Alcohol increases the expression of type 1, but not type 2α corticotropin-releasing factor (CRF) receptor messenger ribonucleic acid in the rat hypothalamus

Molecular Brain Research 52 Ž1997. 78–89

Research report

Alcohol increases the expression of type 1, but not type 2 a corticotropin-releasing factor ž CRF/ receptor messenger ribonucleic acid in the rat hypothalamus Soon Lee, Catherine Rivier

)

Clayton Foundation Laboratories for Peptide Biology, Salk Institute, 10010 N Torrey Pines Road, La Jolla, CA 92037, USA Accepted 25 June 1997

Abstract We investigated the ability of a moderately intoxicating dose of alcohol Ž3 grkg, injected i.p. 3 h earlier. to up-regulate the genetic expression of CRF receptor type 1 ŽCRF-R 1 . and 2 ŽCRF-R 2 a . in the paraventricular nucleus ŽPVN. and supraoptic nucleus ŽSON. of the hypothalamus as well as in the amygdala. The mRNA encoding CRF-R 1 was not constitutively expressed in the PVN or the SON but was present in the amygdala. Alcohol selectively up-regulated CRF-R 1 transcripts in the PVN. Basal levels of CRF-R 2 a transcripts were present in the limbic system and the ventromedial hypothalamic nucleus but were not altered by alcohol. We then determined whether the up-regulation of hypothalamic CRF-R 1 mRNA levels was functionally connected to CRF-dependent pathways. We first showed that the i.c.v. injection of CRF significantly Ž P - 0.01. increased CRF-R 1 but not CRF-R 2 a mRNA levels. We then injected the CRF antagonist, astressin, i.c.v. 30 min prior to alcohol, at a dose previously shown to completely block many CRF-dependent events in the brain, and found that it did not significantly interfere with alcohol-induced gene expression of PVN CRF-R 1. These results indicate that acute alcohol treatment selectively activates CRF-R 1 in the endocrine hypothalamus and that this response does not appear to depend on the stimulation of CRF receptors. In contrast, no up-regulation of CRF-R 1 or CRF-R 2 a gene expression was observed in extrahypothalamic regions thought to participate in the behavioral influence of alcohol. q 1997 Elsevier Science B.V. Keywords: Alcohol; Hypothalamus; Corticotropin-releasing factor ŽCRF. receptor; Rat

1. Introduction Administration of alcohol to rodents induces a vast array of metabolic, behavioral and neuroendocrine responses. While a number of mechanisms have been invoked to explain these effects, one secretagogue has attracted particular attention, viz. corticotropin-releasing factor ŽCRF.. This peptide, originally identified on the basis of its ability to stimulate ACTH secretion w67x, was subsequently found to mediate many homeostatic responses, including those pertaining to the autonomic, cardiovascular and behavioral systems Žsee w66x.. CRF also acts as a neurotransmitter in the brain as well as the periphery and plays a major role in the integration of the overall physiological and behavioral responses of the animal to noxious

) Corresponding author. Fax: q1 Ž619. 552-1546; E-mail: [email protected]

0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 2 6 - X

stimuli Žsee w19,32,56x.. We have previously shown that in rats, alcohol injection up-regulated CRF heteronuclear RNA ŽCRF hnRNA. levels in the paraventricular nucleus ŽPVN. of the hypothalamus w59x, which is the primary site in the brain responsible for ACTH release w65x. We also reported that this activation was necessary for the subsequent release of ACTH in response to the drug w55,58x. CRF initiates its biological effects by binding to plasma membrane receptors which are widely distributed throughout the brain w17x and which mediate CRF-stimulated events, such as increases in intracellular cAMP w6x. Studies using labeled ligands w16x have indicated relatively low CRF levels in the rat PVN and the central nucleus of the amygdala under resting conditions, and the recent cloning of CRF type 1 receptors ŽCRF-R 1 . w12,14x has corroborated these findings w9,18x. A second subtype of CRF receptors has been characterized and called type 2 w36x. Type 2 CRF receptors ŽCRF-R 2 . have two known forms, CRF2 a and CRF2 b , that appear to be confined to subcorti-

