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The acquisition and maintenance of voluntary ethanol drinking in the rat: effects of dopaminergic lesions and naloxone
Behavioural Brain Research 137 (2002) 139 /148 www.elsevier.com/locate/bbr
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The acquisition and maintenance of voluntary ethanol drinking in the rat: effects of dopaminergic lesions and naloxone William J. Shoemaker *, Eva Vavrousek-Jakuba, Cynthia D. Arons, Fung-Cho Kwok Department of Psychiatry, School of Medicine, University of Connecticut Health Center, Farmington Avenue, Farmington, CT 06030, USA Received 26 April 2001; accepted 27 June 2002
Abstract Wistar male rats were microinfused bilaterally with 6-hydroxydopamine or vehicle into the ventral tegmental area. After recovery, ethanol drinking was established using a sucrose-fading paradigm, i.e. rats were given twice a day access to drinks containing increasing amounts of ethanol and decreasing amounts of sucrose. Mean daily intakes at each ethanol/sucrose concentration were similar irrespective of the level of dopamine depletion that, in some animals, reached 80 /90%. The percentage of rats testing as ethanol preferers in a two-bottle choice test also appeared similar in both the lesioned and control groups. After completing the sucrose-fading protocol, all rats were switched to one access per day during which they were presented with a drink containing 10% ethanol with 5% sucrose. Naloxone administration (15 min before the daily access period) decreased ethanol beverage consumption by about 50%, irrespective of the level of dopamine depletion. Total daily water intake was not altered by naloxone. In a two-bottle choice situation, naloxone suppressed intake of an ethanol drink (10% ethanol/5% sucrose), but not the intake of 5% sucrose alone. Thus, a lesion of the dopaminergic cell bodies that results in extensive depletion of dopamine in mesolimbic target regions produced no measurable effect on intake of the sweetened ethanol drinks during the acquisition phase of the sucrose-fading paradigm. Furthermore, during the maintenance phase of drinking, the marked effect of naloxone in inhibiting ethanol beverage ingestion (but not water ingestion or sucrose alone solutions) occurred despite extensive loss of dopaminergic innervation to telencephalic target regions. A preliminary account of these experiments appeared in an abstract form [48] and as an Internet publication [49]. (Supported by NIAAA grants P50-03510 and T32-0720). # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ventral tegmental area; Naloxone administration; Meso-limbic dopamine system
1. Introduction For many years, it has been suggested that the reinforcing properties of ethanol, which are thought to be central to its long-term abuse potential, are mediated in large part by the mesolimbic dopamine system [18,40]. This neural pathway, which consists of dopaminergic neurons in the ventral tegmental area (VTA) and its various targets, notably the nucleus accumbens (NAc), has been similarly implicated in the reinforcing properties of most other drugs of abuse [52,18,20,41]. For
* Corresponding author. Present address: Neurobiology Lab, Psychiatry Department, Room L-4111, University of Connecticut Health Center, Farmington, CT 06030-1410, USA. Tel.: /1-860-6793165; fax: /1-860-679-1296 E-mail address: [email protected] (W.J. Shoemaker).
example, acute administration of ethanol increases extracellular levels of dopamine in the NAc as determined by in vivo microdialysis, an action shared by acute exposure to opiates, cocaine, amphetamines, nicotine, and cannabinoids [6,53]. More than 10 years ago, Larry Reid and his associates demonstrated in a series of studies that morphine would increase alcohol drinking and that naloxone would decrease drinking in a rat model (see review [33]). There were subsequent reports [50,25] demonstrating efficacy of naltrexone in treating alcoholics. Shortly thereafter the FDA approved its use in treatment of alcoholism. Several reviews have detailed the relationship between ethanol and the opioid peptide system [9,10,45,47,12,5]. Recently, Rasmussen et al. [30] conducted a series of studies in Sprague/Dawley rats showing that physiologically meaningful blood ethanol levels acutely increased
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VTA and nuc. accumbens bendorphin levels within 30 min. By 3 h, they observed an increase in medial-basal hypothalamic POMC mRNA levels. These results are consistent with the in vitro work of Gianoulakis [11] showing that similar doses of ethanol produced secretion of bendorphin from mediobasal hypothalamic cultures. Similarly, Sarkar reported increased bendorphin secretion and POMC mRNA in cultured rat hypothalamic neurons following exposure to ethanol [26]. There is some question whether the opiate antagonists such as naloxone actually produce their effects on ethanol consumption by their action on opiate receptors directly or by their indirect effects on dopamine release. For example, endorphinergic-induced locomotor stimulation and reinforcement have been reported to be associated with an increase in dopamine release in the NAc [42]. The present study was designed to address some of the questions regarding the role of dopamine and the opiate system in the acquisition and maintenance of alcohol consumption. It is divided into two experiments. The purpose of Experiment 1 is to determine the effect of bilateral lesion in the VTA using 6-OH-dopamine (6OH-DA) on the acquisition of voluntary ethanol intake in a limited access paradigm. The purpose of Experiment 2 is to compare the effectiveness of naloxone administration on suppression of established ethanol drinking in both VTA-lesioned and in vehicle-treated control animals. Each experimental animal was a subject in both phases of the study.
