1 School of Psychology, University of Sydney, NSW 2006, Australia and 2 School of Psychology, University of New England, Armidale, NSW 2351, Australia
* Author to whom correspondence should be addressed at: Tel: +61 2 9351 3571; Fax: +61 2 9351 8023; E-mail: iain{at}psych.usyd.edu.au
(Received 5 August 2004; first review notified 1 September 2004; in revised form 11 October 2004; accepted 12 October 2004)
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The reinstatement paradigm is an animal model that may help researchers elucidate the causal factors (Shaham et al., 2003). The basic model involves the training of animals to self-administer a drug and then extinguishing this responding through non-reward. The animals are then exposed to certain stimuli (e.g. drugs primes, drug-associated environmental cues or stress) to observe whether those stimuli can reinstate extinguished responding. Recent work has illustrated the ability of small priming doses of alcohol, cues paired with alcohol and stressors such as footshock to reinstate alcohol-seeking behaviour (Le et al., 1998
, 1999
; Liu and Weiss, 2002
, 2003
; Shaham et al., 2000
, 2003
).
We have developed an animal model of alcohol dependence based upon the remarkable appetite that laboratory rats have for beer (Gallate and McGregor, 1999; Gallate et al., 1999
; McGregor et al., 1999
). Rats will drink beer much more than equivalent ethanol solutions in water (McGregor et al., 1999
) and will show clear behavioural and neural signs of withdrawal upon termination of access to beer (Topple et al., 1998
; Gallate et al., 2003
). The motivation for beer in rats is decreased by drugs that have anti-craving properties, including the opioid antagonist naltrexone, the cannabinoid receptor antagonist SR 141716, and SR 141716 and naltrexone combined (Gallate and McGregor, 1999
; Gallate et al., 2004
).
We have also demonstrated a potent stimulatory effect of the cannabinoid receptor agonist CP-55,940 on the motivation for beer (Gallate et al., 1999). Other studies have documented similar stimulatory effects of cannabinoid agonists in rats given access to ethanol solutions in consummatory paradigms (Colombo et al., 2002
, 2004
). Problem drinking is frequently associated with cannabis use in humans (Degenhardt and Hall, 2003
) and therefore it is of some clinical interest to determine whether cannabinoid receptor agonists might promote the desire for alcoholic beverages after a period of abstinence.
Recently, the synthetic cannabinoid receptor agonist HU-210 was found to reinstate extinguished responding for cocaine in rats (De Vries et al., 2001), although in a separate study, the main psychoactive constituent of cannabis,
9-THC, failed to show such an effect (Schenk and Partridge, 1999
). More recently, the synthetic cannabinoids HU-210, WIN-55212-2 and CP-55,940 but not
9-THC were also found to reinstate heroin seeking in rats (De Vries et al., 2003
; Fattore et al., 2003
) In contrast,
9-THC attenuated the increase in methamphetamine-seeking behaviour produced by small priming doses of methamphetamine (Anggadiredja et al., 2004
). Thus
9-THC, in contrast to the synthetic cannabinoid agonists, has so far failed to reinstate drug seeking in any published study. It was therefore the aim of the present study to determine whether
9-THC might reinstate responding in abstinent rats trained previously to self-administer alcohol.
A lick-based beer delivery system was used, as described previously (Gallate and McGregor, 1999; Gallate et al., 1999
; McGregor et al., 1999
). The method used in this study involved the training of rats to lick a tube for small droplets of beer delivered under a VR-10 schedule of reinforcement. Control rats were given equivalent training but were reinforced with either near-beer, a beer-like beverage that contains <0.5% ethanol (Experiment 1), or sucrose solution (Experiment 2). Following a period of extinction, the rats were then tested for the ability of
9-THC and various other stimuli to restore licking at the tube, despite continuing extinction conditions.
The current study also examined effects of the short-acting benzodiazepine drug midazolam. Midazolam shares with 9-THC the ability to stimulate appetite and to cause motor incapacitation at high doses (Cooper and Yerbury, 1986
; Drugan et al., 1996
; Higgs and Cooper, 1998
, 2000
). However, given the widespread use of benzodiazepines in alcohol detoxification it was thought unlikely that midazolam would reinstate extinguished responding for beer. Midazolam has well-documented anxiolytic effects in rats (Dielenberg et al., 1999
) whereas the cannabinoid receptor agonists tend to be anxiogenic (McGregor et al., 1996
; Giuliani et al., 2000
; Arevalo et al., 2001
; Marin et al., 2003
). Thus a differential effect of midazolam and
9-THC on reinstatement might implicate stress in any reinstatement effects seen.
Several recent studies have described the ability of stressors such as footshock to reinstate responding for ethanol in rats (Le et al., 1998, 1999
, 2000
, 2002
; Liu and Weiss, 2002
). However, the effects of other, more ethologically relevant stressors have yet to be examined. Our study and those of others, have documented the ability of cat odour to elicit powerful defensive responses in rats (Blanchard et al., 1990a
,b
; Dielenberg et al., 1999
; Dielenberg and McGregor, 2001
). These defensive responses are attenuated by beer consumption and by injected ethanol (Blanchard et al., 1990b
; Gallate et al., 2003
). In this study, we tested the effects of cat odour to reinstate extinguished responding for beer.
