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Alcohol & Alcoholism Vol. 41, No. 1, pp. 24–32, 2006 Advance Access publication 10 October 2005 doi:10.1093/alcalc/agh217 PHARMACOLOGICAL MANIPULATION OF CB1 RECEPTOR FUNCTION ALTERS DEVELOPMENT OF TOLERANCE TO ALCOHOL KAREN L. NOWAK1,2, K. YARAGUDRI VINOD1,2 and BASALINGAPPA L. HUNGUND1,2,3* 1 New York State Psychiatric Institute, New York, NY, USA, 2Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, USA and 3 College of Physicians and Surgeons, Colombia University, New York, NY, USA (Received 11 July 2005; first review notified 18 August 2005; accepted in revised form 30 August 2005; accepted 9 September 2005; advance access publication 10 October 2005) Abstract — Aims: The current study investigated the efficacy of CB1 receptor-targeted drugs on the development and expression of tolerance to alcohol (EtOH). Methods: An EtOH-inhalation model was used to induce tolerance, as measured by EtOH-induced sedation and hypothermia after a 24 h withdrawal period. Two drug treatment procedures, (i) co-treatment with EtOH and (ii) acute drug administration following chronic EtOH treatment, were used to test the efficacy of CB1 receptor manipulations on EtOH tolerance. Results: The effects of the CB1 receptor agonist CP-55,940 varied depending on paradigm and behavioural measure. Chronic CP55,940 co-treatment blocked tolerance to EtOH-induced hypothermia but not to the sedative effect (sleep time) in EtOH-exposed mice. However, chronic CP-55,940 administration alone resulted in tolerance to the sedative effect of a challenge dose of EtOH in control mice. Acute CP-55,940 administration after chronic alcoholization blocked the development of tolerance to EtOH-induced sedation compared to the EtOH alone exposed group, but induced tolerance to the hypothermic effects of EtOH in control mice. Chronic blockade of CB1 receptor function by SR141716A resulted in tolerance to both the sedative and hypothermic effects of EtOH in control mice, but had no effect on EtOH-exposed mice. Conclusions: The data support a role for the endocannabinoid (EC) system in EtOH tolerance/dependence and suggest that drugs targeted against EC system could be therapeutically useful in treating alcohol-related disorders. INTRODUCTION resulted in decreased preference. There are also innate differences in the cannabinoid system in mouse strains Alcoholism is a psychiatric disorder characterized by impaired selected for EtOH preference. Significant differences were control over drinking, leading to tolerance, physical dependfound in the density, binding affinity and function of CB1 ence, uncontrollable craving, and relapse. The effects of alcoreceptors in brain synaptic plasma membranes (SPM) of hol (EtOH) are mediated via several intracellular signal EtOH-preferring C57BL/6 and EtOH-avoiding DBA/2 mice transduction pathways, involving many known classical (Hungund and Basavarajappa, 2000; Basavarajappa and neurotransmitters and ion channels. There is now evidence to Hungund, 2001). support a role for the endocannabinoid (EC) system in many Little information exists, however on the role of the aspects of EtOH-drinking related behaviours. CB1 receptor function in behavioural tolerance to EtOH. Manipulation of CB1 receptors results in alterations in Compelling evidence in favour of a role of the EC system in EtOH consumption and reward value. Mice lacking CB1 tolerance comes from evidence of neuroadaptive changes receptors (CB1 KO) have a low preference for EtOH that occur following chronic EtOH exposure. Studies from our (Hungund et al., 2003; Poncelet et al., 2003; Wang et al., laboratory have previously demonstrated increased levels of 2003; Naassila et al., 2004). Antagonism of CB1 receptors two naturally occurring cannabimimetic brain constituents, by SR141716A results in decreased EtOH intake and arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol decreased motivation to consume EtOH (Arnone et al., in cultured neuronal cells chronically exposed to EtOH 1997; Colombo et al., 1998; Gallate and McGregor, 1999; (Basavarajappa and Hungund, 1999a; Basavarajappa Rodriguez de Fonseca et al., 1999; Freedland et al., 2001; et al., 2000). Upregulation