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Fig. 1. Representative example of the distribution of mRNA encoding CRF-R 1 in the rat brain after the injection of the vehicle, alcohol Ž3 grkg i.p.. or alcohol preceded by the CRF antagonist astressin Ž15 m g i.c.v., 30 min prior to alcohol.. Brains were obtained 3 h after alcohol treatment. Dark-field photomicrographs taken from nuclear emulsion dipped 30-m m coronal sections hybridized with a w 33 Pxriboprobe for CRF-R 1 mRNA in the PVN. Note the absence of any positive signal for the mRNA encoding CRF-R 1 in the PVN of vehicle-treated rats and a strong signal in this hypothalamic region after alcohol treatment. III, 3rd ventricle. Magnification =350.

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S. Lee, C. RiÕierr Molecular Brain Research 52 (1997) 78–89

mRNA levels in the amygdala, an area that responds to alcohol with increased neuronal activity w13x and which modulates PVN function w3,4,20,40x, do not appear to be altered by physical stimuli such as immobilization stress w41x. In view of the stimulatory influence of alcohol on the HPA axis w57x, we investigated the possibility that a moderately intoxicating dose of alcohol Ž3 grkg. might influence steady-state CRF receptors mRNA levels in the neuroendocrine hypothalamus. In addition, we were interested in determining whether alcohol would up-regulate CRF receptors mRNA in extrahypothalamic areas, such as the limbic system, that are thought to be important for non-endocrine effects of alcohol that involve CRF-dependent pathways w45,50x. We show here that acute alcohol exposure significantly augmented steady-state mRNA levels of CRF-R 1 , but not CRF-R 2 , in the PVN. In contrast, there were no changes in the SON or in extrahypothalamic regions, such as the amygdala and hippocampus. Because CRF can stimulate its own synthesis via an ultra-short loop mechanism w49x and because, as mentioned above, acute alcohol increases hypothalamic CRF biosynthesis w59x, we tested the hypothesis that alcohol-induced increased mRNA levels of CRF-R 1 in the PVN might be secondary to an up-regulation of CRF-dependent pathways. As a first step, we determined whether the injection of CRF into the brain ventricles altered CRF-R 1 signals and found, as was reported by other investigators while our own work was in progress w26,42x, that this was the case. However, when we

Fig. 2. Statistical analysis of the effect of alcohol on PVN CRF-R 1 mRNA levels, showing a statistically significant Ž P - 0.01. alcohol-induced increase in CRF-R 1 mRNA levels in the pPVN, but not in the mPVN, and the inability of prior blockade of CRF receptors to interfere with this response. Each bar represents the mean"S.E.M. percentage of vehicle values Ž ns 4–6 ratsrgroup.. O.D., optical density. ) ) P - 0.01 and y P ) 0.05.

cal structures, including the amygdala and the ventromedial nucleus of the hypothalamus Žin the case of CRF-R 2 a . and the choroid plexus Žfor CRF-R 2 b . w9,34,36,53x. At present, the general consensus is that CRF-R 1 , whose anatomical distribution shows a high degree of correlation with that of CRF itself, represent the primary neuroendocrine receptors while type 2 receptors may preferentially be involved in autonomic and behavioral actions of central CRF w9x. A variety of homeostatic threats, including immune, osmotic, emotional and physical challenges, have recently been shown to up-regulate CRF-R 1 mRNA in the PVN w34,37,38,41,47,52,54x. Changes in osmolarity, which may have relevance in alcohol paradigms, also increase CRF-R 1 transcripts in the supraoptic nucleus ŽSON. w37x while

Fig. 3. Statistical analysis of the effect of alcohol on amygdala CRF-R 1 mRNA levels. Alcohol treatment or pre-treatment with astressin Ž15 m g i.c.v., 30 min prior to alcohol. had no statistically significant effect Ž P ) 0.05. on CRF-R 1 mRNA levels in the basolateral nucleus of amygdala. Each bar represents the mean"S.E.M. percentage of vehicle values Ž ns 4–7 ratsrgroup..

S. Lee, C. RiÕierr Molecular Brain Research 52 (1997) 78–89

examined the role played by alcohol-induced increases in hypothalamic CRF levels in mediating the up-regulation of PVN CRF-R 1 , we found that the potent CRF antagonist astressin w24x injected into the brain ventricle 30 min prior to alcohol treatment, failed to alter alcohol-induced up-regulation of CRF-R 1 transcripts.

