Servicio de Psiquiatría y Unidad de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain
* Author to whom correspondence should be addressed at: Servicio de Psiquiatria, Edificio Materno-Infantil, 6° Planta, 613-A, Hospital Universitario 12 de Octubre, Avda de Cordoba s/n, E-28041 Madrid, Spain. Tel.: +91 390 8252; Fax: +91 390 8538; E-mail: jmanzanares6{at}terra.es
(Received 6 November 2003; first review notified 7 January 2004; in revised form 12 March 2004; accepted 18 March 2004)
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ABSTRACT |
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INTRODUCTION |
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The Fawn Hooded strain of rat presents a high preference for ethanol intake (10% v/v) in a two-bottle free-choice situation (Rezvani et al., 1990) that may be related to decreased brain opioid function (Cowen et al., 1998
). Considering that administration of cannabinoid receptor agonists enhances endogenous opioid activity (Manzanares et al., 1999
), we hypothesize that animals with low opioid expression and greater vulnerability to alcohol may have impaired cannabinoid receptor function in key regions of the brain.
The aim of the present study, was to compare cannabinoid CB1 receptor function between Fawn Hooded and Wistar rats. To this purpose, cannabinoid-stimulated [35S]-GTPS binding and cannabinoid CB1 receptor gene expression were examined in selected brain regions of both strains.
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MATERIALS AND METHODS |
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Ethanol intake protocol
Forty-one rats of each strain had continuous access to two bottles containing either water or ethanol solution (10% v/v). Ethanol intake was measured every day. Data for daily ethanol intake were expressed in g/kg/day. The position of bottles were interchanged at random to avoid development of position preference. Rats remained under these conditions for 30 days. Another set of 10 Wistar and Fawn Hooded rats never exposed to alcohol (naïve rats) were killed by decapitation and their brains were quickly removed and frozen over dry ice.
Histology
Coronal brain sections (12 µm) from rats that received only water (naïve rats), were cut at different levels containing the regions of interest. The first level included the cingulate cortex (Cg) caudate-putamen (CPu) and nucleus accumbens (Acc). The second level included the ventromedial hypothalamic nucleus (VMN), amygdaloid area (AMG), hippocampal fields (CA1, CA2, CA3) and dentate gyrus (DG) and the third level included the substantia nigra pars reticulata (SNr). Sections were obtained according to Paxinos and Watson (1997) and mounted onto gelatin-coated slides and stored at 80°C until the day of the assay.
In-situ hybridization histochemistry
In-situ hybridization histochemistry (ISHH) was performed as described previously (Young et al., 1986) using three synthetic oligonucleotide probes complementary to cannabinoid CB1 receptor gene (bases 451, 349396, 952999). Oligonucleotide probes were labelled using terminal deoxytransferase (Boehringer, Madrid, Spain) to add a 35S-labelled deoxyATP (1000 Ci/mmol; Amersham, Madrid, Spain) tail to the 3' end of the probes. Labelled probes were purified by Mini Quick-Spin oligo columns (Roche, Barcelona, Spain). The probes (in 50 µl 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 mol/l NaCl, 0.015 mol/l 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. In order to control for imaging enhancement variables, each set of slides was apposed to the same film (Kodak BioMax MR-1; Amersham) in individualized cassettes for 10 days (Cg, CPu) and 12 days for VMN and fields of the hippocampus.
