- Email: [email protected]
Repeated administration of Δ9-tetrahydrocannabinol produces a differential time related responsiveness on proenkephalin, proopiomelanocortin and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat
Neuropharmacology 38 (1999) 433 – 439
Repeated administration of D9-tetrahydrocannabinol produces a differential time related responsiveness on proenkephalin, proopiomelanocortin and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat Javier Corchero, Jorge Manzanares *, Jose´ A. Fuentes Departamento de Farmacologı´a, Facultad de Farmacia and Unidad de Cartografı´a Cerebral, Instituto Pluridisciplinar, A6. Juan XXIII, 1 Uni6ersidad Cornplutense de Madrid, 28040 Madrid, Spain
Abstract The purpose of the present study was to explore the time related effects of repeated administration of D9-tetrahydrocannabinol on opioid and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat. By using in situ hybridization histochemistry, the effects of D9-tetrahydrocannabinol (THC, 5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of repeated administration on; (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN. The results revealed that, in most of the hypothalamic and pituitary regions examined, repeated cannabinoid administration upregulates opioid and corticotropin releasing factor gene expression. However, the onset, the degree of magnitude of gene expression reached and the time related effects produced by repeated administration with D9-tetrahydrocannabinol are dependent upon the brain and pituitary regions examined. Taken together, the results of the present study suggest that cannabinoids produce a time related differential responsiveness in opioid and corticotropin releasing factor gene expression, in areas of the hypothalamus and pituitary that may be related, at least in part, to a molecular integrative response to behavioral, endocrine and neurochemical alterations that occur in cannabinoid drug abuse. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: D9-tetrahydrocannabinol; Proenkephalin; Proopiomelanocortin; Corticotropinreleasing factor; mRNA; Hypothalamus; Pituitary
1. Introduction D9-Tetrahydrocannabinol (THC), the major psychoactive compound of Cannabis sati6a preparations (marijuana, hashish) produces a great variety of pharmacological effects that include antinociception, anxiety-like behavior, hypothermia, depression of motor activity, and alterations in the secretion of pituitary hormones (Dewey, 1986). It has been found that in the brain THC acts via a G-protein coupled receptor (CB1) (Matsuda et al., 1990) whose putative endogenous ligands have been identified as arachidonic acid derivatives (Devane et al., 1992). Sn-2 arachidonyl glicerol has been recently described as a second endogenous * Corresponding author. Tel.: +34-1-3943271; fax: 3943264; e-mail: [email protected].
+34-1-
ligand for cannabinoid receptors, present in the brain in greater amounts than anandamide (Stella et al., 1997). However, a large body of evidence indicates that many of the behavioral and neurochemical effects produced by acute or chronic administration with cannabinoids may involve an interaction between the opioid and the cannabinoid neuronal systems. In recent years, our group and others, have shown that; (1) THC and anandamide modulate the expression of physical signs of opioid dependence (Vela et al., 1995), (2) the antiemetic effect of nabilone, a THC synthetic derivative, is antagonized by the opioid receptor antagonist naloxone (Rang and Dale, 1991), (3) antinociception induced by THC can be blocked by a- or k-opioid receptor antagonists or selective antibodies against the endogenous k ligand dynorphin (Reche et al., 1996a,b), and (4) inhibition of opioid degrading enzymes may
0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 8 ) 0 0 1 9 5 - 6
434
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
potentiate THC induced antinociception (Reche et al., 1998). These results suggest that administration of THC may increase the release of endogenous opioids. Indeed, recently, it has been shown that administration of CP55,940, a selective cannabinoid receptor agonist produces a significant release of dynorphin B in the spinal cord concurrent with the production of antinociception (Pugh et al., 1997). Furthermore, our group has reported that subchronic or chronic administration of THC or cannabinoid receptor agonists increases proopiomelanocortin gene expression in the arcuate nucleus of the hypothalamus (Corchero et al., 1997a), prodynorphin and proenkephalin gene expression in the spinal cord (Corchero et al., 1997b) and proenkephalin gene expression in various forebrain and hypothalamic regions of the rat (Manzanares et al., 1998). Stress behavior induced by drug abuse has been related to an increase in the secretion of corticotropin or corticosterone in the plasma. Administration with cannabinoid agonists caused a significant increase in the secretion of corticotropin (Dewey, 1986; Weidenfeld et al., 1994; Rubio et al., 1995; Rodriguez de Fonseca et al., 1996) that appears to be mediated via a central mechanism involving an increase in the release of corticotropin releasing factor (Weidenfeld et al., 1994). However, the molecular mechanisms by which cannabinoids regulate the hypothalamic pituitary adrenal axis (HPA) are still unknown. The present study was undertaken to explore the molecular interaction between the opioid or the corticotropin releasing factor and the cannabinoid system. Time of onset, differential responsiveness and degree of magnitude obtained after repeated administration with THC on opioid and corticotropin releasing factor gene expression were determined in selected regions of the hypothalamus and pituitary gland of male rats. By using in situ hybridization histochemistry, the effects of D9-tetrahydrocannabinol (5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of administration on: (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN.
2. Materials and methods
2.1. Animals Adult male Sprague – Dawley rats, weighing 200–225 g, were obtained from Interfauna Iberica Laboratories (San Feliu de Codines, Barcelona, Spain) and maintained under conditions of controlled temperature
(239 1°C) and lighting (lights on 08:00–20:00 h), with food and water provided ad libitum. All experiments included in this study were performed following the highest standards of humane animal care, monitoring health care and minimizing pain and suffering, in accordance with the National and International Laws for the Care and Use of Laboratory Animals.
2.2. Drug D9-Tetrahydrocannabinol was dissolved in saline:ethanol:cremophor (18:1:1) and administered (5 mg/kg per day; i.p.; 1 ml/kg) for 1, 3, 7, and 14 days. Twenty four hours after the last injection rats were killed by decapitation and brains and pituitaries were quickly removed and frozen over dry ice. In previous experiments performed in our laboratory (data not shown) 24 h after the last injection, no differences were found in proenkephalin, proopiomelanocortin or corticotropin releasing factor gene expression when compared with the mRNA values of rats treated with vehicle at 1, 3, 7 or 14 day, in any of the brain or pituitary regions examined. Therefore, in the present work rats were treated with vehicle for 7 days, the time point that is in the middle of the whole time course study.
