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Time and region-dependent effect of adrenalectomy on neuropeptide gene expression in rat hippocampus and striatum

Time and region-dependent effect of adrenalectomy on neuropeptide gene expression in rat hippocampus and striatum
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  MOLECULAR AND CELLULAR NEUROSCIENCES 2,485-&o (1991) Time- and Region-Dependent Effect of Adrenalectomy on Neuropeptide Gene Expression in Rat Hippocampus and Striatum TERESA IGLESIAS, T* MARLEEN A. E. VERBEECK, ~ RONALD E. DE KLOET&~ WINARDI SUTANTO,~~ Jo& A. FUENTES,* AND J. PETER H. BURBACH$,’ §Rudolf Magnus Institute, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherkm.&; and *Pharmacological Department, Medical School, Complutense University, 28040 Madrid, Spain Received for publication September 11, 1991 Seven days after removal of the adrenals in rats, the messenger RNA levels of preproenkephalin (ENK), pre- prodynorphin (DYN), cholecystokinin (CCK), and neu- ropeptide Y (NPY) were measured in hippocampus, striatum, and hypothalamus. Adrenalectomy (ADX) in the morning, when endogenous corticosterone levels were low, resulted ‘7 days later in a decrease of ENK mRNA and DYN mRNA levels in the hippocampus (4 1.3 + 4.3 and 41.9 + 5.7%, respectively) and in the striatum (32.1 t 6.6 and 31.2 + 12.9%, respectively), but no change was observed in the ENK mRNA content of the hypothalamus. When ADX was performed in the evening the opioid mRNA levels were not changed in these brain areas 7 days after ADX. Pretreatment with a single dose of corticosterone before surgery in the morning produced high corticosterone levels similar to those in the evening and prevented the decrease of ENK mRNA in the hip- pocampus. The decrease in hippocampal DYN mRNA and in striatal ENK mRNA and DYN mRNA persisted. CCK mRNA and NPY mRNA were not changed in any of the experimental groups in any of the three examined brain areas. This study demonstrates that ADX decreases opioid gene expression in the rat hippocampus and stria- turn. The effect of ADX on hippocampal ENK mRNA lev- els that persists for at least 7 days postsurgery is inde- pendent of the circulating corticosterone level at the time of surgery. 0 1991 Academic PWS, 131~ INTRODUCTION Glucocorticoids are important for the central regulation of the stress response (1). Stress is known to stimulate i To whom correspondence should be addressed at Rudolf Magnus Institute, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands, fax 30/896034. r Present address: Department of Pharmacology, University of Leuven, Lauven, Belgium. 3 Present address: Department of Neuroendocrinology and Pharma- cology, Center for Biopharmaceutical Sciences, Sylvius Laboratorium, University of Leiden, 2300 RA Leiden, The Netherlands. the synthesis and release of enkephalins (2). An increase in central preproenkephalin (ENK) mRNA levels follow- ing stress has been demonstrated (3,4). Foot-shocks (5) and electroconvulsive shocks changed the mRNA levels of ENK and of another member of the opioid system, preprodynorphin (DYN) (6). Several reports point to a regulatory role of glucocorticoids in the activation of opioid gene expression during stress (5, 7-9). Other data suggest that certain forms of stress-induced analgesia can be attenuated by adrenalectomy (ADX) and reinstated by administration of corticosterone, shortly before the analgesic trial (2, 10). Recently, behavioral studies demonstrated a long-last- ing effect of ADX on the antinociceptive effect of mor- phine (9-11). Interestingly, the effect depended on the time of the day the adrenals were removed (10,ll). The response latency after morphine administration was pro- longed threefold in rats that were adrenalectomized in the morning (AM ADX) when compared to rats adre- nalectomized in the evening (PM ADX). This change of sensitivity to opiates persisted for 2 weeks following ADX and was dependent on the actual plasma corticosterone level at the time of ADX. Subsequent experiments showed that the uptake of exogenous [3H]diprenorphine was also greater in the hippocampus and in the striatum 7 days after ADX of rats that had low circulating levels of cor- ticosterone