of AEA and downregulation of Hungund et al., 2002), while the agonists CP-55,940 and N-arachidonyl phosphatidyl ethanolamide, a precursor for WIN 55,212-2 result in dose-dependent increases in consumAEA, have been demonstrated in whole brain plasma memption (Colombo et al., 2002) and increased motivation for branes of mice chronically exposed to EtOH (Hungund et al., EtOH (Gallate et al., 1999). The EtOH deprivation effect is 2002). In addition, downregulation of CB1 receptors and CB1 also blocked by SR141716A treatment in EtOH-preferring receptor agonist stimulated [35S]GTPgS binding in SPM of mice chronically exposed to EtOH have been reported (sP) rats (Serra et al., 2002). Lallemand et al. (2001) have (Basavarajappa et al., 1998; Basavarajappa and Hungund, shown, however, that the effects of SR141716A on EtOH 1999b). consumption depend on the temporal administration of the The present experiments examined CB1 receptor involveantagonist. Rats given the antagonist following chronic ment in behavioural tolerance to EtOH through the activation EtOH administration via inhalation showed a decrease in and blockade of CB1 receptor function. Because Lallemand voluntary EtOH consumption, while administration of et al. (2001) found that alterations in EtOH consumption difSR141716A during chronic alcoholization increased intake. fered depending on the temporal delivery of the CB1 receptor In addition, SR141716A treatment in non-alcoholized rats antagonist used, we used two administration paradigms: (i) coadministration with chronic EtOH inhalation and (ii) adminis*Author to whom correspondence should be addressed at: Nathan Kline tration following chronic alcoholization. EtOH tolerance was Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA. Tel: +1 845 398 5452; Fax: +1 845 398 5451; E-mail: examined using both acute EtOH-induced hypothermia and sedation as measures. [email protected] 24 The Author 2005. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL MATERIALS AND METHODS Animals Seven- to eight-week old Swiss-Webster male mice weighing 25–35 g (Taconic Farms, Germantown, NY) were used in the following experiments (N = 8–12/group). Mice were housed in groups under standard laboratory conditions (12:12 light/dark cycle; 23 ± 1 C) with food and water available ad libitum. All mice were treated in accordance with Institutional and National Institute of Health guidelines for the use and care of laboratory mice. Drugs Solutions (20% v/v) of EtOH were prepared in 0.9% saline. An aliquot of CP-55,940 (Tocris, Ellisville, MO) was dissolved in 1% dimethyl sulphoxide and 1% Tween 80 and mixed in saline and was injected at a volume of 1 ml/kg (0.4 mg/kg). The SR141716A was a kind gift from Sanofi Pharmaceuticals (Montpellier, France) and RBI as part of the Chemical Synthesis Program of the NIMH, contract #1MH30003RBINIMH. An aliquot of SR141716A was similarly prepared for a final injection concentration of 3 mg/kg. Pyrazole (Sigma-Aldrich, St Louis, MO) was dissolved in saline and injected at a volume of 10 ml/kg (68 mg/kg). The dose of SR141716A used in the present experiments was chosen based on substantial findings of the effects of SR141716A on voluntary EtOH consumption and EtOH preference (Arnone et al., 1997; Gallate and McGregor, 1999; Rodriguez de Fonseca et al., 1999; Freedland et al., 2001; Lallemand et al., 2001). In addition, SR141716A has been shown to have a relatively flat dose–response curve, with 1 and 3 mg/kg having effects on EtOH and sucrose consumption, while higher doses result in competing behaviours such as excessive scratching and grooming (Compton et al., 1996; Darmani et al., 2000; Jarbe et al., 2002). The dose of CP-55,940 was similarly chosen based on the literature and pilot studies conducted in this laboratory (Rubino et al., 1997; Rubino et al., 2000; Biscaia et al., 2003). Chronic EtOH inhalation procedure Mice were made EtOH-tolerant by continuous exposure to EtOH vapours following the procedure described below. The infusion chambers were 52 · 34 · 27 cm and made of Lucite. A syringe pump delivered absolute EtOH at 25.5 mg/ min onto a gauze wick in a flask. An air pump delivered air at 2–3 l/min through the flask and into the chamber at