2.3. ln situ hybridization histochemistry Brain sections (six slides/area; two sections/slide) were cut at 20 mm at the level of the PVN, ARC, VMN, and pituitary sections (four slides/area; six sections/ slide) from the AL and IL. Sections were mounted onto gelatin-coated slides and stored at −80°C until the day of the assay. ISHH was performed as described previously (Young et al., 1986) using synthetic 48-bases oligonucleotide probes complementary to proenkephalin (NEN-Dupont, NEP-502, Madrid, Spain), corticotropin releasing factor (NEN-Dupont, NEP-554, Madrid, Spain) and to the nucleotides 96-134 of the rat proopiomelanocortin gene (obtained from the Advanced Biotechnology Center, Charing Cross and Westminster Medical School, London, England). Oligonucleotide probes were labelled using terminal deoxytransferase (Boehringer, Madrid, Spain) to add an a 35S-labeled deoxyATP (1000 Ci mmol − 1; Amersham, Madrid, Spain) tail to the 3% end of the probes. The probes (in 50 m1 of hybridization buffer) were applied to each section and left overnight at 37°C for hybridization. Following hybridization, sections were washed four times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2 (1X saline sodium citrate, SSC) at 55°C, followed by two 30 min washes in 1X SSC at room temperature, one brief water dip and were then blown dry with air. The dried slides were apposed to Kodak BioMax MR-1
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
film (Amersham, Madrid, Spain) for 1 h for the intermediate lobe, for 4 h for anterior lobe of the pituitary gland (POMC) and for 7 days for brain regions hybridized for proopiomelanocortin, proenkephalin and corticotropin releasing factor. Autoradiograms were produced with a Macintosh computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Optical densities were calculated from the uncalibrated mode by substracting from each measurement its corresponding background and expressed in grey scale values. The background measurement was taken from an area of the slice with the lowest nonspecific hybridization signal and substracted from the hybridization signal measurement in the same slice. Measurements were pooled from brain or pituitary sections and the values were averaged. Results were presented considering mean control values as 100%. Statistical analyses were performed using one-way analysis of variance followed by the Student Newman–Keul’s test. Differences were considered significant if the probability of error was less than 5%.
3. Results
3.1. Effects of THC on proenkephalin gene expression in the PVN and VMN of the hypothalamus The one-way analysis of variance of the results revealed that repeated administration with THC produced a marked increase of proenkephalin mRNA levels in the PVN (F(4,27) =7.39, P B 0.0006) and in the VMN (F(4,34)=28.50, P B 0.0001). In the PVN, treatment with THC produced a time related increase in proenkephalin mRNA levels reaching a 65% change from vehicle-treated rats on day 14. In contrast, proenkephalin mRNA levels in the VMN increased by day 3(+28%) and day 7 (+38%) and then returned to those of vehicle-treated rats on day 14 (Fig. 1). As shown in Fig. 2, representative autoradiograms of THC-treated rats (at specific time points) used to analyze proenkephalin mRNA levels in the PVN and VMN clearly present a more pronounced intensity in the hybridization signal than those corresponding to vehicle-treated rats.
435
Fig. 1. Time course of effects of repeated injections with D9-tetrahydrocannabinol on PENK mRNA levels in the PVN and VMN in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1); 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines + 1 SEM of PENK mRNA levels in six to eight rats.*, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B0.05) from vehicle-treated rats.
day 7 ( + 23%) and then returned to vehicle-treated values by day 14 (+ 6%). The one-way analysis of variance of the data obtained after repeated administration with THC in the AL revealed statistical significant differences (F(4,35)=19.8, PB 0.0001) in proopiomelanocortin mRNA levels. The post-hoc analysis revealed that proopiomelanocortin mRNA levels in the AL increased + 48% by day 1, and then progressively decreased (−52%) by day 14 (Fig. 3B). In contrast, proopiomelanocortin mRNA levels in the IL decreased (−25%) only on day 14 after administration with THC
3.2. Effects of THC on proopiomelanocortin gene expression in the ARC of the hypothalamus and AL and IL of the pituitary gland As depicted in Fig. 3A, repeated administration with THC produced a marked increase in proopiomelanocortin mRNA levels in the ARC of the hypothalamus (F(4,33)=21.3, P B0.0001) by day 3 (+ 27%) and
Fig. 2. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on PENK mRNA. Representative autoradiograms of coronal brain sections at the level of the PVN and VMN of the rat hypothalamus. Slides were apposed to film (Kodak, Biomax, MR-1) for 7 days. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
436
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
Fig. 3. Time course of effects of repeated injections with D9-tetrahydrocannabinol on POMC mRNA levels in the ARC (A), AL (B) and IL (C) in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1; 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines +1 SEM. of POMC mRNA levels in six to eight rats.*, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B 0.05) from vehicle-treated rats.
(F(4,33)= 3.90, PB 0.0118) and no changes were found at any of the other time points examined (Fig. 3C). Representative autoradiograms at time points where statistical significance was found in proopiomelanocortin mRNA levels between THC- and vehicle-treated group of the ARC, AL and IL are shown in Fig. 4.
3.3. Effects of THC on corticotropin releasing factor gene expression in the PVN of the hypothalamus Repeated administration with THC markedly increased (F(4,28)= 5.69, P B0.0023) corticotropin releasing factor mRNA levels in the PVN. The post-hoc analysis indicated that a statistical significant increase was only found after 3 days of THC-administration ( +85%). However, although not statistically significant, a +34% increase was found on day 1, (+51%) on day 7, and ( + 22%) on day 14 (Fig. 5). Representative autoradiograms at time points where statistical significance was found in corticotropin releasing factor mRNA levels between THC- and vehicle-treated groups in the PVN are shown in Fig. 6.
