ACTH reverses this effect. Second, we have demonstrated that adrenalectomy causes an increase in 5-HT,, mRNA as early as 1 h after surgery; this increase precedes the increase in 5-HT,, binding within the same hippocampal regions. Third, we have demonstrated hippocampal neurons which normally express 5-HT,, receptors increase receptor mRNA production in response to adrenalectomy. There is no evidence to suggest that recruitment of previously non-5-HT,, expressing neurons takes place, however. Fourth, this work demonstrates that transcription of 5-HT,, mRNA is increased by as much as 70% after adrenalectomy, and that the effects of adrenalectomy on 5-HT,, transcription and on receptor mRNA accumulation are fully reversed by dexamethasone administration. However, acute administration of high doses of dexamethasone to adrenalectomized or sham-operated animals does not decrease 5-HT,, mRNA below basal levels. Finally, the increase in 5-HT,, binding and mRNA seen after adrenalectomy does not appear to be reflected in increased activity of two of the principal effector pathways to which the receptor is coupled, adenylyl cyclase or phosphoinositol hydrolysis, at least within the time period selected for study. Work from other laboratories suggested that 5-HT,,-mediated responses were under a suppressive regulation of corticosteroid hormones [8,21,28,441. Taken together, these findings could indicate coupling of the 5-HT,, receptor to other second messengers which are influenced by adrenalectomy, or that changes of other components of the signal transduction pathway (e.g. G proteins) are also affected by adrenalectomy. Alternatively, the period of time (24 h) after adrenalectomy we selected could have been too short to detect a functional effect. Our data, as well as reports from other laboratories, indicate that adrenalectomy-induced changes in 5-HT,, receptors occur primarily in the hippocampus, even though the 5-HT,, receptor is widely distributed throughout the brain. A reasonable explanation for this observation is that glucocorticoid modulation is restricted to those cells which express both 5-HT,, mRNA and glucocorticoid receptors. Both 5-HT,, and glucocorticoid receptors are present in high amounts within the hippocampus, particularly the pyramidal cell layer of the CA fields and the granule cell layer of the dentate gyrus [14,24,39]. An important question to consider is whether 5-HT,, and glucocorticoid receptors are colocalized on the same hippocampal cells. The

32

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answer to this question will not be finalized until microscopic demonstration using double antiserum labeling (5-HT,, receptor and corticosteroid receptor) is available. A recent study by Chalmers et al. [13] showed that the mRNAs of type I and II corticosteroid receptors are localized in all hippocampal subregions that express 5-HT,, receptor mRNA. These results strengthen the likelyhood that the two receptors are indeed colocalized. Since both type I and type II receptors are highly concentrated in the hippocampus, and are present in the same regions in which 5-HT,, receptors are found [4,16,23,401, a number of investigators have tried to define which glucocorticoid receptor subtype modulates 5-HT,, transcription. The weight of evidence currently implicates the involvment of a Type 1 (mineralocorticod) receptor, although the results are not entirely unambiguous. De Kloet et al. [18] and Biegon et al. [lo] showed that the dose of corticosterone used to reverse the 5-HT,, receptor up-regulation following adrenalectomy caused full occupancy of type I, but not type II, receptors in the hippocampus. Mendelson and McEwen [34], and Chalmers et al. [13] demonstrated that the dose of corticosterone necessary to reverse the post-adrenalectomy 5-HT,, receptor increase results in full occupancy of type I receptors [18]. Furthermore, Joels et al. [28] demonstrated that corticosterone suppresses 5-HT,, mediated hyperpolarization in CA, pyramidal neurons. This effect could be blocked by RU38486, a selective type I antagonist. All these observations implicate Type I glucocorticoid receptors. Attenuation of gene transcription by corticosteroids has been demonstrated in numerous studies [1,15,22, 37,41,42] and for several genes. Several mechanisms to explain this phenomenon have been proposed. Sakai et al. [41] identified a 34-bp sequence associated with the bovine prolactin gene which selectively interacts with purified glucocorticoid receptors and confers a glucocorticoid-mediated transcriptional repression. This element has been termed a ‘negative’ glucocorticoid response element (nGRE). Adler et al. [l], have identified a 45 amino acid region immediately adjacent to the DNA binding domain of glucocorticoid receptors which also mediates repression of rat prolactin transcription by glucocorticoids, suggesting that hormonereceptor interaction, as well as receptor-DNA interaction, may modulate transcription. The possibility that a repressor can compete with stimulatoty transcription factors for binding to the same or overlapping promoter elements has also been suggested [9]. More recently, Diamond et al. [20] proposed that whether the promoter activity of a gene responds positively or negatively to activation by glucocorticoid receptors appears to be determined by the number and type of transcriptional factors interacting with activated glucocorticoid receptors. Although the 5-HT,, receptor gene

Brain Research 29 (1995) 23-34

has been cloned [2], no description of its promoter region has been published, so at present it is not known whether hormone response elements are present in the promoter region. However, our work and other studies provide presumptive evidence that such elements are likely to be present. Although glucocorticoids clearly play a significant role in regulating hippocampal 5-HT,, expression, they are probably not the sole regulatory factor for the following reasons. First, there is a robust basal level of 5-HT,, expression which occurs in the presence of an intact pituitary-adrenal axis, and according to our results, this is not attenuated, at least acutely (24 h), by supplemental administration of exogenous glucocorticoids. Second, acute or single dose glucocorticoid administration restores the increase in 5-HT,, binding sites and mRNA after adrenalectomy only to control levels [13,34], however, prolonged and high dose treatment is needed to further suppress receptor mRNA levels. Third, 5-HT,, receptors are expressed in neurons in several brain areas, some of which may not express glucocorticoid receptors, or may express low levels. It is likely the case, therefore, that regulation of 5-HT,, transcription is governed by stimulatory and inhibitory factors, and that glucocorticoids function in this latter role in those brain regions where corticosteroid receptors and 5-HT,, receptors are present together. Currently there is no published evidence that the 5HT,, gene promoter contains negative glucocorticoid response elements, nor is there evidence implicating specific factors as enhancers of 5-HT,, transcription, although it would seem likely that such factors must exist.

Acknowledgements This study was supported by a program project grant from the NIMH (MH 39437), by the Spunk Fund, Inc. (P.Z.) and by the endowment fund of the Nancy Pritzker Laboratory. R.D.C. is the recipient of a Research Career Scientist Award from the NIMH (MH 00219).

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