appropriate flow rates to give nominal EtOH vapour concentrations of 10–16 mg/l. EtOH-exposed mice were given a priming dose of EtOH (2 g/kg) in addition to pyrazole before being placed in the chamber on the first day of the procedure. Pyrazole, an EtOH dehydrogenase inhibitor, was given to inhibit EtOH metabolism and stabilize blood EtOH levels. Mice were exposed to EtOH vapours for 72 h, with daily pyrazole injections. Mice remained in either the EtOH- or air-infusion chamber constantly throughout this 72 h period, except for 30–40 min each morning for injections. Control mice treated with a similar dose of pyrazole as the EtOH group, were subjected to identical housing and handling conditions, with the exception of the EtOH pre-exposure and infusion into the chamber. Body weights and blood samples via tail blood 25 extraction were taken daily in the morning between 9 and 10 AM. Blood samples (10 ml) were collected by clipping the tip of the tail and were mixed 10:90 (v/v) with 3.4% perchloric acid. The mixture was centrifuged at 12 000 r.p.m. and the supernatant (50 ml) was used for EtOH estimation by the spectrophotometric enzymatic method as described previously (Lundquist, 1959; Basavarajappa et al., 1998; Basavarajappa and Hungund, 1999b). Briefly, 50 ml aliquots of supernatant were incubated in semicarbazide buffer containing NAD (2 mM) and alcohol dehydrogenase for 30 min. The resultant concentration of NADH was determined at 340 nM and corresponding concentrations of EtOH were determined using a standard curve. Similarly chamber EtOH concentration was determined by analysing aliquots of air at different ports in the chamber and expressed as mg/l of air. Delivery of EtOH was adjusted to give chamber EtOH vapour concentration of 10–12 mg/l of air. The EtOH inhalation model used in the current studies has proven to be a valuable paradigm for studying EtOH tolerance and dependence (Goldstein and Pal, 1971; Blum et al., 1975; Sprague and Craigmill, 1978; Littleton and Little, 1994; Kliethermes et al., 2004). In the current studies, 72 h EtOH exposure resulted in tolerance to the sedative and hypothermic effects of EtOH. Behavioural studies Mice were given a 24 h withdrawal period after 72 h EtOH vapour exposure prior to behavioural testing. Acute EtOH-induced sedation EtOH-induced sedation was measured by determining the time between loss of the righting reflex (LORR) and time to regain the righting reflex after the administration of a challenge dose of EtOH (4 g/kg body wt). After the LORR, the mice were placed on their back in a V-shaped trough and time was recorded. The mice were deemed to have regained the righting reflex if they could right themselves three times within 30 s when again placed on their backs. Hypothermia EtOH-induced hypothermia was assessed by monitoring rectal temperatures, 60 min after a challenge dose of EtOH (2.0 g/kg body wt). Temperature was determined using a digital rectal thermometer (Physiotemp model BAT-12, Physiotemp Instruments, Inc., Clifton, NJ). The lubricated rectal probe was inserted 1.5 cm into the rectum and temperature was allowed to stabilize before recording. Temperatures were recorded immediately prior to the EtOH challenge and 60 min thereafter. The difference in body temperature between pre- and post-injection determined the degree of hypothermia. The challenge doses of EtOH chosen were based upon pilot work in the laboratory, determining 2.0 g/kg EtOH was optimal to reveal the hypothermic effects of EtOH and that 4.0 g/kg produced consistent sedation with which to examine tolerance. Effects of co-administration of CP-55,940 with chronic EtOH exposure, on tolerance to the sedative and hypothermic effects of EtOH Tolerance to the sedative and hypothermic effects of EtOH were assessed following four experimental conditions: (i) EtOH 26 K. L. NOWAK et al. inhalation, (ii) co-administration of the CB1 receptor agonist CP-55,940 and EtOH inhalation, (iii) chronic CP-55,940 treatment, and (iv) vehicle (pyrazole) administration. Groups treated with the CB1 agonist received CP-55,940 (0.4 mg/kg) 24 and 1 h prior to the beginning of the 72-h inhalation protocol and twice daily for 2.5 days. Acute effects of CP-55,940 