The effects of food deprivation were also examined. In a previous study (McGregor et al., 1999), we documented the ability of food deprivation to increase the motivation for beer in rats tested under a progressive ratio schedule of reinforcement. However, food deprivation is known to be effective in reinstating heroin or cocaine seeking in rats (Shalev et al., 2000
, 2003
), whereas similar effects with alcohol have not, to our knowledge, been documented.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rats were housed in groups of eight in large plastic tubs with cage lids in a temperature controlled room (21 ± 1°C), maintained on a reverse 12 h light:12 h dark cycle (lights off at 0830 h). The rats had ad libitum access to standard rat chow and tap water except during the daily sessions conducted in the drink chambers, which began at 09:30 h each day.
The rats were handled every few days for 3 weeks prior to the start of the experiment. All experiments adhered to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the University of Sydney Animal Ethics Committee.
Beverages and drugs
The beer used in all experiments was Birell's Premium (Coopers Ltd, South Australia), a beer-like malt beverage that contains 0.5% ethanol by volume (Topple et al., 1998
; Gallate and McGregor, 1999
; Gallate et al., 1999
). Rats in the near-beer condition used in Experiment 1 were given this solution only. Rats in the beer conditions in Experiments 1 and 2 were given the same solution with 4.0% absolute ethanol (v/v) added as described below. As in previous studies the beverages were decarbonated prior to use by being placed on a magnetic stirrer for
20 min. The beer was presented at room temperature.
In Experiment 2, a sucrose control condition was used. Sucrose solutions were prepared by dissolving standard table sugar (CSR Ltd, Australia) in tap water.
The cannabinoid 9-THC, in ethanol solution (Research Biochemical International, Natick MA) was mixed with 1% Tween 80 (Sigma Chemical Co., MO). The ethanol was then evaporated under a stream of nitrogen gas and the remaining liquid was diluted in 0.9% saline.
9-THC was injected intraperitoneally (i.p.) at a dose of 1 mg/kg in Experiment 1 and at doses of 0.5, 1 and 3 mg/kg in Experiment 2, the doses being derived from a previous reinstatement study with cocaine self-administration (Schenk and Partridge, 1999
). The 0.5 and 1 mg/kg doses are relatively low doses with stimulatory effects on appetite while, the 3 mg/kg dose was anticipated to cause some motoric impairment (McGregor et al., 1998
; Williams and Kirkham, 2002
; Higgs et al., 2003
; Verty et al., 2004
).
Midazolam hydrochloride (Roche Products, Australia) was dissolved in 0.9% saline and used at a dose of 0.5 mg/kg in Experiment 1 and doses of 0.15, 0.5 and 1.5 mg/kg in Experiment 2. These doses of midazolam have been previously shown to stimulate appetite (Cooper and Yerbury, 1986) while, the 1.5 mg/kg dose also causes some motoric impairment (Dielenberg et al., 1999
).
Apparatus
All behavioural testing took place in eight custom drinking chambers as described previously (Gallate and McGregor, 1999). These consisted of standard operant chambers (30 x 50 x 25.5 cm) with metal grid floors. The side walls, back wall and roof of the chambers were aluminium, whereas the front wall (door) was clear Perspex. Each chamber was housed inside a light and sound attenuating wooden box (69 x 71 x 61.5 cm) with a fan fitted in the back wall, which provided low level masking noise.
A glass tube (5 mm diameter), containing three inner stainless steel tubes of 1.5 mm diameter, protruded through the left wall of the chamber 3 cm above the floor. Each lick at this lick-tube closed a circuit between the metal tubes, the rat and the metal grid floor, which was detected and counted by a Macintosh computer running WorkbenchMac data acquisition software (McGregor, 1996
). Beverages were delivered down the lick-tube from a 50 ml syringe that was located above the lick-tube on the outside of the drink chamber, which was connected to the lick-tube by plastic tubing. A solenoid pinch-valve (Knosys, Maryland), also located outside of the drink chamber, was interposed between the syringe reservoir and the lick-tube. When closed, the valve prevented the flow of beverage through the plastic tubing to the lick-tube. Opening of the valve was controlled by software such that a set amount of fluid could be delivered depending on the number of licks emitted by the rat.
The chambers also contained two passive infra-red motion detectors (Jaycar, Sydney) located in the centre of each side wall at the same height as the rat. The detectors were customized so that they were sensitive to small movements of the head and body of a rat, but generally did not trigger when the rat was licking at the lick-tube. The outputs of the detectors were also sent to the computer, which allowed a measurement of the total number of seconds spent moving in each session.
Experiment 1
The 32 rats used in Experiment 1 were divided into two cohorts (n = 16 per stream), with n = 8 near-beer and n = 8 beer group rats in each cohort. The rats in the first cohort were used in tests that involved drugs (9-THC and midazolam), whereas the rats in the second cohort were used in tests that involved stress (food deprivation and cat odour exposure). All rats from both streams (n = 32) were used in an initial test of whether injection of saline could reinstate responding. The experimental design involved four different phases as follows.