WIN-55,2122-stimulated [35S]GTPS binding
Cannabinoid CB1 receptor [35S]GTPS binding was performed as previously described (Sim et al., 1996
). Brain sections at the level of Cg, CPu, Acc, VMN, AMG, fields of the hippocampus (CA1, CA2, CA3) and SNr were mounted onto gelatin-coated slides and rinsed in assay buffer (50 mmol/l TRIS, 3 mmol/l MgCl2, 0.2 mmol/l EGTA, 100 mmol/l NaCl, pH 7.4) at 25°C for 10 min, then incubated with 2 mmol/l GDP in assay buffer for 30 min at 25°C. Sections were then incubated for 2 h at 25°C in assay buffer with 0.04 nmol/l [35S]-GTP
S, 2 mmol/l GDP and the cannabinoid receptor agonist 7.5 µmol/l WIN-55,2122 in 0.5% bovine serum albumin. Basal activity was assessed in the absence of agonist, whereas nonspecific binding was measured in the presence of 10 µmol/l unlabelled GTP
S. Additional brain sections of the naïve rats were incubated with 0.04 nmol/l [35S] GTP
S, 2 mmol/l GDP, WIN-55,2122 and AM251 0.3 µmol/l to test for specificity of agonist receptor activation. After incubation, slides were rinsed twice in 50 mmol/l TRIS buffer, pH 7.4, and once in cold deionized water, air-dried and exposed to film (Kodak BioMax MR-1, Amersham) for 2448 h.
Image analyses quantification
Autoradiograms from in-situ hybridization studies were analysed with a personal computer using the public domain NIH Image program (US National Institutes of Health; http://rsb.info.nih.gov/nih-image). Previous experiments in our laboratory have found that the selected times of exposure to film, in these brain regions and under our experimental conditions (oligonucleotide probe, radioactivity added to each slide, incubation conditions, type of film selected) renders a hybridization signal whose grey levels are linear with the optical density, according to the NIH Image Program. Therefore, optical densities were calculated from the uncalibrated mode of the Image Program by subtracting 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. Measurements were pooled from brain sections and the values were averaged. Results were presented considering mean control values as 100%.
In the autoradiograms from CB1 agonist-stimulated [35S]GTPS binding, optical densities (OD) were calculated by subtracting from each stimulated measurement its correspond-ing basal value, as previously shown (Corchero et al., 1999
). The addition of the CB1 receptor antagonist AM251 completely blocked the signal induced by WIN-55,212, indicating that the effects of WIN-55,212 in these experimental conditions are specific (data not shown).
Statistical analyses
Statistical analyses were performed using Student's t-test when comparing values between Wistar and Fawn Hooded rats. Differences were considered significant if the probability of error was less than 5%.
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RESULTS |
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DISCUSSION |
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Strains of rats (e.g. Fawn Hooded, Sardinian, AA) or mice (e.g. C57BL/6) that exhibit high preference for ethanol have been widely used to investigate the biochemical alterations that increase consumption of alcohol, and to examine the effects of drugs that reduce alcohol intake in preferring rodents (Cowen et al., 1998; Hungund and Basavarajappa, 2000
). The Fawn Hooded rat is an inbred strain considered to be a genetic animal model of comorbid depression and alcoholism (Rezvani et al., 2002
). This strain shows alterations in proenkephalin (Cowen et al., 1998
), preprocholecistokinin (Lodge and Lawrence, 2001
), GABAA receptor (Chen et al., 1998
) and corticotropin-releasing factor (Altemus et al., 1994
) gene expressions that may be related, at least in part, to excessive alcohol consumption.
The results of this study clearly show a lower cannabinoid CB1 receptor function in the strain of rats that display higher preference for alcohol. Indeed, in our Fawn Hooded rats, that presented a preference for 10% (v/v) ethanol four times higher that of Wistar rats, a lower WIN-55,212-stimulated [35S]GTPS binding receptor autoradiography was detected in many areas of the brain [CPu (50%), Cg (43%), Acc (65%), VMN (40%), AMG (35%) and hippocampus (CA1, 25%; CA3, 43%; DG, 37%)]. In addition, the data obtained by in-situ hybridization studies revealed a lower cannabinoid CB1 receptor gene expression in CPu (25%), Cg (50%), VMN (60%) and CA3 field of hippocampus (20%) in Fawn Hooded than in Wistar rats. These results are in agreement with those reported by Hungund and Basavarajappa (2000)
showing that lower density of CB1 receptors and lower affinity were found in brain membranes of C57BL/6 mice (selected for its high preference for alcohol) than in DBA/2 mice.