administration following chronic alcoholization on tolerance to the hypnotic and hypothermic effects of EtOH Mice were exposed to chronic EtOH via inhalation and then given CP-55,940 (0.4 mg/kg) or vehicle 24 h after removal from the chamber. After 1 h, mice were challenged with 4 or 2 g/kg EtOH and sedation and hypothermia, respectively were measured. Effects of chronic SR141716A administration during chronic alcoholization on tolerance to the sedative and hypothermic effects of EtOH Tolerance to the sedative and hypothermic effects of EtOH was assessed in four groups of mice, treated with: (i) EtOH inhalation, (ii) co-administration of the CB1 receptor antagonist SR141716A and EtOH inhalation, (iii) chronic SR141716A treatment, and (iv) vehicle (pyrazole) administration. Groups treated with the CB1 antagonist received SR141716A (3 mg/kg) 24 and 1 h prior to the beginning of the 72-h inhalation protocol and once daily, thereafter. EtOH-induced sedation and hypothermia were measured as stated above. Effects of acute administration of SR141716A following chronic alcoholization on tolerance to the hypnotic and hypothermic effects of EtOH Mice were exposed to chronic EtOH via inhalation and then given either SR141716A (3 mg/kg) or vehicle 24 h after removal from the chamber. After 1 h, mice were challenged with 4 or 2 g/kg EtOH and sedative effect and hypothermia, respectively, were measured. Effects of co-administration of CP-55,940 during chronic alcoholization on tolerance to the sedative effects of EtOH A two-way ANOVA on the amount of time between the loss of and regaining the righting reflex revealed a significant main effect for Group (F1,44 = 26.12, P < 0.001) and Drug (F1,44 = 22.28, P < 0.0001), in addition to a significant Group · Drug interaction (F1,44 = 4.05, P = 0.05). A clear tolerance was demonstrated between EtOH-exposed mice and control mice given vehicle, verifying the efficacy of the inhalation model (mean ± SEM = 30.00 ± 3.47 and 36.73 ± 5.91 vs 75.50 ± 9.51; LSM with Bonferroni adjustment, P < 0.017). The results also suggest that CP-55,940 differentially affected EtOH-exposed and control mice. A significant reduction in the duration of sedation in control mice treated with CP-55,940 relative to vehicle-treated controls (mean ± SEM = 47.33 ± 4.12 vs 86.5 ± 4.56) was observed but produced no significant effect in EtOH-tolerant mice (Fig. 2). Fig. 1. The blood EtOH levels were estimated in mice following chronic treatment with EtOH, EtOH + CP-55,940, and EtOH + SR141716A. There were no significant changes (P < 0.99) in the BEL by the co-administration of either CP-55,940 or SR141716A during alcoholization. Statistical analyses Analyses were conducted using two-way ANOVAs (Group: EtOH or Air · Drug: CP-55,940/SR141716A or Vehicle). Planned comparisons were done to assess whether: (i) the drug (CP-55,940 or SR141716A) affected tolerance, (ii) the drug affected basal levels, and (iii) tolerance developed. The analyses were performed by the least squares means method (LSM) using SAS statistical software (Cary, NC) with Bonferroni adjustments (P < 0.017). One-way ANOVA test was used to compare the EtOH levels. RESULTS The blood EtOH levels The average blood EtOH levels (BELs) during EtOH exposure was 1.68 ± 0.05. The BELs were found to be similar in the EtOH, EtOH ± CP-55,940, and EtOH ± SR141716A groups (P < 0.99). This indicates no effect of co-treatment of either CP-55,940 or SR141716A on the EtOH metabolism during chronic alcoholization (Fig. 1). Fig. 2. EtOH-induced LORR following chronic treatment with EtOH, CP-55,940, CP-55,940 + EtOH, or vehicle. Histogram represents the time (mean ± SEM) between the loss and gain of the righting reflex following a challenge dose of 4 g/kg EtOH. Tolerance to challenge dose of EtOH was demonstrated in EtOH-exposed mice compared to control mice given vehicle (@P < 0.0001). There was a trend towards CP-55,940 reducing the duration of LORR relative to vehicle-treated controls (#P < 0.0001). Similarly, a significant tolerance to challenge dose of EtOH was evident in the EtOH + CP-55,940 group when compared to control mice ($P < 0.0001). CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL Fig. 3. EtOH-induced hypothermia