Phase 1. Pre-exposure. For 22 consecutive days, all rats were given continuous home cage access to either beer or near-beer, depending upon group allocation. They also had continuous access to tap water in a different bottle. Each home cage (containing 8 rats) was thus provided with one drink bottle containing 720 ml of beer or near-beer, and another bottle containing 720 ml of water. Rats in the beer condition initially received near-beer on the first day, then near-beer plus 2% ethanol (v/v) for the next four days, and finally near-beer plus 4% ethanol (v/v) for the remainder of the phase. All bottles were refilled every 24 h, and the positions of the bottles were switched daily to avoid a positional bias.
Phase 2. Self-administration in drink chambers. Home cage access to beer or near-beer was discontinued and daily sessions in the drink chambers were commenced. The rats were placed individually in the drink chambers and were allowed to lick for unlimited amounts of either near-beer or beer (4% v/v) for 30 min each day. The rats were initially trained to lick on a fixed-ratio 3 (FR3) schedule for 9 days. The schedule was then increased to variable ratio 6 (VR6) for 8 days and finally to VR10 for 7 days.
Phase 3. Extinction. On the day following the final VR10 session, the extinction phase commenced. During extinction, the rats were placed in the drinking chambers each day for 30 min with no beverage available. Licking at the lick-tube had no programmed consequence. The criterion for extinction was set at a group average of 10 responses.
When the extinction criterion was reached, all rats were given a vehicle injection immediately before the beginning of the next extinction session. This was done to habituate the animals to the injection procedure prior to drug testing, but also to test whether the mild stress of injection could reinstate alcohol-seeking. Daily injections continued until extinction criteria were regained. The other reinstatement tests commenced later.
Phase 4. Reinstatement tests. For each test in phase 4, the rats were placed in the drinking chambers under extinction conditions for 30 min. Between each test day, there was a baseline day where the rats were also placed in the drinking chambers under extinction conditions but no manipulation was tested.
Food deprivation. The rats were food deprived for 24 h prior to being placed in the drinking chambers. The data from this session were compared with the data obtained from the previous extinction day.
Cat odour. The rats were placed individually in the drinking chambers. A length of cat collar (3 cm long) that had been worn by a domestic cat for 3 weeks was attached by an alligator clip to the back left wall of the drinking chambers at nose height (6 cm from the bottom of the floor) (Dielenberg et al., 1999). After testing, the cat collar pieces were removed from the chambers and the area around the alligator clip was thoroughly cleaned and left to dry overnight. The data for this session were compared with the data obtained on the previous baseline day where the rats were exposed to an unworn cat collar.
9-THC. The rats were injected with
9-THC (1 mg/kg) 10 min prior to being placed in the drinking chambers for 30 min. The data from this session were compared with the data obtained from the previous baseline (with vehicle injection) day.
Midazolam. The rats were injected with midazolam (0.5 mg/kg) 10 min prior to being placed in the drinking chambers. The data from this session were compared with the previous baseline (with vehicle injection) day.
Experiment 2
The approach in Experiment 2 closely followed that of Experiment 1 except that rats in the control group received sucrose throughout the experiment rather than near-beer.
Phase 1. Pre-exposure. The home cage exposure phase involved four cages of 8 rats. Two cages were given access to 1080 ml sucrose per day whereas the other two cages received equivalent access to beer. Rats in the beer condition received access to near-beer for the first 3 days, followed by access to near-beer for 4 days with 2% ethanol added, followed by access to near-beer for 14 days with 4% ethanol added. Rats in the sucrose condition received calorie matched sucrose solutions: 3 days of 5.1% sucrose, followed by 4 days of 7.7% sucrose and then, 10.4% sucrose for the remainder of the experiment. During the home cage pre-exposure phase rats received at least 3 sham i.p. injections to minimize any effects of these injections during the extinction and reinstatement phases.
Phase 2. Self-administration in drink chambers. After the 21 day home cage phase, home cage access to beer or sucrose was withdrawn and rats were then trained to lick for either beer or sucrose on a FR3 schedule for 9 days in 30 min sessions in the drink chambers each day. The reinforcement schedule was then increased to VR6 for 8 days, and finally to VR10 for 7 days.
Phase 3. Extinction. The rats were then placed on extinction for 9 days prior to the start of reinstatement tests. As in the previous experiment, injections of saline were given on the final 4 days of the extinction phase.
Phase 4. Reinstatement tests.
9-THC. A total of 16 rats (8 sucrose and 8 beer) were used to test the reinstating effects of
9-THC at doses of 0, 0.3, 1 and 3 mg/kg. Each rat was tested with each dose of the drug with the order of doses counterbalanced across subjects. The drug was always administered 10 min before the rats were placed in the chambers. One-drug-free day was given in between each reinstatement test with the rats tested as normal on that day to ensure that baseline extinction levels had returned.
Midazolam. The other group of 16 rats (8 sucrose and 8 beer) were used to test the reinstating effects of midazolam at doses of 0, 0.15, 0.5 and 1.5 mg/kg. The drug was given 10 min before the rats were placed in the chambers. Each rat was tested with each dose of the drug with the order of doses counterbalanced across subjects. One drug free day was given in between each reinstatement test.