4. Discussion The results of the present study demonstrate that exposure to THC markedly increases corticotropin-releasing factor gene expression in the PVN of the hypothalamus. Although some similarities were found in the response of opioid and corticotropin releasing factor genes examined after THC, important regional differences have been detected in the degree of magnitude of each specific gene expression regulation and the onset of this process. Interestingly, as a probe of the system dynamics, decreases in proopiomelanocortin mRNA levels were also found at some of the time intervals examined during THC chronic treatment. In addition,
in most of the brain regions examined, administration with THC upregulates proenkephalin and proopiomelanocortin gene expression in a way which follows the response pattern found in previous studies (Corchero et al., 1997a,b; Manzanares et al., 1998). We have previously shown that subchronic and chronic treatment with cannabinoids upregulates proenkephalin gene expression in the PVN and VMN (Manzanares et al., 1998). In the present study, proenkephalin gene expression in the PVN progressively increased after repeated administration with THC reaching a 65% increase on day 14. However, in the VMN the maximum increase was found after 7 days of treatment and then mRNA levels returned to those of vehicle-treated animals by day 14. In this respect, it appears that proenkephalin in the PVN presents a greater sensitivity to THC treatment than in the VMN. In contrast, the onset of the THC action on proenkephalin gene expression in the VMN occurs very rapidly, but unlike in the PVN, prolonged THC treatment progressively downregulates mRNA levels. These differential responses in proenkephalin gene expression between the PVN and VMN after a long exposure to THC may be related to specific transcription factors that differentially affect gene regulation in each brain region and/or are the result of a neuroadaptative distinct responsiveness to cannabinoid administration. Gonadal steroids and physiological stressors affect the regulation of proenkephalin gene expression in the PVN and VMN (Garcı´a-Garcı´a et al., 1998a,b; Lightman, 1988; Lightman and Young, 1988; Romano et al., 1989; Priest et al., 1995, 1997). Using a transgenic model that expresses a human proenkephalin promoter/ bacterial beta-galactosidase fusion gene (ENK-1) (Borsook et al., 1994), Priest and coworkers have demonstrated that stress and estrogen differentially regulate proenkephalin gene expression in the PVN and
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
VMN suggesting that stress and estrogen have sex-specific and site-specific regulatory effects on the expression of a proenkephalin promoter transgene in hypothalamic neurons (Priest et al., 1997). The differential time course response between the PVN and VMN found in this study after exposure to cannabinoids may be related to a distinct regulation of the proenkephalin gene in these hypothalamic nuclei. The results of this study are consistent with a previous report by our laboratory (Corchero et al., 1997a), showing that administration of THC for 5 days increased proopiomelanocortin gene expression in the ARC whereas no effect was observed in the AL of the pituitary. However, in the present study, marked differences have been found in the onset, the degree of magnitude and the pattern that regulates the expression of the proopiomelanocortin gene in the brain and pituitary regions examined after repeated administration with THC. Proopiomelanocortin mRNA levels increased in the ARC of the hypothalamus 3 and 7 days after treatment with THC at a similar range to that observed previously (Corchero et al., 1997a) and progressively returned to vehicle levels on day 14. It is interesting that proopiomelanocortin gene expression in
437
the anterior lobe of the pituitary gland apparently is the most responsive to THC treatment, as it is induced at the earliest time. Indeed, proopiomelanocortin gene expression in the AL of the pituitary gland exhibited the highest responsiveness to THC administration, reaching a 48% increase 1 day after drug administration, although proopiomelanocortin transcriptional activity decreased in a time related manner up to −52% on day 14. However,in the IL of the pituitary the onset of THC action is slow and only a 25% decrease was found on day 14. The regulatory mechanisms by which THC differentially alters proopiomelanocortin gene expression in the hypothalamus and regions of the pituitary are still unclear. This gene processes a number of proteins including b-lipotropin, a, b, and g-melanocyte stimulating hormones, adrenocorticotropin hormone (ACTH) and b-endorphin (Eipper and Mains, 1980). In the anterior pituitary, ACTH and b-lipotropin are the predominant proopiomelanocortin related peptides, whereas in the arcuate nucleus of the hypothalamus and intermediate lobe b-endorphin is the major end product of proopiomelanocortin gene processing (Gramsch et al., 1980; Emerson and Eipper, 1986). It
Fig. 4. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on POMC mRNA. Representative autoradiograms of coronal brain sections at the level of the ARC, AL and IL of the rat. Slides were apposed to film (Kodak, Biomax, MR-1) 1 h for IL, 6 h for AL and 7 days for ARC. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
438
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
Fig. 5. Time course of effects of repeated injections with D9-tetrahydrocannabinol on corticotropin releasing factor mRNA levels in the PVN of the hypothalamus in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1; 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines+ 1 SEM of corticotropin releasing factor mRNA levels in six to eight rats. *, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B0.05) from vehicletreated rats.
has been suggested that proopiomelanocortin gene expression is differentially regulated by corticotropin releasing factor in the ARC as well as in the AL or IL of the pituitary. In vitro corticotropin releasing factor stimulates the release of b-endorphin from hypothalamic slices (Nikolarakis et al., 1986), suggesting that corticotropin releasing factor neurons in the PVN, in addition to releasing their content into the portal vessels, also stimulate proopiomelanocortin neurons in the ARC. Furthermore, chronic activation of corticotropin releasing factor system increases proopiomelanocortin gene expression in the anterior lobe but decreases it in the intermediate lobe (Ho¨llt and Haarman, 1984). The results of the present study suggest that administration of THC differentially modifies the expression of the proopiomelanocortin gene in the brain and pituitary regions examined by a mechanism that may be mediated, at least in part, via activation of the corticotropin releasing factor hypothalamic system. Indeed, repeated
administration of THC rapidly and markedly increased (day 3, + 85%) corticotropin releasing factor gene expression in the PVN and then slowly decreased, until on day 14 mRNA level were 22% higher than vehicle values. The activation of corticotropin releasing factor gene expression could be produced as part of a compensatory response to daily activation of the HPA axis hormone secretion, since administration of cannabinoid receptor agonists increases both adrenocorticotropin releasing hormone and corticosterone secretion in rats (Weidenfeld et al., 1994; Rodriguez de Fonseca et al., 1996). In addition, it has been suggested that long term exposure to cannabinoids leads to neuroadaptative changes that result in an enhanced release of corticotropin releasing factor in the central amygdala as well as an activation of stress-responsive nuclei (Rodriguez de Fonseca et al., 1997). Exposure to different stressors or drug abuse (cocaine, opiates, ethanol) leads to significant alterations in the corticotropin releasing factor gene expression in the PVN of the hypothalamus (Lightman and Young, 1988; Rivier et al., 1990; Rivier and Lee, 1994). Taken together, the differences found in the activation of the corticotropin releasing factor gene expression in this study, may be part of the adaptative response produced by cannabinoid induced anxiety. In conclusion, the results of the present study indicate, that chronic exposure to THC affects in a different time dependent manner endogenous opioid and corticotropin releasing factor gene expression and provides evidence of molecular alterations that may be relevant to further understand a variety of behavioral and neuroendocrine effects that occur in cannabinoid drug abuse.
Acknowledgements This work was supported by the Universidad Complutense of Madrid grant PR294/95-6189, Concerted Action from the European Union grant BMH1-CT-941108 and Spanish Ministry of Education grant DGICYT UE95 0017. J. Corchero is a Predoctoral Fellow supported by the ‘Comunidad Autonoma de Madrid’ and J. Manzanares is a Senior Fellow supported by the Spanish Ministry of Education.