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2. Materials and methods 2.1. Animals Adult male Sprague-Dawley rats Žf 60 days of age., kept under standard laboratory conditions, were implanted

Fig. 4. Representative example of the distribution of hnRNA or mRNA encoding CRF in the rat brain after the injection of the vehicle, alcohol Ž3 grkg i.p.. or alcohol preceded by the CRF antagonist astressin Ž15 m g i.c.v., 30 min prior to alcohol.. Brains were obtained 3 h after alcohol treatment. Autoradiographic photos depict a coronal section hybridized with a 33 P-riboprobe for CRF hnRNA on X-ray film Žleft panel. and dark-field photographs taken from nuclear emulsion dipped 30-m m coronal sections hybridized with a w 35 Sxriboprobe for CRF mRNA in the pPVN Žright panel.. Note the absence of any positive signal for the hnRNA encoding CRF in the PVN of vehicle-treated rats and a strong signal in this hypothalamic region after alcohol treatment. Magnification =350.

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with i.p. cannulae used to inject alcohol as previously described w59x. In order to avoid abdominal distress and the potential ensuing stress, alcohol was diluted with saline so as to achieve a maximum concentration of 20% vrv. Control rats received an i.p. injection of an equivalent volume of saline. Preliminary experiments were carried out to determine the doses of alcohol that would induce reliable and consistent changes, and a concentration of 3 grkg, injected once, was subsequently selected for the results presented here. This dose, which significantly elevates plasma ACTH levels for f 2 h, induces peak blood alcohol levels of G 0.23 mg% wrv w48x. We also conducted preliminary studies to determine peak transcript responses and, on the basis of these results, chose a 3-h time point for the studies illustrated here. For experiments designed to determine the consequence of blocking CRF receptors, the CRF antagonist astressin, which interferes with CRF-dependent pathways in the brain w39,44,64x, was administered i.c.v. 30 min prior to alcohol. A dose of 15 m g in 5 m l was infused at the rate of 1 m lr10 s. All protocols were approved by the Salk Institute IACUC.

Fig. 5. Statistical analysis of the effect of alcohol on PVN CRF mRNA levels. Alcohol treatment Ž3 grkg i.p.. or pre-treatment with astressin Ž15 m g i.c.v., 30 min prior to alcohol. had no significant effect Ž P ) 0.05. on CRF mRNA levels in the parvocellular division of the paraventricular nucleus ŽPVN.. Each bar represents the mean"S.E.M. precentage of vehicle values Ž ns 3–5 ratsrgroup..

2.2. Reagents Astressin, a CRF antagonist that shows relative selectivity for CRF-R 1 w24x and ovine CRF were synthesized by solid-phase methodology w33x and generously provided by Dr. Jean Rivier ŽSalk Institute.. The peptides were dissolved in apyrogenic water and the pH was brought to 7.3–7.4 prior to injection. Control rats were administered apyrogenic water, pH 7.35.

w 33 PxUTP, 1 U RNAsin ŽPromega, Madison, WI. and 10 U SP6 Žfor CRF., T7 Žfor CRF-R 1 and CRF intron. or T3 Žfor CRF-R 2 a . for 60 min at 378C. Unincorporated nucleotides were removed using Quick-Spin columns ŽBoehringer Mannheim, Indianapolis, IN.. A sense probe was used as a control for non-specific signal in some adjacent sections for in situ hybridization. 2.4. In situ hybridization histochemistry

2.3. cRNA probe synthesis and preparation The pGEM4 plasmid containing a 1.2-kb EcoRI fragment of rat CRF cDNA ŽDr. K. Mayo. and pGEM3 plasmid containing an EcoRI fragment of CRF intron ŽDr. S. Watson. were linearized with HindIII. Rat CRF-R 1 cDNA ŽDr. W. Vale., subcloned into pBluescript SK-1 vector ŽStragene, La Jolla., was linearized with BamHI. CRF-R 2 a cRNA probe was produced from a 460-bp cDNA fragment of the CRF-R 2 a ŽNeurocrine, La Jolla. subcloned into pBluescriptSKq and linearized with XbaI. Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl 2 , 36 mM Tris ŽpH 7.5., 2 mM spermidine, 8 mM dithithreitol, 25 mM ATPrGTPrCTP, 5 mm unlabeled UTP, w a- 35 Sx- or