Statistical analysis
Repeated measures two-way analyses of variance (ANOVA) were used to analyse the data in all phases of the experiments. This analysis involved one between subjects factor and one within subjects factor. The between subjects factor was group (near-beer group versus beer group in Experiment 1 and sucrose group versus beer group in Experiment 2). In Experiment 1, analysis of the reinstatement tests involved a within-subjects factor of day, whereby licking and locomotor activity on the preceding baseline day were compared with that on the reinstatement test day.
In Experiment 2, the within-subjects factor was dose and analysis involved comparing licking and locomotor activity under each dose of the drug, with that obtained in the vehicle condition.
When a pronounced positive skew was evident in the data, log transformations were performed on the raw data prior to statistical analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The vehicle injection given on day 5 significantly reinstated responding in both the near-beer and beer groups (F(1,30) = 17.41, P < 0.05). The injection effect also occurred on day 6 (F(1,30) = 13.87, P < 0.05). The rats did not reach the extinction criterion again until day 8.
Reinstatement tests.
Effect of food deprivation. The results can be seen in Fig. 3. Food deprivation had no significant effect on responding in either group (group: F(1,14) = 2.10; deprivation: F(1,14) = 3.08; group by deprivation: F(1,14) = 0.69). Figure 3 (bottom) also indicates that food deprivation had no significant effects on locomotor activity (group: F(1,14) = 0.99; day: F(1,14) = 0.07; group by day: F(1,14) = 0.35).
|
Effects of 9-THC. Figure 3 depicts the results from the reinstatement test with
9-THC (1 mg/kg). This drug produced an overall reinstatement of responding (F(1,14) = 25.61, P < 0.05) (Fig. 3, top). There was no significant main effect of group (F(1,14) = 0.07) and no group by drug interaction effects (F(1,14) = 0.28), suggesting equivalent effects in the beer and near-beer groups. Figure 3 (bottom) shows that
9-THC significantly decreased locomotor activity (F(1,14) = 13.01, P < 0.05). Again, there was no significant main effect of group (F(1,14) = 0.61), or group by drug interaction (F(1,14) = 2.90), although there was a tendency towards a bigger reduction in locomotor activity in the near-beer group.
Effect of midazolam. Figure 3 shows the results of the reinstatement test with midazolam (0.5 mg/kg). Midazolam failed to reinstate responding (group: F(1,14) = 0.23; drug: F(1,14) = 0.22; group by drug: F(1,14) = 0.06). Figure 3 (bottom) shows that midazolam decreased locomotor activity (F(1,14) = 29.26, P < 0.05), with an increased tendency for midazolam to decrease locomotor activity in the beer group as shown by the near significant group by drug interaction (F(1,14) = 4.32, P = 0.056).
Experiment 2
Pre-exposure phase. The average daily home cage consumption of beer (near-beer with 4% added ethanol) and sucrose (10.4%) over the last 14 days of the home cage phase was 298 ± 10 ml and 793 ± 35 ml, respectively (data not shown). By the final day of the pre-exposure phase, the rats in the beer group were consuming an average of 41.4 ml of 4.5% strength beer each per day. This translates to an ethanol intake of 5.11 g/kg/day in the rats, which weighed an average of 456 g.
Self-administration. Figure 4 shows the number of licks and amount of beverage consumed (ml) during each session of self-administration. As the schedule changed from FR3 to VR6 to VR10, the rats significantly increased their responding (Fig. 4, top). Overall, the sucrose group made more responses than the beer group (F(1,30) = 32.68, P < 0.001). The beer group, but not the sucrose group, appeared to increase their consumption over time (Fig. 4, bottom).
|
Reinstatement tests.
Effects of 9-THC. Figure 5 depicts the data from the reinstatement test with
9-THC (0.3, 1 and 3 mg/kg). Analysis showed there was a significant effect for 0.3 mg/kg of
9-THC (F(1,14) = 6.29, P = 0.02) and a highly significant effect for 1 mg/kg of
9-THC (F(1,14) = 32.71, P < 0.001). There was no significant effect for 3 mg/kg of
9-THC. There were no significant group by dose interaction effects, suggesting equivalent effects of
9-THC on beer and sucrose reinstatement (Fig. 5, top).
|
Effect of midazolam. Figure 5 shows the results of the reinstatement test with midazolam. Midazolam at the lowest dose of 0.15 mg/kg caused an increase in licking under extinction relative to vehicle treatment (F(1,14) = 14.27, p < 0.01). There were no significant effects of the other two doses and no group by dose interaction effects, although the effect of the highest (1.5 mg/kg) dose approached significance (Fig. 5, top).
Figure 5 (bottom) shows the effect of midazolam on locomotor activity. Both the 0.5 mg/kg (F(1,14) = 8.29, P < 0.016) and 1.5 mg/kg (F(1,14) = 54.34, P < 0.001) doses decreased locomotor activity relative to vehicle treatment. There were no significant group by dose interaction effects.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In both experiments described in the present study, the rats showed a good appetite for beer in their home cage, typical of our previous studies (Gallate and McGregor, 1999; Gallate et al., 2003
, 2004
). Intake of beer under free access conditions in the home cage was at least 35 ml per rat per day, with beer strongly preferred over water. As in previous studies, home cage consumption of near-beer or sucrose was somewhat higher than for full strength beer, presumably due to palatability or caloric factors, or even hangover factors associated with consumption of large amounts of full strength beer (McGregor et al., 1999
; Gallate and McGregor, 1999
; Gallate et al., 2003
, 2004
).