References Fig. 6. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on corticotropin releasing factor (CRF) mRNA. Representative autoradiograms of coronal brain sections at the level of the PVN of the rat hypothalamus. Slides were apposed to film (Kodak, Biomax, MR-1) for 7 days. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
Borsook, D., Falkowski, O., Burstein, R., Strassman, A., Konradi, C., Dauber, A., Comb, M., Hyman, S.E., 1994. Stress-induced regulation of a human proenkephalin-b-galactosidase fusion gene in the hypothalamus of transgenic mice. Mol. Endocrinol. 8, 116 – 125. Corchero, J., Fuentes, J.A., Manzanares, J., 1997. D9-tetrahydrocannabinol increases proopiomelanocortin gene expression in the
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439 arcuate nucleus of the rat hypothalamus. Eur. J. Pharmacol. 323, 193 – 195. Corchero, J., Avila, M.A., Fuentes, J.A., Manzanares, J., 1997. D9-tetrahydrocannabinol increases prodynorphin and preproenkephalin gene expression in the spinal cord of the rat. Life Sci. 61, PL39 – PL43. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., et al., 1992. Isolation and structure of a brain constituent that binds to the cannabinod receptor. Science 258, 1946–1949. Dewey, W.L., 1986. Cannabinoid Pharmacology. Pharmacol. Rev. 38, 151 – 178. Eipper, B.A., Mains, R.E., 1980. Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1, 1 – 27. Emerson, R.B., Eipper, B.A., 1986. Characterization of pro-ACTH/ endorphin derived peptides in rat hypothalamus. J. Neurosci. 6, 837 – 849. Garcı´a-Garcı´a, L., Harbuz, M., Manzanares, J., Lightman, S.L., Fuentes, J.A., 1998. RU-486 blocks stress-induced enhancement of proenkephalin gene expression in the paraventricular nucleus of rat hypothalamus. Brain Res. 786, 215–218. Garcı´a-Garcı´a, L., Llevelyn-Jones, V., Fernandez Fernandez, I., Fuentes, J.A., Manzanares, J., 1998. Acute and repeated ECS treatment increases CRF, POMC and PENK gene expression in selected regions of the rat hypothalamus. Neuroreport 9, 73 – 77. Gramsch, C., Kleber, G., Ho¨llt, V., Pasi, A., Mehracin, P., Herz, A., 1980. Proopiocortin fragments in human and rat brain: b-endorphin and a-MSH are the predominant peptides. Brain Res. 192, 109 – 119. Ho¨llt, V., Haarman, I., 1984. Corticotropin-releasing factor differentially regulates proopiomelanocortin messenger ribonucleic acid levels in anterior as compared to intermediate pituitary lobes of rats. Biochem. Biophys. Res. Commun. 124, 407–415. Lightman, S.L., 1988. The neuroendocrine paraventricular hypothalamus: receptor, signal transduction, mRNA and neurosecretion. J. Exp. Biol. 139, 31 – 49. Lightman, S.L., Young, W.S. III, 1988. Corticotrophin-releasing factor, vasopressin and pro-opiomelanocortin mRNA responses to stress and opiates in the rat. J. Physiol. 403, 511–523. Manzanares, J., Corchero, J., Romero, J., Fernandez-Ruiz, J.J., Ramos, J.A., Fuentes, J.A., 1998. Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain. Mol. Brain Res. 55, 126–132. Matsuda, L.A., Lolait, S.J., Browstein, M.J., Young, A.C., Bonner, T.I., 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561–564. Nikolarakis, K.E., Almeida, O.F.X., Herz, A., 1986. Stimulation of hypothalamic b-endorphin and dynorphin release by corticotropin-releasing factor (in vitro). Brain Res. 399, 152–155. Priest, C.A., Eckersell, C.B., Micevych, P.E., 1995. Estrogen regulates preproenkephalin-A mRNA levels in the rat ventromedial nucleus: temporal and cellular aspects. J. Neuroendocrinology 2, 251 – 262. Priest, C.A., Borsook, D., Pfaff, D.W., 1997. Estrogen and stress interact to regulate the hypothalamic expression of a human proenkephalin promoter-beta-galactosidase fusion gene in a sitespecific and sex specific manner. J. Neuroendocrinology 9, 317 – 326.
.
439
Pugh G. Jr., Mason, D.J. Jr., Combs V., Welch, S.P., 1997. Involvement of dynorphin B in the antinociceptive effects of the cannabinoid CP 55,940 in the spinal cord. J. Pharmacol. Exp. Ther. 281: 730 – 737. Rang H.P., Dale M.M., 1991 Drugs Affecting Major Organs Systems: The Gastrointestinal Tract. In: Pharmacology, 2nd ed. Churchill Livingstone, Edinburgh, pp. 458 – 459. Reche, I., Fuentes, J.A., Ruiz-Gayo, M., 1996. A role for central cannabinoid and opioid systems in peripheral D9-tetrahydrocannabinol-induced analgesia in mice. Eur. J. Pharmacol. 301, 75–81. Reche, I., Fuentes, J.A., Ruiz-Gayo, M., 1996. Potentiation of D9-tetrahydrocannabinol-induced analgesia by morphine in mice: Involvement of m- and k-opioid receptors. Eur. J. Pharmacol. 318, 11 – 16. Reche I., Ruiz-Gayo, M., Fuentes, J.A., 1998. Inhibition of opioiddegrading enzymes potentiates D-9 tetrahydrocannabinol-induced antinociception in mice, Neuropharmacology 37, 215 – 222. Rivier, C., Imaki, T., Vale, W., 1990. Prolonged exposure to alcohol: effects on CRF mRNA levels, and CRF- and stress-induced ACTH secretion in the rat. Brain Res. 520, 1 – 5. Rivier, C., Lee, S., 1994. Stimulatory effect of cocaine on ACTH secretion: role of the hypothalamus. Mol. Cell Neurosci. 5, 189– 195. Rodriguez de Fonseca, F., Rubio, P., Menzaghi, F., Merlo-Pich, E., Rivier, J., Koob, G.F., Navarro, M., 1996. Corticotropin-releasing factor (CRF) antagonist [D-Phe12, Nle21,38, CaMeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther. 276, 56 – 64. Rodriguez de Fonseca, F., Carrera, M.R.A., Navarro, M., Koob, G.F., Weiss, F., 1997. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276, 2050 – 2054. Romano, G.J., Mobbs, C.V., Howells, R.D., Pfaff, D.W., 1989. Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone. Mol. Brain Res. 5, 51 – 58. Rubio, P., Rodriguez de Fonseca, F., Munoz, R.M., Ariznavarreta, C., Martin-Calderon, J.L., Navarro, M., 1995. Long-term behavioral effects of perinatal exposure to D9-tetrahydrocannabinol in rats: possible role of pituitary adrenal axis. Life Sci. 56, 2169– 2176. Stella, N., Schweitzer, P., Piomelli, D., 1997. A second endogenous cannabinoid that modulates long-trem potentiation. Nature 388, 773 – 778. Vela, G., Ruiz-Gayo, M., Fuentes, J.A., 1995. Anandamide decreases naloxone precipitated withdrawal signs in mice chronically treated with morphine. Neuropharrnacology 34, 665 – 668. Weidenfeld, J., Feldman, S., Mechoulam, R., 1994. Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamus-pituitary-adrenal axis in the rat. Neuroendocrinology 59, 110 – 112. Young, W.S. III, Bonner, T.I., Barnn, M.R., 1986. Mesencephalic dopaminergic neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83, 9827 – 9831.