at the time of surgery (11). From these data it was postulated that ADX arrested opiate responsiveness and, thus, perhaps the activity of the limbic-midbrain opioid system, which appeared correlated with the level of circulating corticosteroids at the time of surgery. The present study was designed to examine the influ- ence of ADX on the mRNA level of two opioid genes ENK and DYN in hippocampus, striatum, and hypo- thalamus and to determine whether the time of ADX re- sults in a long-lasting effect on the expression of opioid genes as it occurs for the level of opiate binding sites and antinociception according to Ratka et al. (10, 11). In ad- dition, two other neuropeptide genes NPY and CCK 485 1044-7431/91 3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.  486 IGLESIAS ET AL. which are involved in the neuroendocrine response to stress (11) were included in the study. Three different conditions of the hypothalamic-pituitary-adrenal axis were examined: AM ADX, when endogenous corticoste- rone levels are low, PM ADX when levels are high, or AM ADX following a single injection of corticosterone to produce high levels of the steroid during ADX (1). The data show a long-term, state-dependent, site- and peptide- specific effect of ADX on neuropeptide gene expression in the rat brain and support the observations from be- havioral and binding studies. MATERIALS AND METHODS Animal-s and Surgery Adult male Wistar rats (200-220 g) were used in all experiments. Animals were housed four or five per cage under standard light (lights on from 0700-1900 h) and temperature conditions (24’0. Standard laboratory food and drinking water were available ad libitum. During 1 week prior to surgery, the rats were handled daily at 0900 h. Bilateral ADX or sham surgery (sham) was performed under ether anesthesia using the dorsal approach under resting conditions, either in the morning (0700-0900 h) or in the evening (1800-2000 h). One group of AM ad- renalectomized rats received a single subcutaneous injec- tion of 10 mg corticosterone/kg body wt 1 h before surgery. ADX animals were given 0.9% NaCl instead of water. Rats were killed on the seventh day after ADX by decap- itation between 0800 and 1000 h, and trunk blood was collected for determination of circulating corticosterone levels by a specific radioimmunoassay to monitor the ef- fectiveness of ADX (12). In all ADX animals in this study the plasma corticosterone levels were below detection (co.5 pg/lOO ml), while sham-operated rats had average levels of 8 pg/lOO ml. Brains were rapidly removed and three areas were dissected: the striatum, the hippocampus, and the hypothalamus. Tissues were frozen in dry ice and then stored at -8O’C until RNA extraction. Isolation of Total Cellular RNA Total RNA was extracted as described previously (13). Frozen tissues of each individual animal were homoge- nized separately at room temperature in 700 ~1 of GTC solution (GTC = 4 M guanidine thiocyanate, 0.1 M @- mercaptoethanol, 25 mM sodium citrate, pH 7.0). The homogenate was extracted with an equal volume of phe- nol-chloroform-isoamyl alcohol (25:24:1 (v/v/v)). After 5 min of centrifugation at lO,OOOg, he aquous phase was collected and RNA was precipitated by addition of 450 ~1 of 45 mM sodium acetate in 100% ethanol, overnight in- cubation at -2O’C, and centrifugation for 20 min at 10,OOOg. After a wash with 70% ethanol, the pellet was dissolved and incubated with 50 pg/ml proteinase K at 37°C for 1 h in 200 ~1 TES buffer (TES = 10 r&f Tris- HCl, 5 mA4 EDTA, 0.5% SDS, pH 7.8). The incubate was again extracted with phenol and precipitated. The quan- tity of RNA was determined spectrophotometrically by uv absorption at 260 nm and the samples were stored at -20°C until use. Blotting Procedures For dot blots two amounts of total RNA were used: 2 and 5 pg from hippocampus, 5 and 20 pg from hypothal- amus, and 0.5 and 2 pg from striatum. The RNA samples were denaturated in 7.4% formaldehyde at 65°C for 15 min and then cooled on ice. The samples were spotted onto a nylon membrane (Hybond-N, Amersham, UK) within a dot blot manifold (Bio-dot microfiltration ap- paratus, Bio-Rad, Richmond, CA) and were filtered through the membrane under slight