When placed on a limited access schedule, rats consumed a considerable amount of beer in short 30 min sessions daily. When reinforcement schedules were changed to FR-6 and VR-10, the rats were motivated to increase the responding to maintain a constant, if not increasing, level of alcohol intake. With the introduction of an extinction schedule, the rats showed a fairly rapid cessation of licking for beer, near-beer and sucrose, with negligible responding evident within 89 days of extinction. This is similar in duration to the extinction periods used in lever-based models of reinstatement.
A number of findings were evident in the various reinstatement tests undertaken. Most notable was the ability of 9-THC to restore licking for beer in abstinent rats. Thus in both Experiments 1 and 2, a clear reinstating effect of
9-THC was evident at the dose of 1 mg/kg. A dose of 0.3 mg/kg also produced an evident effect in Experiment 2. To our knowledge this is the first demonstration of the reinstating effect of THC: previous experiments have failed to obtain significant reinstatement effects with THC on cocaine, heroin and methamphetamine seeking in rats, despite demonstrable effects of synthetic cannabinoid receptor agonists (Schenk and Partridge, 1999
; Fattore et al., 2003
; Anggadiredja et al., 2004
). This suggests an unusually selective effect of THC on alcohol craving versus craving for other drugs.
However, somewhat disappointingly, the reinstating effects of 9-THC obtained were not limited to alcohol-containing beverages. Thus
9-THC also reinstated responding for near-beer in Experiment 1. Given that near-beer contains a residual level of ethanol (<0.5%) we sought to further test the alcohol-specific nature of the effect by using a sucrose solution in Experiment 2. In this experiment,
9-THC clearly reinstated sucrose-seeking as well as beer-seeking behaviour in rats.
The lack of specificity for alcoholic beverages is somewhat perplexing, casting doubt on the possibility that the cannabinoid receptor agonist specifically increases alcohol craving. Interestingly, this parallels our earlier results on the stimulatory effects of cannabinoid receptor agonists on the motivation for beer delivered under a progressive ratio of reinforcement (Gallate et al., 1999). In this study, we found that the synthetic cannabinoid receptor agonist CP-55,940 increased the motivation for beer, as shown by increased break points. However, the motivation to obtain sucrose solutions was also increased by the drug. A subsequent study has recently confirmed that
9-THC, and other cannabinoid receptor agonists, increases the amount of licking made by rats to obtain a palatable 10% sucrose solution (Higgs et al., 2003
). These results are in contrast to the findings of Colombo et al. (2002)
who report that neither CP-55,940 nor WIN-552122 stimulated consumption of sucrose solution in rats during the first 4 h of the dark phase. However, baseline intake of sucrose was very high (
80 ml per session) in this study, perhaps masking a stimulatory effect of the cannabinoids on the motivation for sucrose.
The most obvious explanation of the 9-THC reinstatement effect obtained in the present study is that the drug simply increases appetite for any calorie, containing solution, regardless of alcohol content. This is in line with the well-known appetite stimulatory effects of cannabinoid receptor agonists (Williams et al., 1998
; Koch, 2001
; Williams and Kirkham, 2002
; Verty et al., 2004
). We specifically tested this hypothesis by examining the effects of food deprivation in our model: if the
9-THC simply reflected increased appetite then food deprivation should produce the same effect. Somewhat surprisingly, food deprivation failed to increase beer or near-beer seeking behaviour in Experiment 1. This contrasts with the facilitatory effects of food deprivation on the reinstatement of heroin and cocaine seeking in rats (Shalev et al., 2000
, 2003
), although to our knowledge, an analogous effect on alcohol seeking has yet to be demonstrated. Indeed the present results suggest that food deprivation may have little effect on reinstatement of alcohol seeking, despite our earlier finding that the motivation for beer (delivered under a progressive ratio schedule) is increased by food deprivation (McGregor et al., 1999
).
The lack of involvement of appetite is further suggested by the overall weakness of midazolam to reinstate responding for beer, near-beer or sucrose. At doses similar to those used in the present study, midazolam exerts a powerful orexigenic effect, increasing the consumption of palatable foodstuffs and beverages, including sucrose solutions (Cooper and Yerbury, 1986; Higgs and Cooper, 1998
). However, midazolam failed to affect reinstatement with beer and near-beer in Experiment 1, although it had a weak positive effect on beer and sucrose seeking in Experiment 2. This latter effect is concordant with a recent report that the GABA-A agonist allopregnanolone can reinstate alcohol-seeking behaviour in abstinent rats (Nie and Janak, 2003
) and suggests that further research into the effects of GABA-A receptor ligands on relapse to alcohol seeking may be warranted.