Repeated administration of D9-tetrahydrocannabinol produces a differential time related responsiveness on proenkephalin, proopiomelanocortin and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat Javier Corchero, Jorge Manzanares *, Jose´ A. Fuentes Departamento de Farmacologı´a, Facultad de Farmacia and Unidad de Cartografı´a Cerebral, Instituto Pluridisciplinar, A6. Juan XXIII, 1 Uni6ersidad Cornplutense de Madrid, 28040 Madrid, Spain
Abstract The purpose of the present study was to explore the time related effects of repeated administration of D9-tetrahydrocannabinol on opioid and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat. By using in situ hybridization histochemistry, the effects of D9-tetrahydrocannabinol (THC, 5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of repeated administration on; (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN. The results revealed that, in most of the hypothalamic and pituitary regions examined, repeated cannabinoid administration upregulates opioid and corticotropin releasing factor gene expression. However, the onset, the degree of magnitude of gene expression reached and the time related effects produced by repeated administration with D9-tetrahydrocannabinol are dependent upon the brain and pituitary regions examined. Taken together, the results of the present study suggest that cannabinoids produce a time related differential responsiveness in opioid and corticotropin releasing factor gene expression, in areas of the hypothalamus and pituitary that may be related, at least in part, to a molecular integrative response to behavioral, endocrine and neurochemical alterations that occur in cannabinoid drug abuse. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: D9-tetrahydrocannabinol; Proenkephalin; Proopiomelanocortin; Corticotropinreleasing factor; mRNA; Hypothalamus; Pituitary
1. Introduction D9-Tetrahydrocannabinol (THC), the major psychoactive compound of Cannabis sati6a preparations (marijuana, hashish) produces a great variety of pharmacological effects that include antinociception, anxiety-like behavior, hypothermia, depression of motor activity, and alterations in the secretion of pituitary hormones (Dewey, 1986). It has been found that in the brain THC acts via a G-protein coupled receptor (CB1) (Matsuda et al., 1990) whose putative endogenous ligands have been identified as arachidonic acid derivatives (Devane et al., 1992). Sn-2 arachidonyl glicerol has been recently described as a second endogenous * Corresponding author. Tel.: +34-1-3943271; fax: 3943264; e-mail: [email protected].
+34-1-
ligand for cannabinoid receptors, present in the brain in greater amounts than anandamide (Stella et al., 1997). However, a large body of evidence indicates that many of the behavioral and neurochemical effects produced by acute or chronic administration with cannabinoids may involve an interaction between the opioid and the cannabinoid neuronal systems. In recent years, our group and others, have shown that; (1) THC and anandamide modulate the expression of physical signs of opioid dependence (Vela et al., 1995), (2) the antiemetic effect of nabilone, a THC synthetic derivative, is antagonized by the opioid receptor antagonist naloxone (Rang and Dale, 1991), (3) antinociception induced by THC can be blocked by a- or k-opioid receptor antagonists or selective antibodies against the endogenous k ligand dynorphin (Reche et al., 1996a,b), and (4) inhibition of opioid degrading enzymes may
0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 8 ) 0 0 1 9 5 - 6
434
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
potentiate THC induced antinociception (Reche et al., 1998). These results suggest that administration of THC may increase the release of endogenous opioids. Indeed, recently, it has been shown that administration of CP55,940, a selective cannabinoid receptor agonist produces a significant release of dynorphin B in the spinal cord concurrent with the production of antinociception (Pugh et al., 1997). Furthermore, our group has reported that subchronic or chronic administration of THC or cannabinoid receptor agonists increases proopiomelanocortin gene expression in the arcuate nucleus of the hypothalamus (Corchero et al., 1997a), prodynorphin and proenkephalin gene expression in the spinal cord (Corchero et al., 1997b) and proenkephalin gene expression in various forebrain and hypothalamic regions of the rat (Manzanares et al., 1998). Stress behavior induced by drug abuse has been related to an increase in the secretion of corticotropin or corticosterone in the plasma. Administration with cannabinoid agonists caused a significant increase in the secretion of corticotropin (Dewey, 1986; Weidenfeld et al., 1994; Rubio et al., 1995; Rodriguez de Fonseca et al., 1996) that appears to be mediated via a central mechanism involving an increase in the release of corticotropin releasing factor (Weidenfeld et al., 1994). However, the molecular mechanisms by which cannabinoids regulate the hypothalamic pituitary adrenal axis (HPA) are still unknown. The present study was undertaken to explore the molecular interaction between the opioid or the corticotropin releasing factor and the cannabinoid system. Time of onset, differential responsiveness and degree of magnitude obtained after repeated administration with THC on opioid and corticotropin releasing factor gene expression were determined in selected regions of the hypothalamus and pituitary gland of male rats. By using in situ hybridization histochemistry, the effects of D9-tetrahydrocannabinol (5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of administration on: (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN.
2. Materials and methods
2.1. Animals Adult male Sprague – Dawley rats, weighing 200–225 g, were obtained from Interfauna Iberica Laboratories (San Feliu de Codines, Barcelona, Spain) and maintained under conditions of controlled temperature
(239 1°C) and lighting (lights on 08:00–20:00 h), with food and water provided ad libitum. All experiments included in this study were performed following the highest standards of humane animal care, monitoring health care and minimizing pain and suffering, in accordance with the National and International Laws for the Care and Use of Laboratory Animals.
2.2. Drug D9-Tetrahydrocannabinol was dissolved in saline:ethanol:cremophor (18:1:1) and administered (5 mg/kg per day; i.p.; 1 ml/kg) for 1, 3, 7, and 14 days. Twenty four hours after the last injection rats were killed by decapitation and brains and pituitaries were quickly removed and frozen over dry ice. In previous experiments performed in our laboratory (data not shown) 24 h after the last injection, no differences were found in proenkephalin, proopiomelanocortin or corticotropin releasing factor gene expression when compared with the mRNA values of rats treated with vehicle at 1, 3, 7 or 14 day, in any of the brain or pituitary regions examined. Therefore, in the present work rats were treated with vehicle for 7 days, the time point that is in the middle of the whole time course study.