following chronic treatment with EtOH, CP-55,940, CP-55,940 + EtOH, or vehicle. Graph represents (Mean ± SEM) drop in body temperature ( C) at 60 min post-injection following a challenge dose of 2 g/kg EtOH. There was no significant effect of CP-55,940 on the development of tolerance to the hypothermic effects of a challenge dose of EtOH in mice chronically exposed to EtOH. @P < 0.017 compared to control groups. Co-administration of CP-55,940 with EtOH exposure resulted in significant decrease in the body temperature compared to the EtOH group ($P < 0.001). Chronic administration of CP-55,940 to EtOH-naı̈ve mice showed a higher decrease in the body temperature compared to the EtOH group (xP < 0.038). Effects of co-administration of CP-55,940 during chronic alcoholization on tolerance to the hypothermic effects of EtOH Changes in body temperature 60 min post-injection, analysed by two-way ANOVA, did not reveal a significant main effect of Group (F1,37 = 0.80, P > 0.05) nor Drug (F1,37 = 0.124, P > 0.05). There was, however, a significant Group · Drug interaction (F1,37 = 11.37, P < 0.01). As above, tolerance was demonstrated between EtOH-exposed and control mice given vehicle (mean ± SEM = –1.33 ± 0.39 vs –2.74 ± 0.31 C; Bonferroni adjusted to P < 0.017). There was no significant difference between control mice given the drug vs vehicle. However, CP-55,940 significantly blocked the development of tolerance to the hypothermic effect of EtOH (mean ± SEM = –3.0 ± 0.18 vs –1.33 ± 0.39 C), as there was a significant difference between EtOH-exposed mice given CP-55,940 vs EtOH and vehicle (Fig. 3). Effects of acute administration of CP-55,940 after chronic alcoholization on tolerance to the sedative effects of EtOH A two-way ANOVA on the amount of time between the loss of and regaining the righting reflex revealed a significant main effect for Group (F1,24 = 35.69, P < 0.001) and Drug (F1,24 = 8.4, P < 0.007). In addition, there was a significant Group · Drug interaction (F1,24 = 5.17, P < 0.03). EtOHexposed mice given vehicle had a lower latency of LORR (57%; P < 0.01) than control mice given vehicle, suggesting a tolerance to the ataxic effects of EtOH (mean ± SEM = 42.1 ± 10.49 vs 98.8 ± 6.3). There was a trend of decreased tolerance by the CP-55,940 treatment when compared to the vehicle-treated group (P < 0.09). However, increased sedative effect was evident in EtOH-exposed mice treated with 27 Fig. 4. EtOH-induced LORR following chronic alcoholization and acute CP-55,940 treatment. Histogram represents the time (mean ± SEM) between the loss and gain of the righting reflex following a challenge dose of 4 g/kg EtOH in mice treated with CP-55,940 or vehicle 1 h prior to behavioural testing. EtOH-exposed mice showed tolerance to the ataxic effects of EtOH (@P < 0.01). Acute CP-55,940 treatment showed a trend towards blockade of tolerance when compared to the vehicle-treated group (#P < 0.09). The CP-55,940 treatment to the EtOH group reduced the development of tolerance when compared to EtOH-exposed mice ($P < 0.001). CP-55,940 when compared to the EtOH exposed group (P < 0.001; Fig. 4). Effects of acute administration of CP-55,940 after chronic alcoholization on tolerance to the hypothermic effects of EtOH Changes in body temperature 60 min post-injection were analysed using a two-way ANOVA. There was not a significant main effect of Group (F1,38 = 0.54, P > 0.05), but there was a significant Drug effect (F1,38 = 24.38, P < 0.0001) and a significant Group · Drug interaction (F1,38 = 11.09, P < 0.01). A clear tolerance was demonstrated between EtOH-exposed vs. control mice given vehicle, verifying the efficacy of the inhalation model (mean ± SEM = –0.95 ± 0.30 vs –2.19 ± 0.20 C; Bonferroni adjusted P < 0.017). The mice exposed to EtOH + CP-55,940 showed less of a drop in body temperature compared to control group indicative of tolerance to hypothermic effect of EtOH (Fig. 5). Control mice treated with an acute dose of CP-55,940 were also shown to be tolerant to the hypothermic effects of EtOH (mean ± SEM = 0.57 ± 0.39 vs –2.19 ± 0.20 C; CP-55,940 vs vehicle controls, P < 0.0001), suggestive of rapid cross-tolerance to CP-55,940. Effects of co-administration of SR141716A during chronic alcoholization on tolerance to the sedative effects of EtOH A two-way ANOVA on the amount of time between the loss of and regaining the righting reflex revealed a significant main effect for both Group (F1,42 = 12.33, P < 0.001) and Drug (F1,42 = 6.94, P < 0.01). There was no significant Group · Drug interaction (F3,42 = 2.33, P > 0.05). Tolerance to the sedative effect of EtOH was demonstrated in EtOH-exposed vs control mice given vehicle as evidenced by a 51% reduction in sedation (mean ± SEM = 36.73 ± 5.91 vs 75.50 ± 9.51). 