Three hours after vehicle or alcohol treatment, rats were deeply anesthetized with chloral hydrate, a drug that does not increase immediate-early genesrpeptides mRNA levels. They were then perfused transcardially with saline followed by 4% paraformaldehyder0.1 M borate buffer, pH 9.5. The brains were removed and post-fixed in 4% paraformaldehyde for 4–5 days, then placed overnight in 10% sucroser4% paraformaldehyder0.1 M borate buffer. They were cut into 30-m m coronal slices obtained at 120-m m intervals throughout the hypothalamus and stored at y208C in a cryoprotectant solution Ž50% 0.1 M phosphate-buffered saline, 30% ethylene glycol and 20% glycerol. until histochemical analysis. Brains from control and experimental animals belonging to the same experiment

Fig. 6. Representative example of the distribution of the mRNA encoding CRF-R 2 a in a coronal section of the rat brain, obtained 3 h after the injection of the vehicle, alcohol Ž3 grkg i.p.. or alcohol preceded by the CRF antagonist astressin Ž15 m g i.c.v., 30 min prior to alcohol.. Autoradiographic photos displaying brain sections hybridized with a w 33 Pxriboprobe for CRF-R 2 a mRNA on X-ray film. Constitutive CRF-R 2 a mRNA expression was found in the amygdala, the hippocampus, the supraoptic nucleus and the ventromedial nucleus of the hypothalamus ŽVMH.. These signals were unaltered after alcohol treatment or pre-treatment with astressin Žsee Table 1.. Note the absence of any hybridization signals in the vehicle-treated rats.

S. Lee, C. RiÕierr Molecular Brain Research 52 (1997) 78–89

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were always analyzed in the same hybridization experiment. Hybridization histochemical localization of each transcript was carried out using 35 S- or 33 P-labeled cRNA probes. Protocols for riboprobe synthesis, hybridization and autoradiographic localization of mRNA signals were adapted from Simmons et al. w63x. All solutions were treated with diethylpyrocarbonate ŽDep. C. and sterilized in order to prevent RNA degradation. Sections mounted onto gelatin- and poly-L-lysine-coated slides were desiccated under vacuum overnight, fixed in 4% paraformaldehyde for 30 min and digested by proteinase K Ž10 m grml in 50 mM Tris-HCl ŽpH 7.5. and 5 mM EDTA at 378C for 25 min.. Thereafter, brain sections were rinsed in sterile Dep. C water followed by a solution of 0.1 M triethanolamine ŽTEA; pH 8.0., acetylated in 0.25% acetic anhydride in 0.1 M TEA and dehydrated through graded concentrations of alcohol Ž50, 70, 95 and 100%.. After vacuum drying for a minimum of 2 h, 90 m l hybridization mixture Ž10 7 cpmrml. was spotted on each slide, sealed under a coverslip and incubated at 608C overnight in a slide warmer. Coverslips were then removed and the slides rinsed in 4 = SSC Ž1 = SSC: 0.15 M NaCl, 15 mM trisodium citrate buffer, pH 7.0. at room temperature. Sections were digested by RNase A Ž20 m grml, 378C, 30 min., rinsed in descending concentrations of SSC Ž2 = , 1 = , 0.5 = SSC., washed in 0.1 = SSC for 15 min at 658C and dehydrated through graded concentrations of alcohol. After being dried under the vacuum, sections were exposed at 48C to X-ray film ŽKodak. for 15–72 h, defatted in xylene and dipped in NTB2 nuclear emulsion ŽKodak; diluted 1 : 1 with distilled water.. Slides were exposed for 6–64 days, developed in D19 developer ŽKodak. for 3.5 min at 158C and fixed in rapid fixer ŽKodak. for 6 min. Thereafter, tissues were rinsed in running distilled water, counterstained with thionin Ž0.25%., dehydrated through graded concentrations of alcohol, cleared in xylene and coverslipped with DPX. 2.5. QuantitatiÕe analysis of in situ hybridization results Semiquantitative densitometric analysis of hybridization signals for RNAs of interest was carried out in nuclear emulsion-dipped slides. Brain paste standards containing serial dilutions of w 35 SxUTP or w 33 PxUTP, used for quantification of mRNA signal, were prepared concurrently to ensure that optical density was found within the linear range of the standard curve w11x. In addition, analyses with emulsion-coated slides were carried out with 2–3 different exposure times in order to confirm that signals were not saturated. Densitometric analyses of autoradiographic signals was done over the confines of cells within the PVN using a Leitz optical system coupled to a Macintosh II computer and Image software Žversion 1.60, W Rasband, NIH.. Dark-field measurements for the parvo- and magnocellular divisions of the PVN ŽpPVN and mPVN. were obtained separately. We also analyzed the supraoptic nucleus ŽSON. and the amygdala in all the sections. Gray