2.3. ln situ hybridization histochemistry Brain sections (six slides/area; two sections/slide) were cut at 20 mm at the level of the PVN, ARC, VMN, and pituitary sections (four slides/area; six sections/ slide) from the AL and IL. Sections were mounted onto gelatin-coated slides and stored at −80°C until the day of the assay. ISHH was performed as described previously (Young et al., 1986) using synthetic 48-bases oligonucleotide probes complementary to proenkephalin (NEN-Dupont, NEP-502, Madrid, Spain), corticotropin releasing factor (NEN-Dupont, NEP-554, Madrid, Spain) and to the nucleotides 96-134 of the rat proopiomelanocortin gene (obtained from the Advanced Biotechnology Center, Charing Cross and Westminster Medical School, London, England). Oligonucleotide probes were labelled using terminal deoxytransferase (Boehringer, Madrid, Spain) to add an a 35S-labeled deoxyATP (1000 Ci mmol − 1; Amersham, Madrid, Spain) tail to the 3% end of the probes. The probes (in 50 m1 of hybridization buffer) were applied to each section and left overnight at 37°C for hybridization. Following hybridization, sections were washed four times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2 (1X saline sodium citrate, SSC) at 55°C, followed by two 30 min washes in 1X SSC at room temperature, one brief water dip and were then blown dry with air. The dried slides were apposed to Kodak BioMax MR-1
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
film (Amersham, Madrid, Spain) for 1 h for the intermediate lobe, for 4 h for anterior lobe of the pituitary gland (POMC) and for 7 days for brain regions hybridized for proopiomelanocortin, proenkephalin and corticotropin releasing factor. Autoradiograms were produced with a Macintosh computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Optical densities were calculated from the uncalibrated mode by substracting from each measurement its corresponding background and expressed in grey scale values. The background measurement was taken from an area of the slice with the lowest nonspecific hybridization signal and substracted from the hybridization signal measurement in the same slice. Measurements were pooled from brain or pituitary sections and the values were averaged. Results were presented considering mean control values as 100%. Statistical analyses were performed using one-way analysis of variance followed by the Student Newman–Keul’s test. Differences were considered significant if the probability of error was less than 5%.
3. Results
3.1. Effects of THC on proenkephalin gene expression in the PVN and VMN of the hypothalamus The one-way analysis of variance of the results revealed that repeated administration with THC produced a marked increase of proenkephalin mRNA levels in the PVN (F(4,27) =7.39, P B 0.0006) and in the VMN (F(4,34)=28.50, P B 0.0001). In the PVN, treatment with THC produced a time related increase in proenkephalin mRNA levels reaching a 65% change from vehicle-treated rats on day 14. In contrast, proenkephalin mRNA levels in the VMN increased by day 3(+28%) and day 7 (+38%) and then returned to those of vehicle-treated rats on day 14 (Fig. 1). As shown in Fig. 2, representative autoradiograms of THC-treated rats (at specific time points) used to analyze proenkephalin mRNA levels in the PVN and VMN clearly present a more pronounced intensity in the hybridization signal than those corresponding to vehicle-treated rats.
435
Fig. 1. Time course of effects of repeated injections with D9-tetrahydrocannabinol on PENK mRNA levels in the PVN and VMN in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1); 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines + 1 SEM of PENK mRNA levels in six to eight rats.*, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B0.05) from vehicle-treated rats.
day 7 ( + 23%) and then returned to vehicle-treated values by day 14 (+ 6%). The one-way analysis of variance of the data obtained after repeated administration with THC in the AL revealed statistical significant differences (F(4,35)=19.8, PB 0.0001) in proopiomelanocortin mRNA levels. The post-hoc analysis revealed that proopiomelanocortin mRNA levels in the AL increased + 48% by day 1, and then progressively decreased (−52%) by day 14 (Fig. 3B). In contrast, proopiomelanocortin mRNA levels in the IL decreased (−25%) only on day 14 after administration with THC
3.2. Effects of THC on proopiomelanocortin gene expression in the ARC of the hypothalamus and AL and IL of the pituitary gland As depicted in Fig. 3A, repeated administration with THC produced a marked increase in proopiomelanocortin mRNA levels in the ARC of the hypothalamus (F(4,33)=21.3, P B0.0001) by day 3 (+ 27%) and
Fig. 2. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on PENK mRNA. Representative autoradiograms of coronal brain sections at the level of the PVN and VMN of the rat hypothalamus. Slides were apposed to film (Kodak, Biomax, MR-1) for 7 days. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
436
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
Fig. 3. Time course of effects of repeated injections with D9-tetrahydrocannabinol on POMC mRNA levels in the ARC (A), AL (B) and IL (C) in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1; 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines +1 SEM. of POMC mRNA levels in six to eight rats.*, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B 0.05) from vehicle-treated rats.
(F(4,33)= 3.90, PB 0.0118) and no changes were found at any of the other time points examined (Fig. 3C). Representative autoradiograms at time points where statistical significance was found in proopiomelanocortin mRNA levels between THC- and vehicle-treated group of the ARC, AL and IL are shown in Fig. 4.
3.3. Effects of THC on corticotropin releasing factor gene expression in the PVN of the hypothalamus Repeated administration with THC markedly increased (F(4,28)= 5.69, P B0.0023) corticotropin releasing factor mRNA levels in the PVN. The post-hoc analysis indicated that a statistical significant increase was only found after 3 days of THC-administration ( +85%). However, although not statistically significant, a +34% increase was found on day 1, (+51%) on day 7, and ( + 22%) on day 14 (Fig. 5). Representative autoradiograms at time points where statistical significance was found in corticotropin releasing factor mRNA levels between THC- and vehicle-treated groups in the PVN are shown in Fig. 6.
4. Discussion The results of the present study demonstrate that exposure to THC markedly increases corticotropin-releasing factor gene expression in the PVN of the hypothalamus. Although some similarities were found in the response of opioid and corticotropin releasing factor genes examined after THC, important regional differences have been detected in the degree of magnitude of each specific gene expression regulation and the onset of this process. Interestingly, as a probe of the system dynamics, decreases in proopiomelanocortin mRNA levels were also found at some of the time intervals examined during THC chronic treatment. In addition,