vacuum. For Northern blots 25 and 10 pg RNA from hippocam- pus and hypothalamus and 16 and 4 pug RNA from stria- turn were used. RNA samples were denaturated with glyoxal and dimethyl sulfoxide in 10 &phosphate buffer pH 6.5, at 55°C for 60 min, cooled on ice, loaded on a 1.4% agarose-10 mM phosphate gel, and run at 10 V/cm for 3 h. Transfer of RNA from the gel to the nylon mem- brane was carried out overnight in 25 mM phosphate buffer, pH 6.5. Both dot blots and Northern blots were air-dried and subsequently baked at 80°C for 2 h. Hybridizations All probes were 32P-labeled to a specific activity of about 1 X 10’ cpm/gg by the random primer method (14). The probe for rat ENK mRNA was a 935-bp SmuI-Sac1 cDNA fragment containing the entire coding sequence (15). The rat NPY probe was the 280-bp AuaI-XbaI fragment of exon 2 of the rat gene (16); the rat DYN probe was the 1700-bp BglI-BamHI genomic fragment containing the main exon (17), the rat cholecystokinin (CCK) probe was a 535-bp fragment of a cDNA clone (18) and the rat @- actin (ACT) probe was the PstI fragment of a cDNA clone (approximately 1500 bp) (19). Probes were hybridized separately. Filters were prehybridized at 43-5O”C, depending on the probe, in 50% (v/v) formamide, 6 X SSC (1X SSC = 0.15 M NaCl, 0.015 sodium citrate, pH 7.0), 0.5% defatted milk powder, 1 mg/ml denatured herring sperm DNA, and 5% (w/v) dextran sulfate for at least 6 h. Denaturated 32P-labeled DNA probes were added to the prehybridi- zation mix to a final concentration of at least lo6 cpm/ ml and incubated overnight. The blots were washed twice in 2X SSC, 1% SDS at room temperature for 10 min; twice in 0.5X SSC, 1% SDS at 50°C for 10 min; and once in 0.1X SSC, 0.1% SDS, 2 mMEDTA at 60°C for 10 min. The filters were then exposed to X-ray film (Kodak X- OMAT) with an intensifying screen at -80°C for various lengths of time. For rehybridization with another probe  EFFECT OF ADRENALECTOMY ON NEUROPEPTIDE GENE EXPRESSION 487 ENK DYN ACT FIG. 1. Northern blot analysis of ENK, DYN, and actin (ACT) mRNAs in hippocampus and striatum of sham operated and adrenal- ectomixed rats. Twenty-five micrograms of total RNA from hippocampus (I, Hippoc.) and 16 pg total RNA from striatum (II; Striat.) from sham operated (lane l), AM ADX (lane Z), and PM ADX (lane 3) rats were analyzed as described under Materials and Methods. The exposure times of the autoradiograms for the ENK mRNA signal in hippocampus and in striatum were 120 and 6 h, respectively. The DYN and ACT mRNA signals were observed after 24 and 15 h of exposure. The length of ENK mRNA was approximately 1506 nt, of DYN mRNA approximately 2400 nt, and of ACT mRNA approximately 2200 nt. the blots were stripped in 1 mM EDTA-5 mM Tris, pH 8.0, at 95°C for 20 min and checked for remaining probe by autoradiography. Quantification of RNA The optical density of the lanes on the films of hybrid- ized Northern blots were determined with a one-dimen- sional densitometric scanner (Zeiss KM3, Oberkochen, West Germany). Optical densities of autoradiograms of hybridized dot blots were analyzed with a microtiter plate reader (EIA reader 2550, Bio-Rad, Richmond, CA). The range of total RNA on the filters in combination with the exposure times allowed a linear relationship between the quantities of the mRNAs and the relative densitometric values of the hybridization signal. The actin mRNA con- tent was used as an internal standard. The levels of neu- ropeptide mRNAs were normalized to the @actin mRNA content. The average value of the control group was taken as 100%. Levels were determined for each individual an- imal. Statistical analysis was performed by MANOVA. A level of P < 0.05 was considered significant. rats when compared to the sham and PM ADX animals (Fig. 1). Effect of Adrenalectomy in the Morning (AM ADX) To quantify the response to AM ADX, dot blots were used in another series of experiments. Neuropeptide mRNA levels were expressed as a ratio to the @actin mRNA content. Significant changes in the @actin mRNA content expressed per microgram of total RNA were not observed. Significant decreases of 41% (P < 0.01) and 42% (P < 0.05) in ENK and DYN mRNA levels, respec- tively, were detected in the