level measurements were taken under dark-field illumination of hybridized sections over the medial pPVN, mPVN, SON or amygdala, as defined by redirected sampling from the corresponding Nissl-stained sections under bright-field images. Data were expressed in gray scale values of 1–256. All gray level measurements were corrected for background. Signals were measured in both sides of the brain and mean values for all animals Ž4–6rgroup. were determined in 3–4 sections for each rat throughout the PVN. 2.6. Statistical analysis RIA results were analyzed by repeated measured analysis of variance and multiple comparisons were performed using the Duncan multiple range test. Data of in situ hybridization experiments are expressed as percentage of control reading and were analyzed by one-way analysis of variance, and post-hoc comparisons using Scheffe´ test ŽStatview II.. All brain sections hybridized at the same time were included in a single statistical test.

3. Results 3.1. Effect of alcohol treatment on CRF-R 1 and CRF transcripts and interaction with astressin Fig. 1 illustrates a representative example of the distribution of the CRF-R 1 gene expression in coronal sections of the PVN of a male Sprague-Dawley rat injected with the vehicle, alcohol Ž3 grkg i.p.. or alcohol preceded by the potent CRF antagonist astressin Ž15 m g i.c.v., y30 min.. As previously reported w38,46,52,54x, we found no constitutive CRF-R 1 transcripts in the prmPVN or the SON of vehicle-treated rats while these signals were readily found in the amygdala Žbasolateral and medial nuclei.. Alcohol treatment significantly Ž P - 0.01. increased mRNA levels of CRF-R 1 in the pPVN but not in the mPVN Žsee Fig. 2 for statistical analysis of the results. or the amygdala ŽFig. 3.. When we examined the importance of activation of CRF receptors in this response, we observed that pre-treatment with astressin did not significantly interfere with the alcohol-induced up-regulation of pPVN CRF-R 1 mRNA levels. We also confirmed here our previously published results w59x indicating that acute alcohol treatment significantly increased CRF hnRNA but not mRNA levels in the pPVN. The expression of the CRF intron was not significantly decreased by astressin ŽFigs. 4 and 5.. 3.2. Effect of alcohol on CRF-R 2 a transcripts and interaction with astressin As previously reported w9,34,36,53x, constitutive CRFR 2 a signals were primarily found in the amygdala, the hippocampus and the ventromedial nucleus of the hypo-

S. Lee, C. RiÕierr Molecular Brain Research 52 (1997) 78–89 Table 1 Statistical analysis of the effect of alcohol on CRF-R 2 a mRNA levels Region

Treatment

Relative mRNA levelsa Ž% vehicle.

VMH

Vehicle Alcohol Alcoholqastressin Vehicle Alcohol Alcoholqastressin

100.0"11.2 108.0"4.9 103.9"13.0 100.0"6.6 84.9"8.9 101.3"8.0

SON

a

Values are based on gray level measurements and are expressed as mean"S.E.M. percentage of vehicle values; ns 3–7 ratsrgroup. VMH, ventromedial hypothalamus nucleus; SON, supraoptic nucleus.