in most of the brain regions examined, administration with THC upregulates proenkephalin and proopiomelanocortin gene expression in a way which follows the response pattern found in previous studies (Corchero et al., 1997a,b; Manzanares et al., 1998). We have previously shown that subchronic and chronic treatment with cannabinoids upregulates proenkephalin gene expression in the PVN and VMN (Manzanares et al., 1998). In the present study, proenkephalin gene expression in the PVN progressively increased after repeated administration with THC reaching a 65% increase on day 14. However, in the VMN the maximum increase was found after 7 days of treatment and then mRNA levels returned to those of vehicle-treated animals by day 14. In this respect, it appears that proenkephalin in the PVN presents a greater sensitivity to THC treatment than in the VMN. In contrast, the onset of the THC action on proenkephalin gene expression in the VMN occurs very rapidly, but unlike in the PVN, prolonged THC treatment progressively downregulates mRNA levels. These differential responses in proenkephalin gene expression between the PVN and VMN after a long exposure to THC may be related to specific transcription factors that differentially affect gene regulation in each brain region and/or are the result of a neuroadaptative distinct responsiveness to cannabinoid administration. Gonadal steroids and physiological stressors affect the regulation of proenkephalin gene expression in the PVN and VMN (Garcı´a-Garcı´a et al., 1998a,b; Lightman, 1988; Lightman and Young, 1988; Romano et al., 1989; Priest et al., 1995, 1997). Using a transgenic model that expresses a human proenkephalin promoter/ bacterial beta-galactosidase fusion gene (ENK-1) (Borsook et al., 1994), Priest and coworkers have demonstrated that stress and estrogen differentially regulate proenkephalin gene expression in the PVN and
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
VMN suggesting that stress and estrogen have sex-specific and site-specific regulatory effects on the expression of a proenkephalin promoter transgene in hypothalamic neurons (Priest et al., 1997). The differential time course response between the PVN and VMN found in this study after exposure to cannabinoids may be related to a distinct regulation of the proenkephalin gene in these hypothalamic nuclei. The results of this study are consistent with a previous report by our laboratory (Corchero et al., 1997a), showing that administration of THC for 5 days increased proopiomelanocortin gene expression in the ARC whereas no effect was observed in the AL of the pituitary. However, in the present study, marked differences have been found in the onset, the degree of magnitude and the pattern that regulates the expression of the proopiomelanocortin gene in the brain and pituitary regions examined after repeated administration with THC. Proopiomelanocortin mRNA levels increased in the ARC of the hypothalamus 3 and 7 days after treatment with THC at a similar range to that observed previously (Corchero et al., 1997a) and progressively returned to vehicle levels on day 14. It is interesting that proopiomelanocortin gene expression in
437
the anterior lobe of the pituitary gland apparently is the most responsive to THC treatment, as it is induced at the earliest time. Indeed, proopiomelanocortin gene expression in the AL of the pituitary gland exhibited the highest responsiveness to THC administration, reaching a 48% increase 1 day after drug administration, although proopiomelanocortin transcriptional activity decreased in a time related manner up to −52% on day 14. However,in the IL of the pituitary the onset of THC action is slow and only a 25% decrease was found on day 14. The regulatory mechanisms by which THC differentially alters proopiomelanocortin gene expression in the hypothalamus and regions of the pituitary are still unclear. This gene processes a number of proteins including b-lipotropin, a, b, and g-melanocyte stimulating hormones, adrenocorticotropin hormone (ACTH) and b-endorphin (Eipper and Mains, 1980). In the anterior pituitary, ACTH and b-lipotropin are the predominant proopiomelanocortin related peptides, whereas in the arcuate nucleus of the hypothalamus and intermediate lobe b-endorphin is the major end product of proopiomelanocortin gene processing (Gramsch et al., 1980; Emerson and Eipper, 1986). It
Fig. 4. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on POMC mRNA. Representative autoradiograms of coronal brain sections at the level of the ARC, AL and IL of the rat. Slides were apposed to film (Kodak, Biomax, MR-1) 1 h for IL, 6 h for AL and 7 days for ARC. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
438
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439
Fig. 5. Time course of effects of repeated injections with D9-tetrahydrocannabinol on corticotropin releasing factor mRNA levels in the PVN of the hypothalamus in the rat. Rats were injected with D9-tetrahydrocannabinol (5 mg/kg per day; 1, 3, 7 and 14 days; i.p.) or with its vehicle (saline:ethanol:cremophor; 18:1:1; 1 ml/kg per day, 7 days) and were decapitated 24 h after the last administration. Symbols represent the means and vertical lines+ 1 SEM of corticotropin releasing factor mRNA levels in six to eight rats. *, values from D9-tetrahydrocannabinol-treated animals that are significantly different (P B0.05) from vehicletreated rats.
has been suggested that proopiomelanocortin gene expression is differentially regulated by corticotropin releasing factor in the ARC as well as in the AL or IL of the pituitary. In vitro corticotropin releasing factor stimulates the release of b-endorphin from hypothalamic slices (Nikolarakis et al., 1986), suggesting that corticotropin releasing factor neurons in the PVN, in addition to releasing their content into the portal vessels, also stimulate proopiomelanocortin neurons in the ARC. Furthermore, chronic activation of corticotropin releasing factor system increases proopiomelanocortin gene expression in the anterior lobe but decreases it in the intermediate lobe (Ho¨llt and Haarman, 1984). The results of the present study suggest that administration of THC differentially modifies the expression of the proopiomelanocortin gene in the brain and pituitary regions examined by a mechanism that may be mediated, at least in part, via activation of the corticotropin releasing factor hypothalamic system. Indeed, repeated
administration of THC rapidly and markedly increased (day 3, + 85%) corticotropin releasing factor gene expression in the PVN and then slowly decreased, until on day 14 mRNA level were 22% higher than vehicle values. The activation of corticotropin releasing factor gene expression could be produced as part of a compensatory response to daily activation of the HPA axis hormone secretion, since administration of cannabinoid receptor agonists increases both adrenocorticotropin releasing hormone and corticosterone secretion in rats (Weidenfeld et al., 1994; Rodriguez de Fonseca et al., 1996). In addition, it has been suggested that long term exposure to cannabinoids leads to neuroadaptative changes that result in an enhanced release of corticotropin releasing factor in the central amygdala as well as an activation of stress-responsive nuclei (Rodriguez de Fonseca et al., 1997). Exposure to different stressors or drug abuse (cocaine, opiates, ethanol) leads to significant alterations in the corticotropin releasing factor gene expression in the PVN of the hypothalamus (Lightman and Young, 1988; Rivier et al., 1990; Rivier and Lee, 1994). Taken together, the differences found in the activation of the corticotropin releasing factor gene expression in this study, may be part of the adaptative response produced by cannabinoid induced anxiety. In conclusion, the results of the present study indicate, that chronic exposure to THC affects in a different time dependent manner endogenous opioid and corticotropin releasing factor gene expression and provides evidence of molecular alterations that may be relevant to further understand a variety of behavioral and neuroendocrine effects that occur in cannabinoid drug abuse.
Acknowledgements This work was supported by the Universidad Complutense of Madrid grant PR294/95-6189, Concerted Action from the European Union grant BMH1-CT-941108 and Spanish Ministry of Education grant DGICYT UE95 0017. J. Corchero is a Predoctoral Fellow supported by the ‘Comunidad Autonoma de Madrid’ and J. Manzanares is a Senior Fellow supported by the Spanish Ministry of Education.
References Fig. 6. Effects of repeated administration of D9-tetrahydrocannabinol and vehicle on corticotropin releasing factor (CRF) mRNA. Representative autoradiograms of coronal brain sections at the level of the PVN of the rat hypothalamus. Slides were apposed to film (Kodak, Biomax, MR-1) for 7 days. Experimental design was as indicated in Fig. 1. Bar represents 1 mm.