2. Materials and methods
2.2. 6-OH-dopamine lesion surgery Surgery was performed under aseptic conditions. Rats were pretreated with the monoamine oxidase inhibitor, pargylline (50 mg/kg, i.p. Sigma Chemical Company, pargylline /HCl, 800 mg/16 ml saline) and with the noradrenaline uptake inhibitor desipramine (25 mg/kg, i.p., desipramine /HCl, Sigma Chemical Company, dissolved in saline, 400 mg/32 ml) 30 min prior to the administration of the anesthetic (sodium pentobarbital, The Butler Company, Columbus OH, 65 mg/kg body weight, i.p.). Anesthetized rats were positioned in the Kopf stereotaxic apparatus with the incisor bar adjusted to /3.3 mm from the intraaural line [27]. Craniotomy holes for the VTA placement were drilled bilaterally with the following coordinates (from bregma): 5.0 mm posterior and 9/0.9 mm lateral. The microinjections of the neurotoxin (6-OH-DA hydrobromide, Sigma Chemical Company, 6 mg/ml, fresh solution made daily, kept on ice until used) or of the same volume of the vehicle solution (Hank’s balanced salt solution containing 0.2% ascorbic acid, pH adjusted to 7.4, [21]) for control animals, were made using a CMA/12 microdialysis probe attached to CMA/100 pump (BAS, West Lafayette, IN; old probes from which the dialysis membrane was stripped were used) at a flow rate adjusted to 0.4 ml/min. The probe was lowered to the depth of 8.2 mm from bregma. The total volume of the solution delivered to each side of the brain was 1 ml (equal to 4 mg 6-OH-DA base). The infusion probe was left in place for an additional 2 min before removal to assure dispersion of the neurotoxin. Neurotoxin doses were the same as described by others [24]. Average weight at surgery was 3939/6.8 g. A ‘no surgery’ (NS) control group of 3 rats was also included in the experimental design. These animals were given all presurgery injections including the anesthetic, but had no craniotomy surgery.
2.1. Animals Three-month-old male Wistar rats were purchased from Harlan Industries, Indianapolis. All rats were housed in suspended metal cages (2 rats/cage) in the AAALAC-accredited Center for Laboratory Animal Care at the University of Connecticut Health Center. The animal room was maintained at 20 8C with a 12-h light/dark cycle (lights on 6 a.m.). Pelleted diet (Prolab Rat-Mouse-Hamster diet, Agway, Syracuse, NY) and water were available at all times. After lesion surgery all rats were returned to the same room, but were housed singly in polypropylene plastic cages with wood shavings for bedding. This housing arrangement was maintained throughout the remainder of the study. Each experimental animal was a subject in both Experiment 1 and 2. The overall length of time from surgery until sacrifice was 3 months.