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thalamus ŽFig. 6, Table 1. and the SON ŽTable 1. while, as also discussed by others w53x, these receptors were not detectable in the hypothalamic PVN. Alcohol administration failed to induce significant increases in CRF-R 2 a transcripts in the brain areas in which these transcripts were already present, nor did CRF-R 2 a mRNA appear in regions where it was not detected under basal conditions. 3.3. Effect of the i.c.Õ. injection of CRF on CRF-R 1 and CRF-R 2 a transcripts The i.c.v. injection of the vehicle did not cause the appearance of CRF-R 1 transcripts in the pPVN or in the

Fig. 7. Representative example of the distribution of the mRNA encoding CRF-R 1 in a coronal section rat brain, obtained 2–4 h after the injection of the vehicle or CRF Ž1 m g i.c.v... Autoradiographic photos displaying brain sections hybridized with a w 33 Pxriboprobe for CRF-R 1 mRNA on X-ray film. CRF-R 1 mRNA signal was not detectable in the PVN of the vehicle-treated rats. The i.c.v. injection of CRF significantly increased CRF-R 1 mRNA levels in the PVN.

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SON and did not alter basal levels in the amygdala ŽFig. 7.. In contrast, the i.c.v. injection of 1 m g CRF significantly Ž P - 0.01. increased CRF-R 1 mRNA levels in the pPVN, a finding also reported by others w26,42x. We found steady-state CRF-R 2 a mRNA levels in the same regions as described above and no further changes were observed following i.c.v. CRF treatment.

4. Discussion We show here that CRF-R 1 transcripts, which were virtually undetectable in control rats, became significantly up-regulated in the pPVN of rats acutely administered a moderately intoxicating dose of alcohol. These results agree with reports illustrating the ability of other homeostatic threats, such as immobilization stress w29,41x and endotoxemia w34,46,54x, to stimulate CRF-R 1 expression in the neuroendocrine hypothalamus. Stimulation of sensory terminals in the peritoneum can activate the PVN wsee in particular w21,28,60xx. However, it is unlikely that this mechanism is responsible for the influence of alcohol on hypothalamic CRF and CRF-R 1 levels because we have recently observed comparable responses in rats admnistered the drug via an intragastric cannula w48x. In view of the influence of alcohol on osmolarity on one hand w51x and of the up-regulation of CRF-R 1 transcripts in the SON of rats injected with hypertonic saline on the other hand w37x, it was of interest to note that CRF-R 1 mRNA levels were not increased in the SON of rats acutely exposed to this drug. While we did not measure plasma ACTH levels in the present work, we have conducted many experiments indicating that peak hormone response takes place within 15–30 min of drug treatment w57–59x. This means that the appearance of CRF-R 1 transcripts in the PVN is significantly delayed in comparison with the time-frame of ACTH secretion, an observation that suggests that increases in CRF-R 1 transcripts are probably not related to the immediate ACTH response to alcohol. Similar findings obtained in other stress paradigms w15,54x have suggested that changes in CRF-R 1 synthesis may be important in amplifying andror strengthening CRF-dependent responses. Indeed, the observation that the transcription of CRF-R 1 occurs at least 1 h after the appearance of hypothalamic CRF hnRNA and c-fos mRNA, has led several investigators to propose that increases in these receptors might either represent a functional adaptation of the HPA axis to stress – i.e. help prepare the organism for subsequent challenges – or result from the eventual decreases in corticosterone and the feedback imposed by this steroid w37,42x. The ability of CRF to stimulate its own transcription w49x as well as that of its type 1 receptors in the endocrine hypothalamus w26,42x, and the presence of CRF-containing fibers that connect with PVN CRF perikarya originating from within the PVN itself w62x or from other hypothalamic nuclei w2x, have indicated that