The present study shows for the first time a lower regional level of cannabinoid receptor function and lower cannabinoid CB1 receptor gene expression in the brain of Fawn Hooded than in Wistar rats. The brain areas examined in the present study (CPu, Cg, Acc, VMN, AMG and fields of hippocampus) were selected because of their potential role in the neurobiological mechanisms involved in drug dependence (González et al., 2002; Herrera et al., 2003
; Tang et al., 2003
). For instance, CPu has been involved in motor and emotional behaviour response to withdrawal syndromes induced by drugs of abuse (Oliva et al., 2003
). Alterations in opioid gene expression in CPu and Acc have also been related to greater vulnerability to opiates and alcohol. Indeed, lower proenkephalin gene expression in this region was found in Lewis (displaying a higher vulnerability to morphine self-administration) than in Fischer rats (Martin et al., 1999
) and in Fawn Hooded than in WistarKyoto rats (Cowen et al., 1998
). Ventromedial nucleus exhibits the highest level of cannabinoid receptor and cannabinoid receptor gene expression in the hypothalamus of the rat (Herkenham et al., 1991
) and is thought to regulate ingestive (Jamshidi and Taylor, 2001
) and reproductive behaviours (Li et al., 1997
). Interestingly, it has been recently reported that CPu and VMN of the hypothalamus are key sites involved in cannabinoid-opioid interactions, as naloxone inhibited THC-induced Fos immunoreactivity in both regions (Allen et al., 2003
). The hippocampus is the main brain nucleus in which cannabinoids probably exert their effects on memory and cognition (Herkenham et al., 1991
; Ameri, 1999
). Activation of cannabinoid receptors in the hippocampus inhibits neurotransmitter release (acetylcholine, GABA, glutamate and noradrenaline) (Doherty and Dingledine, 2003
), which presumably contributes to the short- and long-term plasticity underlying memory alterations that often occur after prolonged exposure to ethanol. Indeed, recent work has posited common molecular and cellular substrates of addiction and memory, suggesting a close relationship between brain circuits involved in the regulation of drug dependence and learning and memory (Nestler, 2002
).
The fact that alterations in cannabinoid receptor function were found in the strain of rats presenting higher vulnerability to alcohol consumption strengthens the notion that manipulations of cannabinoid CB1 receptor may affect alcohol intake. Indeed, cannabinoid receptor agonists such as WIN-55,2122 and CP-55,940 increase voluntary ethanol intake in Sardinian alcohol-preferring (sP) rats (Colombo et al., 2002) whereas the cannabinoid receptor antagonist SR-141,716 A reduced voluntary ethanol intake in C57BL/6 (Arnone et al., 1997
), alcohol self-administering Long Evans rats (Freedland et al., 2001
) and Sardinian alcohol-preferring (sP) rats (Colombo et al., 1998
).
Overall, the results of this study show lower cannabinoid receptor function in many brain areas of the Fawn Hooded compared to Wistar rats, suggesting that CB1 cannabinoid receptors play a key role in the regulation of voluntary alcohol intake. Furthermore, these findings strongly support the CB1 cannabinoid receptor as a new pharmacological target to treat alcohol dependence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Allen, K. V., McGregor, I. S., Hunt, G. E., Singh, M. E. and Mallet, P. E. (2003) Regional differences in naloxone modulation of Delta(9)-THC induced Fos expression in rat brain. Neuropharmacology 44, 264274.[CrossRef][ISI][Medline]
Altemus, M., Smith, M. A., Diep, V., Aulakh, C. S. and Murphy, D. L. (1994) Increased mRNA for corticotrophin releasing hormone in the amygdala of fawn-hooded rats: a potential animal model of anxiety. Anxiety 95, 251257.