Borsook, D., Falkowski, O., Burstein, R., Strassman, A., Konradi, C., Dauber, A., Comb, M., Hyman, S.E., 1994. Stress-induced regulation of a human proenkephalin-b-galactosidase fusion gene in the hypothalamus of transgenic mice. Mol. Endocrinol. 8, 116 – 125. Corchero, J., Fuentes, J.A., Manzanares, J., 1997. D9-tetrahydrocannabinol increases proopiomelanocortin gene expression in the
J. Corchero et al. / Neuropharmacology 38 (1999) 433–439 arcuate nucleus of the rat hypothalamus. Eur. J. Pharmacol. 323, 193 – 195. Corchero, J., Avila, M.A., Fuentes, J.A., Manzanares, J., 1997. D9-tetrahydrocannabinol increases prodynorphin and preproenkephalin gene expression in the spinal cord of the rat. Life Sci. 61, PL39 – PL43. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., et al., 1992. Isolation and structure of a brain constituent that binds to the cannabinod receptor. Science 258, 1946–1949. Dewey, W.L., 1986. Cannabinoid Pharmacology. Pharmacol. Rev. 38, 151 – 178. Eipper, B.A., Mains, R.E., 1980. Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1, 1 – 27. Emerson, R.B., Eipper, B.A., 1986. Characterization of pro-ACTH/ endorphin derived peptides in rat hypothalamus. J. Neurosci. 6, 837 – 849. Garcı´a-Garcı´a, L., Harbuz, M., Manzanares, J., Lightman, S.L., Fuentes, J.A., 1998. RU-486 blocks stress-induced enhancement of proenkephalin gene expression in the paraventricular nucleus of rat hypothalamus. Brain Res. 786, 215–218. Garcı´a-Garcı´a, L., Llevelyn-Jones, V., Fernandez Fernandez, I., Fuentes, J.A., Manzanares, J., 1998. Acute and repeated ECS treatment increases CRF, POMC and PENK gene expression in selected regions of the rat hypothalamus. Neuroreport 9, 73 – 77. Gramsch, C., Kleber, G., Ho¨llt, V., Pasi, A., Mehracin, P., Herz, A., 1980. Proopiocortin fragments in human and rat brain: b-endorphin and a-MSH are the predominant peptides. Brain Res. 192, 109 – 119. Ho¨llt, V., Haarman, I., 1984. Corticotropin-releasing factor differentially regulates proopiomelanocortin messenger ribonucleic acid levels in anterior as compared to intermediate pituitary lobes of rats. Biochem. Biophys. Res. Commun. 124, 407–415. Lightman, S.L., 1988. The neuroendocrine paraventricular hypothalamus: receptor, signal transduction, mRNA and neurosecretion. J. Exp. Biol. 139, 31 – 49. Lightman, S.L., Young, W.S. III, 1988. Corticotrophin-releasing factor, vasopressin and pro-opiomelanocortin mRNA responses to stress and opiates in the rat. J. Physiol. 403, 511–523. Manzanares, J., Corchero, J., Romero, J., Fernandez-Ruiz, J.J., Ramos, J.A., Fuentes, J.A., 1998. Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain. Mol. Brain Res. 55, 126–132. Matsuda, L.A., Lolait, S.J., Browstein, M.J., Young, A.C., Bonner, T.I., 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561–564. Nikolarakis, K.E., Almeida, O.F.X., Herz, A., 1986. Stimulation of hypothalamic b-endorphin and dynorphin release by corticotropin-releasing factor (in vitro). Brain Res. 399, 152–155. Priest, C.A., Eckersell, C.B., Micevych, P.E., 1995. Estrogen regulates preproenkephalin-A mRNA levels in the rat ventromedial nucleus: temporal and cellular aspects. J. Neuroendocrinology 2, 251 – 262. Priest, C.A., Borsook, D., Pfaff, D.W., 1997. Estrogen and stress interact to regulate the hypothalamic expression of a human proenkephalin promoter-beta-galactosidase fusion gene in a sitespecific and sex specific manner. J. Neuroendocrinology 9, 317 – 326.
.
439
Pugh G. Jr., Mason, D.J. Jr., Combs V., Welch, S.P., 1997. Involvement of dynorphin B in the antinociceptive effects of the cannabinoid CP 55,940 in the spinal cord. J. Pharmacol. Exp. Ther. 281: 730 – 737. Rang H.P., Dale M.M., 1991 Drugs Affecting Major Organs Systems: The Gastrointestinal Tract. In: Pharmacology, 2nd ed. Churchill Livingstone, Edinburgh, pp. 458 – 459. Reche, I., Fuentes, J.A., Ruiz-Gayo, M., 1996. A role for central cannabinoid and opioid systems in peripheral D9-tetrahydrocannabinol-induced analgesia in mice. Eur. J. Pharmacol. 301, 75–81. Reche, I., Fuentes, J.A., Ruiz-Gayo, M., 1996. Potentiation of D9-tetrahydrocannabinol-induced analgesia by morphine in mice: Involvement of m- and k-opioid receptors. Eur. J. Pharmacol. 318, 11 – 16. Reche I., Ruiz-Gayo, M., Fuentes, J.A., 1998. Inhibition of opioiddegrading enzymes potentiates D-9 tetrahydrocannabinol-induced antinociception in mice, Neuropharmacology 37, 215 – 222. Rivier, C., Imaki, T., Vale, W., 1990. Prolonged exposure to alcohol: effects on CRF mRNA levels, and CRF- and stress-induced ACTH secretion in the rat. Brain Res. 520, 1 – 5. Rivier, C., Lee, S., 1994. Stimulatory effect of cocaine on ACTH secretion: role of the hypothalamus. Mol. Cell Neurosci. 5, 189– 195. Rodriguez de Fonseca, F., Rubio, P., Menzaghi, F., Merlo-Pich, E., Rivier, J., Koob, G.F., Navarro, M., 1996. Corticotropin-releasing factor (CRF) antagonist [D-Phe12, Nle21,38, CaMeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther. 276, 56 – 64. Rodriguez de Fonseca, F., Carrera, M.R.A., Navarro, M., Koob, G.F., Weiss, F., 1997. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276, 2050 – 2054. Romano, G.J., Mobbs, C.V., Howells, R.D., Pfaff, D.W., 1989. Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone. Mol. Brain Res. 5, 51 – 58. Rubio, P., Rodriguez de Fonseca, F., Munoz, R.M., Ariznavarreta, C., Martin-Calderon, J.L., Navarro, M., 1995. Long-term behavioral effects of perinatal exposure to D9-tetrahydrocannabinol in rats: possible role of pituitary adrenal axis. Life Sci. 56, 2169– 2176. Stella, N., Schweitzer, P., Piomelli, D., 1997. A second endogenous cannabinoid that modulates long-trem potentiation. Nature 388, 773 – 778. Vela, G., Ruiz-Gayo, M., Fuentes, J.A., 1995. Anandamide decreases naloxone precipitated withdrawal signs in mice chronically treated with morphine. Neuropharrnacology 34, 665 – 668. Weidenfeld, J., Feldman, S., Mechoulam, R., 1994. Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamus-pituitary-adrenal axis in the rat. Neuroendocrinology 59, 110 – 112. Young, W.S. III, Bonner, T.I., Barnn, M.R., 1986. Mesencephalic dopaminergic neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83, 9827 – 9831.