CRF synthesis is regulated through an ultra-short looppositive feedback mechanism Žsee in particular w5x.. The presence of CRF-R 1 on CRF perikarya w54x thus suggests that alcohol, through specific increases in the transcription of these receptors, could induce a positive feedback serving to amplify the CNS effects of this peptide. In this regard, it is of interest that in both humans and laboratory animals, the repeated use of various drugs produces a hypersensitivity to the behavioral effects of these drugs that persists for long periods of time, a phenomenon thought to be mediated at least in part by CRF w7,8x. These observations led us to consider the possibility that alcohol might increase levels of either CRF itself andror of CRF-R 1 , through a CRF-dependent mechanism. We reasoned that if this was the case, interfering with of the action of CRF by a specific receptor antagonist should prevent the stimulatory effect of the drug on CRFrCRF-R 1 synthesis. Using this method, Imaki et al. w27x and Mansi et al. w42x showed that blockade of CRF receptors partially attenuated restraint-induced c-fos as well as CRF-induced CRF-R 1 mRNA expression in the PVN. Our hypothesis proved incorrect because astressin did not significantly decrease the up-regulation of CRF-R 1 transcripts induced by acute alcohol treatment. It is unlikely that our results were due to the use of too small a dose of astressin because on the basis of a similar protocol, several other investigators have found significant decreases in CRF-dependent responses, such as stress-induced emotionality w64x, gastro-intestinal functions w43x and seizure w39x. Our data thus suggest that either astressin was unable to block the type of CRF receptors that are important for the effect of alcohol, or that the drug influences CRF-R 1 through mechanisms not mediated by CRF. In this respect, the possibility that neurotransmitters might increase levels of hypothalamic CRF-R 1 independently of CRF is worthy of investigation. Prostaglandins ŽPGs., which are released by alcohol w22x, are one example, as blockade of their synthesis prevents the stimulatory effect of endotoxemia on these receptors w34x while the central injection of PGE 2 up-regulates PVN CRF-R 1 gene expression w35x. Another potential signal is noradrenaline, whose levels may be influenced by alcohol w1x. Whether this bioamine influences CRF-R 1 mRNA levels and whether this effect depends on hypothalamic CRF for this effect, remains to be determined. Finally, acute alcohol treatment may increase levels of glutamate and serotonin Žsee for example w61,69x. which could alter levels of CRF-R 1 mRNA in the hypothalamus. Alcohol not only stimulates the activity of the HPA axis, it also exerts a wide spectrum of effects within the central nervous system, several of which implicate CRF. For example the amygdala is a brain region known for its role in regulating behavioral and emotional response, such as tolerance and dependence to alcohol and other drugs w25,45,50x. CRF present in the amygdala, where it is thought to act as a neurotransmitter w23x, is considered important for many of these responses Žsee w31x.. While

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our model only used one acute alcohol injection, it was of interest to note that at least during the initial phase of drug exposure, no extrahypothalamic brain regions involved in alcohol reinforcement showed increased CRF-R 1 transcripts. Similarly, we had failed to observe any up-regulation of CRF mRNA or hnRNA levels outside the PVN in rats acutely administered alcohol w59x. It will therefore be important to determine whether long-term treatment with the drug can elicit an increased synthesis of CRF receptors in brain areas other than those directly associated with the control of ACTH release, particularly those thought to be responsible for reinforcing the reward property of alcohol. At present, the role of brain CRF-R 2 a , for which the newly characterized peptide urocortin is believed to be an important endogenous ligand w68x, remains unclear. Abundantly expressed in the ventromedial hypothalamus w10,52x, these receptors may be involved, in particular, in the regulation of energy balance w53x. In addition, their presence in the limbic system has led to the hypothesis that they may modulate the type of autonomic, behavioral and reinforcing actions of central CRF w9x that are induced by alcohol w30,45,50x. It is therefore of interest to note the lack of influence of acute alcohol on the pattern of expression of CRF-R 2 a , a finding in keeping with the present consensus that transcription of the gene encoding for these receptors is not altered by manipulations that influence CRF-R 1 mRNA levels w34,42,46x. The fact that CRF-R 2 a transcripts did not increase or appear in brain areas that are either targeted by alcohol or represent a substrate for its multifaceted effect, suggests that the reinforcing properties of this drug, as well as its influence on mood and behavior, probably do not primarily depend on the activation of CRF-dependent circuitries. In conclusion, we have shown that the acute injection of alcohol significantly up-regulated CRF-R 1 mRNA levels in the rat PVN but not in extrahypothalamic regions, such as the amygdala. The inability of the potent CRF antagonist astressin to attenuate this response, suggests that neurotransmitters other than CRF may participate in the activation of neurons expressing CRF and CRF-R 1.

Acknowledgements We are indebted to S. Johnson, J. Janas, B. d’Arc, Y. Haas and H.S. Wong for excellent technical assistance, and to Dr. Jean Rivier for the gift of astressin. Research supported by NIH Grant AA06420.

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