Arnone, M., Maruani, J., Chaperon, F., Thiebot, M. H., Poncelet, M., Soubrie, P. and Le Fur, G. (1997) Selective inhibition of sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology 132, 104106.[CrossRef][ISI][Medline]
Basavarajappa, B. S., Cooper, T. B. and Hungund, B. L. (1998) Chronic ethanol administration down-regulates cannabinoid receptors in mouse brain synaptic plasma membrane. Brain Research 793, 212218.[CrossRef][ISI][Medline]
Basavarajappa, B. S. and Hungund, B. L. (1999) Down-regulation of cannabinoid receptor agonist-stimulated 35GTP gamma S binding in synaptic plasma membrane from chronic ethanol exposed mouse. Brain Research 815, 8997.[CrossRef][ISI][Medline]
Chen, F., Rezvani, A., Jarrott, B. and Lawrence, A. J. (1998) Distribution of GABAA receptors in the limbic system of alcohol-preferring and nonpreferring rats: in situ hybridization histochemistry and receptor autoradiography. Neurochemistry International 32, 143151.[CrossRef][ISI][Medline]
Colombo, G., Agabio, R., Fa, M., Guano, L., Lobina, C., Loche, A., Reali, R. and Gessa, G. L. (1998) Reduction of voluntary ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol and Alcoholism 33, 126130.[Abstract]
Colombo, G., Serra, S., Brunetti, G., Gomez, R., Melis, S., Vacca, G., Carai, M. M., and Gessa, L. (2002) Stimulation of voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats. Psychopharmacology 159, 181187.[CrossRef][ISI][Medline]
Corchero, J., Romero, J., Berrendero, F., Fernandez-Ruiz, J., Ramos, J. A., Fuentes, J. A. and Manzanares, J. (1999) Time-dependent differences of repeated administration with Delta9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by mu-opioid and CB1-cannabinoid receptors in the caudate-putamen. Molecular Brain Research 6, 148157.
Cowen, M. S., Rezvani, A., Jarrott, B. and Lawrence, A. J. (1998) Distribution of opioid peptide gene expression in the limbic system of Fawn-Hooded (alcohol-preferring) and Wistar-Kyoto (alcohol-non-preferring) rats. Brain Research 796, 323326.[CrossRef][ISI][Medline]
Doherty, J. and Dingledine, R. (2003) Functional interactions between cannabinoid and metabotropic glutamate receptors in the central nervous system. Current Opinion in Pharmacology 3, 4653.[CrossRef][ISI][Medline]
Freedland, C. S., Sharpe, A. L., Samson, H. H. and Porrino, L. J. (2001) Effects of SR141716A on ethanol and sucrose self-administration. Alcohol: Clinical and Experimental Research 25, 277282.[ISI][Medline]
González, S., Fernández-Ruiz, J., Sparpaglione, V., Parolaro, D. and Ramos, J. A. (2002) Chronic exposure to morphine, cocaine or ethanol in rats produced different effects in brain cannabinoid CB1 receptor binding and mRNA levels. Drug and Alcohol Dependence 66, 7784.[CrossRef][ISI][Medline]
Herrera, D. G., Yague, A. G., Johnsen-Soriano, S., Bosch-Morell, F., Collado-Morente, L. Muriach, M., Romero, F. J. and Garcia-Verdugo, J. M. (2003) Selective impairment of hippocampal neurogenesis by chronic ethanolism: protective effects of an antioxidant. Proceedings of the National Academy of Sciences of the United States of America 100, 79197924.