28 K. L. NOWAK et al. Fig. 5. EtOH-induced hypothermia following chronic alcoholization and acute CP-55,940 treatment. Graph represents (mean ± SEM) drop in body temperature ( C) at 60 min post-injection following a challenge dose of 2 g/kg EtOH in mice treated with CP-55,940 or vehicle 1 h prior to behavioural testing. Tolerance to challenge dose of EtOH was demonstrated in EtOHexposed mice compared to control mice given vehicle (@P < 0.011). Acute CP-55,940 treatment showed a rapid cross-tolerance to the hypothermic effect of EtOH in alcoholized ($P < 0.043) and non-alcoholized mice (#P < 0.0001). Fig. 6. EtOH-induced LORR following chronic treatment with EtOH, SR141716A, SR141716A + EtOH, or vehicle. Histogram represents the time (mean ± SEM) between the loss and gain of the righting reflex following a challenge dose of 4 g/kg EtOH. Tolerance to challenge dose of EtOH was demonstrated in EtOH-exposed mice compared to control mice given vehicle (@P < 0.009). Chronic SR141716A treatment showed a development of tolerance to the sedative effect of EtOH in alcoholized ($P < 0.0001) and nonalcoholized mice (#P < 0.051) compared to control groups. There was a trend toward SR141716A reducing the duration of LORR compared to vehicletreated controls. Planned comparisons of simple main effects using least square means and Bonferroni correction (P < 0.017) are suggestive, yet not conclusive, of SR141716A reducing the duration of sedation in control mice relative to vehicle-treated controls (mean ± SEM = 28.72 ± 7.54 and 44.00 ± 7.03 vs 75.50 ± 9.51) without affecting EtOH-exposed mice (Fig. 6). Fig. 7. EtOH-induced hypothermia following chronic treatment with EtOH, SR141716A, SR141716A + EtOH, or vehicle. Graph represents (mean ± SEM) drop in body temperature ( C) following a challenge dose of 2 g/kg EtOH in mice treated with SR141716A or vehicle during chronic alcoholization and control mice given the antagonist or vehicle. Tolerance to challenge dose of EtOH was demonstrated in EtOH-exposed mice compared to control mice given vehicle (@P < 0.012). There was a trend toward SR141716A attenuating hypothermia compared to alcoholized ($P < 0.0002) and nonalcoholized mice ($P < 0.004) compared to control group. Effects of co-administration of SR141716A during chronic alcoholization on tolerance to the hypothermic effects of EtOH Changes in body temperature 60 min post-injection were analysed, revealing significant main effects for both Group (F1,43 = 7.13, P < 0.01) and Drug (F1,43 = 10.22, P < 0.01), but no significant Group · Drug interaction (F1,43 = 1.51, P > 0.05). Tolerance was demonstrated between EtOHexposed and control mice given vehicle, verifying the efficacy of the inhalation model (mean ± SEM = –2.0 ± 0.39 vs –3.13 ± 0.33 C; Bonferroni adjusted P < 0.012). As detailed above, planned comparisons are only suggestive of the fact that SR141716A attenuated hypothermia in control mice, but not EtOH-exposed mice, relative to vehicle-treated controls (mean ± SEM = –1.84 ± 0.26 vs –3.13 ± 0.33 C), suggesting cross-tolerance to SR141716A (Fig. 7). Effects of acute administration of SR141716A following chronic alcoholization on tolerance to the sedative effects of EtOH Analysis of sedative effect of challenge dose of EtOH revealed a significant effect of Group (F1,41 = 22.52, P < 0.0001), but no significant Drug effect (F1,41 = 0.01, P > 0.05), nor a significant Group · Drug interaction. (F1,41 = 1.17, P > 0.05). The mice exposed to EtOH alone or EtOH + SR141716A showed a similar reduction in sedation, indicative of tolerance to the sedative effects of EtOH, relative to either SR141716Atreated or control mice (Fig. 8). Effects of