Stressors other than food deprivation are known to exert a potent reinstating effect with alcohol, and this invites another possible explanation of the 9-THC effect. Cannabinoids such as THC are known to have aversive and anxiogenic effects in rats across a wide range of animal models (McGregor et al., 1996
; Giuliani et al., 2000
; Arevalo et al., 2001
; Marin et al., 2003
). This stressor-like effect of THC might therefore underlie the reinstating effects of
9-THC. The present study did not examine the effects of footshock, although it was evident that the very mild stress of injection in Experiment 1 caused a transient increase in responding under extinction (Fig. 2, insert). This suggests that stress may have the ability to increase responding for beer or near-beer under some circumstances. A weak but nonetheless significant stress-induced reinstatement of sucrose seeking has been reported in at least one previous study (Buczek et al., 1999
). Thus a stressor-like effect of
9-THC in promoting beer, near-beer and sucrose seeking cannot be ruled out.
However, it was interesting to note that the exposure to cat odour failed to reinstate beer or near-beer seeking behaviour, indicating that this ethological stressor has different properties to footshock, or indeed 9-THC. Rats exposed to cat odour in the drinking chambers stopped licking completely and showed a significant reduction in locomotor activity. Previous analyses have suggested that exposure to predator threat cues inhibits normal non-defensive housekeeping behaviours (grooming, exploration, feeding and reproduction) whereas defensive behaviours such as freezing, hiding and risk assessment predominate (Blanchard et al., 1990a
). Although we know of no previous published studies of predator odour effects on reinstatement, a recent review notes an unpublished study in which fox odour failed to affect reinstatement of heroin seeking in rats (Shaham et al., 2000
). A general consensus in the literature suggests that reinstatement effects may be both stressor-specific and drug-specific (Lu et al., 2003
): a notion that the current study supports.
Some further insight into the effects of 9-THC in the present study might be obtained by considering the neural substrates of reinstatement effects (see Shaham et al., 2003
for recent review). The bed nucleus of the stria terminalis (BNST) and its projections from the amygdala, appear to play a vital role in reinstatement effects (Erb and Stewart, 1999
; Shaham et al., 2000
). It is perhaps not coincidental that cannabinoids including
9-THC strongly activate these two regions in Wistar rats (McGregor et al., 1998
; Arnold et al., 2001
; Allen et al., 2003
).
Some limitations in the present study should be acknowledged. First, the relatively short period of beer pre-exposure may not have been sufficient to engender strong dependence on alcohol. Reinstatement effects are known to differ depending upon the duration of exposure to ethanol (Liu and Weiss, 2002, 2003
), and presumably whether repeated withdrawals to ethanol have been experienced. It would therefore be of some interest to repeat the present study in rats that have had several months of free access to beer. Clearly, future studies might also usefully assess the effects of other cannabinoid receptor agonists and also determine the ability of cannabinoid receptor antagonists such as SR 141716 to block the effects of
9-THC. The effects of SR 141716 on cue and stress-induced reinstatement to alcohol seeking are also worthy of investigation.
In conclusion, the present study provides some intriguing evidence that 9-THC is able to increase craving for alcoholic beverages in abstinent animals. However, the effect is non-specific in as much as craving for other non-alcoholic beverages is also affected. Moreover, the exact mechanism underlying the effect may require considerable further study although it almost certainly does not involve a simple cannabinoid modulation of appetite.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anggadiredja, K., Nakamichi, M., Hiranita, T., Tanaka, H., Shoyama, Y., Watanabe, S. and Yamamoto, T. (2004) Endocannabinoid system modulates relapse to methamphetamine seeking: possible mediation by the arachidonic acid cascade. Neuropsychopharmacology 29, 14701478.[CrossRef][ISI][Medline]
Arevalo, C., de Miguel, R. and Hernandez-Tristan, R. (2001) Cannabinoid effects on anxiety-related behaviours and hypothalamic neurotransmitters. Pharmacology Biochememsitry, and Behavior 70, 123131.[CrossRef]
Arnold, J. C., Topple, A. N., Mallet, P. E., Hunt, G. E. and McGregor, I. S. (2001) The distribution of cannabinoid-induced Fos expression in rat brain: differences between the Lewis and Wistar strain. Brain Research 921, 240255.[CrossRef][ISI][Medline]
Blanchard, R. J., Blanchard, D. C., Rodgers, J. and Weiss, S. M. (1990a) The characterization and modelling of antipredator defensive behavior. Neuroscience and Biobehavioral Reviews 14, 463472.[ISI][Medline]
Blanchard, R. J., Blanchard, D. C., Weiss, S. M. and Meyer, S. (1990b) The effects of ethanol and diazepam on reactions to predatory odors. Pharmacology, Biochemistry, and Behavior 35, 775780.[CrossRef][ISI][Medline]