Herkenham, M., Lynn, A. B., Johnson, M. R., Melvin, L. S., de Costa, B. R. and Rice, K. C. (1991) Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. Journal of Neuroscience 11, 563583.[Abstract]
Hungund, B. L. and Basavarajappa, B. S. (2000) Distinct differences in the cannabinoid receptor binding in the brain of C57BL/6 and DBA/2 mice, selected for their differences in voluntary ethanol consumption. Journal of Neuroscience Research 60, 122128.[ISI][Medline]
Hungund, B. L., Szakall, I., Adam, A., Basavarajappa, B. S. and Vadasz, C. (2003) Cannabinoid CB1 receptor knockout mice exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus accumbens. Journal of Neurochemistry 84, 698704.[CrossRef][ISI][Medline]
Jamshidi, N. and Taylor, D. A. (2001) Anandamide administration into the ventromedial hypothalamus stimulates appetite in rats. British Journal of Pharmacology 134, 11511154.
Li, Y., McGivern, R. F., Nagahara, A. H. and Handa, R. J. (1997) Alterations in the estrogen sensitivity of hypothalamic proenkephalin mRNA expression with age and prenatal exposure to alcohol. Molecular Brain Research 47, 215222.[ISI][Medline]
Lodge, D. J. and Lawrence, A. J. (2001) Comparative analysis of the central CCK system in Fawn Hooded and Wistar Kyoto rats: extended localisation of CCK-A receptors throughout the rat brain using a novel radioligand. Regulatory Peptides 15, 191201.[CrossRef]
Manzanares, J., Corchero, J., Romero, J., Fernandez-Ruiz, J. J., Ramos, J. A. and Fuentes, J. A. (1999) Pharmacological and biochemical interactions between opiates and cannabinoids. Trends in Pharmacological Sciences 20, 287294.[CrossRef][ISI][Medline]
Martin, S., Manzanares, J., Corchero, J., García-Lecumberri, C., Crespo, J. A., Fuentes, J. A. and Ambrosio, E. (1999) Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self administration in Fischer 344 and Lewis rats. Brain Research 821, 350355.[CrossRef][ISI][Medline]
Nestler, E. J. (2002) Common molecular and cellular substrates of addiction and memory. Neurobiology of Learning and Memory 78, 637647.[CrossRef][ISI][Medline]
Oliva, J. M., Ortiz, S., Palomo, T. and Manzanares, J. (2003) Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice. Journal of Neurochemistry 85, 94104.[CrossRef][ISI][Medline]
Paxinos, G. and Watson, C. (1997) The Rat Brain in Sterotaxic Coordinates. Academic Press, New York.
Poncelet, M., Maruani, J., Calassi, R. and Soubrie, P. (2003) Overeating, alcohol and sucrose consumption decrease in CB1 receptor deleted mice. Neuroscience Letters 12, 216218.[CrossRef]
Rezvani, A. H., Overstreet, D. H. and Janowsky, D. S. (1990) Genetic serotonin deficiency and alcohol preference in the fawn hooded rats. Alcohol and Alcoholism 25, 573575.[ISI][Medline]
Rezvani, A. H., Parsian, A. and Overstreet, D. H. (2002) The Fawn-Hooded (FH/Wjd) rat: a genetic animal model of comorbid depression and alcoholism. Psychiatry Genetics 12, 116.[CrossRef]
Tang, A., George, M. A., Randall, J. A. and Gonzales, R. A. (2003) Ethanol increases extracellular dopamine concentrations in the ventral striatum in C57BL/6 mice. Alcoholism: Clinical and Experimental Research 27, 10831089.[ISI][Medline]
Sim, L. J., Hampson, R. E., Deadwyler, S. A. and Childers, S. R. (1996) Effects of chronic treatment with 9-tetrahydrocannabinol on cannabinoid-stimulated [35S]-GTP
S autoradiography in rat brain. Journal of Neuroscience 16, 80578066.
Wang, G., Liu, J., Harvey-White, J., Zimmer, A. and Kunos, G. (2003) Endocannabinoid signaling via cannabinoid receptor 1 is involved in ethanol preference and its age dependent decline in mice. Proceedings of the National Academy of Sciences of the United States of America 100, 13931398.
Young, W. S. 3rd, Bonner, T. and Brann, M. (1986) Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proceedings of the National Academy of Sciences of the United States of America 83, 98279831.[Abstract]