hippocampus of the AM ADX group (Fig. 2). This effect was observed 1 week after sur- gery and therefore indicated a long-lasting effect of ADX. In the striatum of the AM ADX group, a decrease of 32% (P < 0.01) in the level of ENK mRNA was observed (Fig. 3). The level of DYN mRNA decreased by 31%, but this change was not statistically significant (Fig. 3). In the hypothalamus, AM ADX did not influence the level of ENK mRNA (Fig. 4). Expression of DYN mRNA levels was not determined in this brain area; the level of this mRNA was to low to allow accurate quantitation. The levels of the other investigated neuropeptide mRNAs, i.e., CCK mRNA and NPY mRNA were not influenced in any of these brain areas by AM ADX (Figs. 2-4). Effect of Adrenalectomy in the Evening (PM ADX) In PM ADX rats, which were operated when the levels of circulating corticosterone were high (1, 10, ll), no sig- RESULTS DYN neucwptide rrR CCK WY 4A in hippocamxls 0 ADX pm . h- Expression of ENK and DYN Genes Initial analyses on Northern blots of total RNA of striatum and hippocampus showed clear signals of ENK mRNA and DYN mRNA of approximately 1500 and 2400 nt, respectively (Fig. 1). The ENK mRNA was very abun- dant in the striatum. DYN mRNA levels were roughly similar in the hipocampus and striatum. Northern blots also indicated a decrease in the level of ENK mRNA and DYN mRNA in hippocampus and in striatum of AM ADX FIG. 2. Effect of adrenalectomy on levels of neuropeptide mRNAs in the hippocampus. For each individual animal the neuropeptide mRNA level was expressed in an arbitrary unit representing the ratio to the fl- actin mRNA content and was derived from two different RNA concen- trations on dot blots. The average value from sham operated rats was taken as 100% (n = 5) (black bars). Statistical comparisons between these values and the average values from AM ADX rats (n = 6) (hatched bars), from AM ADX rats pretreated with a single injection of corti- costerone (prey bar, ADX+), and from PM ADX rats (n = 6) (open bars) were made by MANOVA. Statistically significant differences are **P < 0.01, different from sham, and ADX+, and PM ADX, *P < 0.05, different from sham and PM ADX, ‘P < 0.05, different from sham and AM ADX.  IGLESIAS ET AL. 88 i 150 125 0 6 100 $ 75 g 50 2 P 25 0 M DYN Tili h FIG. 3. Effect of adrenalectomy on levels of neuropeptide mRNAs in the striatum. The figure shows the neuropeptide mRNA levels as a ratio to the &a&in mRNA content in sham rats (black bars), AM ADX rats (hatched bars), AM ADX rats pretreated with a single dose of cor- ticosterone before surgery (grey bars, ADX+), and PM ADX rats (open bars). For details see the legend to Fig. 2. Statistically significant dif- ferences are **P < 0.01, different from sham and PM ADX; *P < 0.05, different from sham and PM ADX; 'P < 0.05, different from sham and ADx+. nificant changes in mRNA levels of ENK, DYN, CCK, and NPY were detected in the hippocampus, when com- pared with the sham operated group, 7 days after the op- eration (Fig. 2). Similarly, the levels of ENK mRNA, DYN mRNA, and NPY mRNA in the striatum were not sig- nificantly different from the sham group (Fig. 3). However, the CCK mRNA level increased significantly by 33% (P c 0.05). None of the determined neuropeptide mRNA levels changed by PM ADX in the hypothalamus (Fig. 4). Effect of a Single Corticosterone Injection of AM ADX Rats A single injection of corticosterone given 1 h before adrenalectomy was chosen to raise the steroid levels to those of PM ADX rats (11). This treatment had a differ- ential effect on the opioid peptide mRNA levels as mea- sured 7 days later. ENK mRNA levels in the hippocampus were not significantly different from sham operated and PM ADX rats (Fig. 2), whereas in the striatum a signif- icant reduction occurred (Fig. 3). DYN mRNA levels were lower in both hippocampus and striatum compared to sham operated rats, but did not reach the criterion of significance (P < 0.05) in the latter area. Opioid mRNA levels did not change in the hypothalamus (Fig. 4). The treatment caused an increase in hippocampal NPY mRNA levels (Fig. 2) and a small decrease in striatal CCK mRNA levels (Fig. 3). DISCUSSION This study demonstrates that ADX influences the