acute administration of SR141716A following chronic alcoholization on tolerance to the hypothermic effects of EtOH Analysis of the change in body temperature at 60 min by twoway ANOVA revealed a significant Group effect (F3,39 = 10.02, P < 0.01). There was not a significant effect of Drug CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL Fig. 8. EtOH-induced LORR following chronic alcoholization and acute SR141716A treatment. Histogram represents the time (mean ± SEM) between the loss and gain of the righting reflex following a challenge dose of 4 g/kg EtOH in mice treated with SR141716A or vehicle 1 h prior to behavioural testing. Mice exposed to EtOH (@P < 0.0011) and EtOH + SR141716A (#P < 0.0002) had reduced LORRs compared to control and SR141716Atreated groups, respectively, indicative of tolerance to the ataxic effects of EtOH relative to control mice. Fig. 9. EtOH-induced hypothermia following chronic alcoholization and acute SR141716A treatment. Graph represents (mean ± SEM) drop in body temperature ( C) at 60 min post-injection following a challenge dose of 2 g/kg EtOH in mice treated with SR141716A or vehicle 1 h prior to behavioural testing. Tolerance to challenge dose of EtOH was demonstrated in EtOH-exposed mice compared to control mice given vehicle (@P < 0.061). There was significant reduction in the hypothermic effect of EtOH in acute SR141716A-treated group compared to SR141716A group (#P < 0.013). (F1,39 = 2.19, P >0.05), nor a significant Group · Drug interaction (F1,39 = 0.12, P > 0.05). There was no differential effect of SR141716A on tolerance to the hypothermic effects of EtOH (Fig. 9). DISCUSSION Studies from this laboratory have reported neuroadaptive changes in the CB1 receptor system that occur following chronic EtOH exposure, suggesting a role for this system in 29 EtOH tolerance (Basavarajappa et al., 1998; Basavarajappa and Hungund, 1999a; Basavarajappa and Hungund, 1999b; Basavarajappa et al., 2000). The current studies investigated the efficacy of CB1 receptor-targeted drugs in modulating the development and expression of tolerance to EtOH in Swiss-Webster mice. The results indicate that it is possible to modulate EtOH tolerance with both the CB1 receptor agonist and the antagonist and that results depend upon temporal administration of the drugs. The effects of CP-55,940 varied depending on paradigm and behavioural measure. When chronic CP-55,940 administration resulted in tolerance to the sedative effects of a challenge dose of EtOH in control mice, but did not differentially affect tolerance in EtOH-exposed mice. This finding is consistent with reported studies of cross-tolerance between cannabinoids and EtOH (Sprague and Craigmill, 1976; Siemens and Doyle, 1979; da Silva et al., 2001). When measuring EtOH-induced hypothermia, tolerance to hypothermic effect was observed in mice exposed to EtOH chronically. However, chronic CP-55,940 treatment during chronic alcoholization blocked tolerance to the hypothermic effects of EtOH. Acute CP-55,940 administration after chronic alcoholization had a significant effect on EtOH-induced sedation when compared to the EtOH-exposed mice, suggesting a blockade of expression of tolerance to sedative effect of EtOH. However, there was a trend towards reduction in the development of tolerance in mice treated with CP-55,940 compared to the vehicletreated group. Conversely, administration of the agonist after chronic alcoholization resulted in tolerance to the hypothermic effects of EtOH in control mice, and showed a trend toward attenuating hypothermia in EtOH-exposed mice as well, suggesting that mice displayed rapid cross-tolerance between the agonist and EtOH. A possible explanation, though speculative, for the disparate effects for EtOH-induced hypothermia and hypnosis under both paradigms could be due to differential effects of the agonist on CB1 receptor function in specific brain regions controlling these behaviours. The CB1 receptors are widely distributed throughout the brain, with significantly higher densities in the basal ganglia and cerebellum, areas known to be involved in motor coordination and motor function, and the hypothalamus, which is implicated in thermoregulation (Moldrich