Buczek, Y., Le, A. D., Wang, A., Stewart, J. and Shaham, Y. (1999) Stress reinstates nicotine seeking but not sucrose solution seeking in rats. Psychopharmacology 144, 183188.[CrossRef][ISI][Medline]
Ciccocioppo, R., Angeletti, S. and Weiss, F. (2001) Long-lasting resistance to extinction of response reinstatement induced by ethanol-related stimuli: role of genetic ethanol preference. Alcoholism: Clinical and Experimental Research 25, 14141419.[CrossRef][ISI][Medline]
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]
Colombo, G., Serra, S., Vacca, G., Gessa, G. L. and Carai, M. A. (2004) Suppression by baclofen of the stimulation of alcohol intake induced by morphine and WIN 55,2122 in alcohol-preferring rats. European Journal of Pharmacology 492, 189193.[CrossRef][ISI][Medline]
Cooper, S. J. and Yerbury, R. E. (1986) Midazolam-induced hyperphagia and FG 7142-induced anorexia: behavioural characteristics in the rat. Pharmacology, Biochemistry, and Behavior 25, 99106.[CrossRef][ISI][Medline]
De Vries, T. J., Homberg, J. R., Binnekade, R., Raaso, H. and Schoffelmeer, A. N. (2003) Cannabinoid modulation of the reinforcing and motivational properties of heroin and heroin-associated cues in rats. Psychopharmacology 168, 164169.[CrossRef][ISI][Medline]
De Vries, T. J., Shaham, Y., Homberg, J. R., Crombag, H., Schuurman, K., Dieben, J., Vanderschuren, L. and Schoffelmeer, A. N. M. (2001) A cannabinoid mechanism in relapse to cocaine seeking. Nature Medicine 7, 11511154.[CrossRef][ISI][Medline]
Degenhardt, L. and Hall, W. (2003) Patterns of co-morbidity between alcohol use and other substance use in the Australian population. Drug and Alcohol Review 22, 713.[CrossRef][ISI][Medline]
Dielenberg, R. A., Arnold, J. C. and McGregor, I. S. (1999) Low-dose midazolam attenuates predatory odor avoidance in rats. Pharmacology, Biochemistry, and Behavior 62, 197201.[CrossRef][ISI][Medline]
Dielenberg, R. A. and McGregor, I. S. (2001) Defensive behavior in rats towards predatory odors: a review. Neuroscience and Biobehavioral Reviews 25, 597609.[CrossRef][ISI][Medline]
Drugan, R. C., Coyle, T. S., Healy, D. J. and Chen, S. (1996) Stress controllability influences the ataxic properties of both ethanol and midazolam in the rat. Behavioral Neuroscience 110, 360367.[CrossRef][ISI][Medline]
Erb, S. and Stewart, J. (1999) A role for the bed nucleus of the stria terminalis, but not the amygdala, in the effects of corticotropin-releasing factor on stress-induced reinstatement of cocaine seeking. Journal of Neuroscience 19, RC35.[Medline]
Fattore, L., Spano, M. S., Cossu, G., Deiana, S. and Fratta, W. (2003) Cannabinoid mechanism in reinstatement of heroin-seeking after a long period of abstinence in rats. European Journal of Neuroscience 17, 17231726.[ISI][Medline]
Gallate, J. E., Mallet, P. E. and McGregor, I. S. (2004) Combined low dose treatment with opioid and cannabinoid receptor antagonists synergistically reduces the motivation to consume alcohol in rats. Psychopharmacology 173, 210216.[CrossRef][ISI][Medline]
Gallate, J. E. and McGregor, I. S. (1999) The motivation for beer in rats: effects of ritanserin, naloxone and SR 141716. Psychopharmacology 142, 302308.[CrossRef][ISI][Medline]
Gallate, J. E., Morley, K. C., Ambermoon, P. and McGregor, I. S. (2003) The consequences of beer consumption in rats: acute anxiolytic and ataxic effects and withdrawal-induced anxiety. Psychopharmacology 166, 5160.[ISI][Medline]
Gallate, J. E., Saharov, T., Mallet, P. E. and McGregor, I. S. (1999) Increased motivation for beer in rats following administration of a cannabinoid CB1 receptor agonist. European Journal of Pharmacology 370, 233240.[CrossRef][ISI][Medline]
Giuliani, D., Ferrari, F. and Ottani, A. (2000) The cannabinoid agonist HU 210 modifies rat behavioural responses to novelty and stress. Pharmacological Research 41, 4753.[ISI][Medline]
Higgs, S. and Cooper, S. J. (1998) Effects of benzodiazepine receptor ligands on the ingestion of sucrose, intralipid, and maltodextrin: an investigation using a microstructural analysis of licking behavior in a brief contact test. Behavioral Neuroscience 112, 447457.[CrossRef][ISI][Medline]
Higgs, S. and Cooper, S. J. (2000) The effect of the dopamine D2 receptor antagonist raclopride on the pattern of licking microstructure induced by midazolam in the rat. European Journal of Pharmacology 409, 7380.[CrossRef][ISI][Medline]
Higgs, S., Williams, C. M. and Kirkham, T. C. (2003) Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after delta(9)-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716. Psychopharmacology 165, 370377.[ISI][Medline]
Koch, J. E. (2001) Delta(9)-THC stimulates food intake in Lewis rats: effects on chow, high-fat and sweet high-fat diets. Pharmacology, Biochemistry, and Behavior 68, 539543.[CrossRef][ISI][Medline]
Le, A. D., Harding, S., Juzytsch, W., Fletcher, P. J. and Shaham, Y. (2002) The role of corticotropin-releasing factor in the median raphe nucleus in relapse to alcohol. Journal of Neuroscience 22, 78447849.