mRNA levels of the ENK and DYN opioid genes. This effect depended on the time of the day when the adrenals were removed. The level of ENK mRNA and DYN mRNA in hippocampus and in striatum was lower in rats adre- nalectomized in the morning (AM ADX) than in sham operated rats (sham). The level of ENK mRNA and DYN mRNA of rats adrenalectomized in the evening (PM AXD) was not different from that of the sham group, but was significantly higher than that of the AM ADX group. This difference in opioid gene expression seemed to de- pend on the circulating corticosterone level at the time of ADX: the group with low basal plasma corticosterone levels had low ENK and DYN mRNA levels in the brain 1 week after surgery. In the hypothalamus the expression of the two opioids was not affected by the removal of the adrenals. The mRNA level of the two other investigated neuropeptides CCK and NPY did not change in these brain areas after ADX. Therefore, the influence of AM ADX was not general for all ENK- and DYN-expressing neurons, nor for all neuropeptides expressed in the stria- turn and hippocampus. It should be realized that the four neuropeptide mRNAs are located differently in the rat hippocampus. DYN mRNA is almost exclusively localized in the dentate gyrus (20). ENK mRNA is also predomi- nantly present in this region (21), while CCK and NPY are primarily expressed in the pyramidal cell layers (22, 23). Therefore, differential effects of ADX on different cell types in the hippocampus may play a role in the effects observed here. Since long-term ADX leads to selective loss of dentate gyrus granule cells (24), we did not pursue the effect of ADX at time intervals longer than 7 days postsurgery. Moreover, the finding that corticosterone administration prior to ADX increases subsequently ENK mRNA, but not DYN mRNA, argues against altered pep- tide mRNA levels due to neuronal degeneration. Finally, the decreases in ENK mRNA and DYN mRNA are not 175 150 125 0 z 100 { 75 r 5 50 e 25 0 EM CCK newqaeptide mRNA in t FIG. 4. Effect of adrenalectomy on levels of neuropeptide mRNAs in the hypothalamus. The figure shows the neuropeptide mRNA levels as a ratio to the B-actin mRNA content in sham rats (black bars), AM ADX rats (striped bars), AM ADX rats pretreated with a single doss of corticosterone before surgery (grey bars, ADX+), and PM ADX rats (open bars). For details see the legend to Fig. 2.  EFFECT OF ADRENALECTOMY ON NEUROPEPTIDE GENE EXPRESSION 489 restricted to the hippocampus, which further minimizes a predominant role of possible cell loss in altered mRNA levels. A decrease in ENK mRNA level in the hippocampus after ADX has been reported (25-27). In the hippocampus, which is an important action site for glucocorticoids (l), the ENK mRNA level was shown to increase after treat- ment with corticosterone (26) or dexamethasone (28). In this brain area ENK mRNA and DYN mRNA have been found to decrease after electroconvulsive shocks (6), but to respond differently to electrical stimulation (20). In the striatum, however, stress by electroconvulsive shock, increases the level of both ENK and DYN mRNA (6). These data together with our findings indicate that al- though ENK mRNA and DYN mRNA respond differently depending on brain region and treatment, they are re- sponsive in the same way to ADX in the hippocampus and striatum. Several authors have described the response of the mRNA level of ENK and DYN in neurons of the para- ventricular nucleus (PVN) of the hypothalamus to cor- ticosterone treatment or stress (3,4). Swanson and Sim- mons (29) showed that corticosterone decreased ENK mRNA in the parvocellular neurosecretory neurons of ADX rats, but increased such levels in certain magno- cellular neurons of the PVN. In our experiments we have not detected a change in mRNA levels of ENK and DYN in the medial basal hypothalamus. Accordingly, this large size of the dissected tissue used for our analyses may have masked steroid effects on peptide gene expression in sub- sets of hypothalamic neurons. Our data on the hypo- thalamus, therefore, does not allow us to conclude about the possibility that subsets are influenced by ADX, rather it shows that the changes found in striatum and hippo- campus