and Wenger, 2000). Regarding CP-55,940 co-administration, chronic exposure with either EtOH or CP-55,940 has been shown to increase endogenous cannabinoid content, specifically in the limbic forebrain, and to desensitize the CB1 receptors (Sim-Selley et al., 2002). Changes in the degree of endogenous tone could alter the sensitivity of various neural circuits to both agonists and antagonists. Alternatively, neurotransmitter systems within different brain regions that regulate the behavioural effects of EtOH may be differentially affected by CB1 receptor manipulations. For example, inhibition of serotonin reuptake and blockade of dopamine and NMDA receptors are known to potentiate cannabinoid-evoked hypothermia (Davies and Graham, 1980; Malone and Taylor, 1998; Nava et al., 2000; Rawls et al., 2002). Thus, differential modulation of one or more neurotransmitter systems with CP-55,940 may explain the effects observed. The behavioural syndromes elicited by EtOH and CB1 receptor agonists only partially overlap (Wiley and Martin, 30 K. L. NOWAK et al. 2003). Although both elicit hypothermia, CB1 agonists do not result in the LORR. This may be one explanation for the differential effects of both acute and chronic CP-55,940 on olerance to EtOH-induced hypothermia and ataxia. There are indeed numerous reports of pharmacological and genetic manipulations differentially affecting EtOH-induced hypothermia and the sedation (Syapin et al., 1987; Boehm et al., 2003; Cowen et al., 2003). In addition, the effective dose range of CP-55,940 for a variety of behaviours unlike that of the antagonist SR141716A, is broad. For example, 3 mg of CP-55,940 increases voluntary EtOH intake in selectivelybred sP rats (Colombo et al., 2002), whereas Genn et al. (2004) demonstrated that 40 mg of CP-55,940 resulted in anxiogenic effects, but 10 and 20 mg were ineffective. Although the current experiments demonstrate a role for CP-55,940 in the development and expression of tolerance to the sedative and hypothermic effects of EtOH, a thorough dose–response study is necessary to fully elucidate the effects of this CB1 receptor agonist. The results are suggestive, yet not conclusive, of chronic blockade of the CB1 receptor function by SR141716A reducing the duration of sedation and attenuating hypothermia in control mice relative to vehicle-treated controls. This would suggest that chronic SR141716A treatment results in crosstolerance to EtOH. When SR141716A was given following chronic alcoholization, however, there was no effect of the drug. Mice exposed to EtOH showed clear tolerance to both the sedative and hypothermic effects of EtOH, whether treated with the antagonist or vehicle. Although SR141716A is known to antagonize many of the physiological responses to cannabinoid receptor agonists in vivo, it is intriguing that it produced cross-tolerance to EtOH similar to that of the agonist CP-55,940. Several recent studies have ascribed different pharmacological properties to SR141716A. It has been shown to have inverse agonist properties (Bouaboula et al., 1997; Landsman et al., 1997; MacLennan et al., 1998; Rubino et al., 2000) and there is evidence of a partial agonistlike profile (De Vry and Jentzsch, 2004). The fact that chronic treatment with CP-55,940 resulted in a similar increase in tolerance to the sedative effects of EtOH suggests that SR141716A may act as a partial agonist under the experimental conditions used in the current study. A recent study has demonstrated that co-administration of SR141716A had similar effects on the acquisition of morphine sensitization as that of CP-55,940 co-administration. (Norwood et al., 2003). This study also demonstrated cross-sensitization between CP-55,940 and morphine in rats administered both the agonist and antagonist simultaneously. Alternatively, there is a possibility that SR141716A may produce the observed effects through another non-CB1-receptor-mediated mechanism. Several recent reports suggest the possible presence of a non-CB1 receptor in the brain (Breivogel et al., 2001; Hajos et al., 2001). However, further studies are needed to clearly understand the mechanism of action of SR141716A. 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