Le, A. D., Harding, S., Juzytsch, W., Watchus, J., Shalev, U. and Shaham, Y. (2000) The role of corticotrophin-releasing factor in stress-induced relapse to alcohol-seeking behavior in rats. Psychopharmacology 150, 317324.[CrossRef][ISI][Medline]
Le, A. D., Poulos, C. X., Harding, S., Watchus, J., Juzytsch, W. and Shaham, Y. (1999) Effects of naltrexone and fluoxetine on alcohol self-administration and reinstatement of alcohol seeking induced by priming injections of alcohol and exposure to stress. Neuropsychopharmacology 21, 435444.[CrossRef][ISI][Medline]
Le, A. D., Quan, B., Juzytch, W., Fletcher, P. J., Joharchi, N. and Shaham, Y. (1998) Reinstatement of alcohol-seeking by priming injections of alcohol and exposure to stress in rats. Psychopharmacology 135, 169174.[CrossRef][ISI][Medline]
Liu, X. and Weiss, F. (2002) Additive effect of stress and drug cues on reinstatement of ethanol seeking: exacerbation by history of dependence and role of concurrent activation of corticotropin-releasing factor and opioid mechanisms. Journal of Neuroscience 22, 78567861.
Liu, X. and Weiss, F. (2003) Stimulus conditioned to foot-shock stress reinstates alcohol-seeking behavior in an animal model of relapse. Psychopharmacology 168, 184191.[CrossRef][ISI][Medline]
Lu, L., Shepard, J. D., Scott Hall, F. and Shaham, Y. (2003) Effect of environmental stressors on opiate and psychostimulant reinforcement, reinstatement and discrimination in rats: a review. Neuroscience and Biobehavioral Reviews 27, 457491.[CrossRef][ISI][Medline]
Marin, S., Marco, E., Biscaia, M., Fernandez, B., Rubio, M., Guaza, C., Schmidhammer, H. and Viveros, M. P. (2003) Involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940 in male rats. Pharmacology, Biochemistry, and Behavior 74, 649656.[CrossRef][ISI][Medline]
McGregor, I. S. (1996) Using Strawberry Tree WorkbenchMac and Workbench PC software for data acquisition and control in the animal learning laboratory. Behavior Research Methods, Instruments, and Computers 28, 3848.[ISI]
McGregor, I. S., Arnold, J. C., Weber, M. F., Topple, A. N. and Hunt, G. E. (1998) A comparison of Delta(9)-THC and anandamide induced c-fos expression in the rat forebrain. Brain Research 802, 1926.[CrossRef][ISI][Medline]
McGregor, I. S., Issakidis, C. N. and Prior, G. (1996) Aversive effects of the synthetic cannabinoid CP 55,940 in rats. Pharmacology, Biochemistry, and Behavior 53, 657664.[CrossRef][ISI][Medline]
McGregor, I. S., Saharov, T., Hunt, G. E. and Topple, A. N. (1999) Beer consumption in rats: the influence of ethanol content, food deprivation, and cocaine. Alcohol 17, 4756.[CrossRef][ISI][Medline]
Nie, H. and Janak, P. H. (2003) Comparison of reinstatement of ethanol- and sucrose-seeking by conditioned stimuli and priming injections of allopregnanolone after extinction in rats. Psychopharmacology 168, 222228.[CrossRef][ISI][Medline]
Schenk, S. and Partridge, B. (1999) Cocaine-seeking produced by experimenter-administered drug injections: dose-effect relationships in rats. Psychopharmacology 147, 285290.[CrossRef][ISI][Medline]
Shaham, Y., Erb, S. and Stewart, J. (2000) Stress-induced relapse to heroin and cocaine seeking in rats: a review. Brain Research Reviews 33, 1333.[CrossRef][ISI][Medline]
Shaham, Y., Shalev, U., Lu, L., De Wit, H. and Stewart, J. (2003) The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology 168, 320.[CrossRef][ISI][Medline]
Shalev, U., Highfield, D., Yap, J. and Shaham, Y. (2000) Stress and relapse to drug seeking in rats: studies on the generality of the effect. Psychopharmacology 150, 337346.[CrossRef][ISI][Medline]
Shalev, U., Marinelli, M., Baumann, M. H., Piazza, P. V. and Shaham, Y. (2003) The role of corticosterone in food deprivation-induced reinstatement of cocaine seeking in the rat. Psychopharmacology 168, 170176.[CrossRef][ISI][Medline]
Topple, A. N., Hunt, G. E. and McGregor, I. S. (1998) Possible neural substrates of beer-craving in rats. Neuroscience Letters 252, 99102.[CrossRef][ISI][Medline]
Verty, A. N., McFarlane, J. R., McGregor, I. S. and Mallet, P. E. (2004) Evidence for an interaction between CB1 cannabinoid and melanocortin MCR-4 receptors in regulating food intake. Endocrinology 145, 32243231.
Williams, C. M. and Kirkham, T. C. (2002) Observational analysis of feeding induced by Delta9-THC and anandamide. Physiology and Behavior 76, 241250.[CrossRef][ISI][Medline]
Williams, C. M., Rogers, P. J. and Kirkham, T. C. (1998) Hyperphagia in pre-fed rats following oral delta9-THC. Physiology and Behavior 65, 343346.[CrossRef][ISI][Medline]
|