are not universal for all ENK- and DYN-ex- pressing systems. There is direct evidence for positive regulation of the ENK gene by glucocorticoids in other cellular systems (30-32). The ENK gene expression is positively regulated by glucocorticoids alone or synergistically with other cel- lular activators, such as CAMP, in cell lines expressing both ENK mRNA and glucocorticoid receptors (32). In general glucocorticoids exert their regulatory influence on neuropeptide gene expression through binding to a specific glucocorticoid responsive element (GRE) (33) of the sensitive gene. Two GRE-like motifs exist in the 5’- flanking region and in intron A of the rat ENK gene (32, 34). The region-dependent effect on ENK gene expression can be due to cell-specific differences in the transacting factors that regulate expression, such as the corticosteroid receptors. The presence of a functional GRE in the pro- moter of the DYN genes CCK and NPY has not been addressed directly. Our findings indicate that a GRE may exist in the DYN gene. It has also been shown that NPY mRNA levels can be upregulated by dexamethasone treatment, as has been shown in the NPY -expressing cell lines NGlO&15 and PC12 (35), and a decrease in NPY mRNA levels has been observed in hypothalamus and striatum after 4 days of ADX (36). The postsurgery period seems the most prominent difference between this and our study. Although we did not detect effects of ADX on NPY gene expression, the NPY gene is a candidate for a glucocorticoid-sensitive neuropeptide gene. In the hippocampus and in the striatum, opioid mRNA levels are decreased after AM ADX, but not after PM ADX. Thus, the results revealed a persistent state-de- pendent effect of ADX, suggesting that ADX alters ac- tivity in these brain regions. However, only the hippo- campal ENK mRNA levels depended on the circulating corticosterone level at the time of ADX. Previous studies have shown that also the central responsiveness to ex- ogenous opioids is under a similar long-lasting state-de- pendent control of glucocorticoids. In these experiments Ratka et al. (10, 11) showed that AM ADX resulted in a considerable decrease in antinociceptive efficiency of ICV given morphine and /3-endorphin. Moreover, exogenous t3H]diprenorphine was retained in larger amounts in lim- bit and striatal regions following AM ADX, suggesting larger availability of opioid receptors. These observations can be taken as indicators for the ADX-induced hypoac- tivity of opioid synthesis and release in the brain regions; the present study indeed revealed reduced opioid mRNA levels under these conditions. Substitution by injection of glucocortiocoids before, and hereby mimicking the PM situation, reduced the antinociceptive activity of endog- enous opiates and reduced [3H]diprenorphine uptake. As shown in the present study, the highest hippocampal ENK mRNA levels occurred when plasma corticosterone levels were high in the PM ADX condition and after AM ADX preceded by corticosterone administration. Although hippocampal ENK mRNA is not likely to be physiolog- ically related to antinociceptive activity and receptor binding of opioids, the ADX- and steroid-induced changes observed in this and previous studies indicate that an opioid system can be modulated in a state-dependent manner at the levels of gene expression, receptor sites, and biological response. In conclusion, this study provides evidence for a time- and region-specific decrease of endogenous opioid mRNAs in ADX rats. In agreement with the behavioral data, the changes in opioid mRNAs also indicate a selective long- lasting regulatory role of glucocorticoids in the activation of opioid systems. ACKNOWLEDGMENTS We are grateful to Dr. J. Douglass for the gift of the rat preprody- norphin clone, to Dr. S. Sabol for the rat preproenkephalin clone, to Dr. H. Persson for the rat neuropeptide Y clone, and to Dr. J. Dixon for the rat cholecystokinin clone. T. Iglesias was supported by the Eu- ropean Training Programme (ETP) in Brain and Behaviour of the Eu- ropean Science Foundation, Strassbourg, France, and by the Stichting Farmacologisch Studiefonds, Utrecht, The Netherlands.
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