3. Experiment 1 3.1. Acquisition of voluntary ethanol drinking in ‘twice-aday home cage limited access paradigm’ A modified version of ‘Samson’s sucrose fading procedure’ [13,39] was used to induce a stable pattern of ethanol drinking. Rats remained in their home cages during each access and had their regular water bottles and food available at all times. Experimental solutions were presented twice a day (at 10:00 h and at 14:00 h), for 20 min, 5 days per week. The drinks were offered at room temperature in small plastic bottles placed on wire cage tops. Intake was assessed from the difference in bottle weight before and after each access period (with corrections applied for drip). The individual bottles were
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stored overnight in a cold room and were cleansed and filled with fresh solutions at least once a week. The first experimental solution contained 20% sucrose with no ethanol added. Thereafter the composition of the experimental solution was changed so that it contained increasing amounts of ethanol and decreasing amounts of sucrose. The final solution contained 10% ethanol (v/ v) and 3% sucrose (w/v). Individual steps and the length of time at each step of the ‘sucrose-fading’ protocol are outlined in Table 1. The 6-OH-DA group, the vehicleinjected group and the NS controls received their first ethanol-containing drink 2 weeks after surgery. All solutions were made using 95% grain-based ethanol that was obtained as a generous gift from Heublein Company (Hartford, CT). Ethanol intakes were calculated as g ethanol per kg body weight (animals were weighed at weekly intervals). 3.2. Ethanol preference test Ethanol preference was assessed on day 4 of stage 10 (Table 1). Two solutions were offered to each animal simultaneously: (a). 10% ethanol in 5% sucrose and (b). 5% sucrose. The length of the access was 20 min. The test was given in the morning and a second time in the afternoon of the same day. The position of the experimental bottles was switched between each individual test session. At the start of the test, each cage was fitted with a freshly clean wire cage top. The top was placed in the opposite orientation to the usual placement (the trough for the food and water towards the back of the cage). This was done to eliminate any lingering odors and/or the placement-associated cues. Preference for ethanol was assessed by calculating the percent of ethanol containing drink consumed relative to the total volume drunk as an average of both sessions. Rats drinking 51% or more of their total fluid intake during that session
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from the bottle containing ethanol were coded as preferers.
4. Experiment 2
4.1. Maintenance of voluntary ethanol drinking and naloxone administration At the end of Experiment 1 all rats were switched to a ‘once-a-day’ limited access protocol. The drink contained 10% ethanol in 5% sucrose and it was given 5 times per week, for 20 min each morning. After 1 week on this protocol all rats were injected with saline (i.p., 0.25 ml) 15 min before access to the drink. Following 2 days of saline injections all rats received injections of naloxone for 4 consecutive days (Naloxone/HCl, Sigma Chemical Company, St. Louis, MO). Naloxone was dissolved in saline and it was given intraperitoneally, 15 min before the access to the drink, at a dose of 1 mg/kg body weight. The first round of naloxone treatment was followed by 2 days of saline injections. In addition to ethanol intake during the 20-min access, water consumption over 24 h was also measured. Four days after completion of the first naloxone trial, a second round of naloxone injections was started to test whether naloxone effect is specific to ethanol. On the second day of the new trial, the rats were given two bottles to drink from: 10% ethanol/5% sucrose and 5% sucrose alone. The intake from each bottle was recorded. Naloxone was then discontinued and 4 days later the same two-bottle choice paradigm was repeated, except that the rats were given saline injections instead of naloxone prior to the access of the two bottles.
Table 1 Ethanol presentation protocol: drink composition and length of time on the specified drink Drink stage
1 2 3 4 5 6 7 8 9 10
Drink composition % Ethanol (w/v)
% Sucrose (w/v)
0 5 5 5 5 5 5 10 10 10
20 20 15 10 7.5 5 3 5 3 5
Number drinks per day
Number of days
2 2 2 2 2 2 2 2 2 2
10 2 2 2 2 2 2 10 2 8
The protocol describes drinking experience of all animals prior to the start of naloxone experiment. Drinks were given at 10:00 h and at 14:00 h, 5 days a week, for 20 min during each access.
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4.2. Brain tissue analysis for catecholamine, indoleamine and protein levels Rats were sacrificed by decapitation following an anesthetic dose of sodium pentobarbital (65 mg/kg body weight). Brains were rapidly removed, placed on an icecold inverted Petri dish, cleansed of membranes, and dissected under a magnifying glass. Six regions of the brain were collected: ventral striatum (including the NAc and tuberculus olphactorium), caudate-putamen, hypothalamus, amygdala, medial prefrontal cortex, and VTA. Bilateral regions were pooled and all tissues immediately frozen on dry ice. All tissues were stored at /70 8C until assay. For assay, samples were thawed on ice, minced in 1 ml of 0.2 N perchloric acid, and sonicated for 1/1.5 min using Kontes sonicator fitted with a microprobe (Vineland, NJ; both power and tune dial set at 4). One hundred microliters (100 ml) of the homogenate was set aside for later protein analysis using BCA Protein Reagent kit from Pierce (Rockford, IL). The rest of the homogenate (900 ml) was processed as described by Lyness [22] by centrifugation (15 min at 10 000 /g ) followed by a filtration of the supernatant using 0.45 mm Millipore filters (Waters, Milford, MA). Supernatant filtrates were kept frozen at /70 8C until injected directly without further purification into the highpressure liquid chromatography system (HPLC). Norepinephrine, dopamine, homovanilic acid, dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindolacetic acid were detected in a single chromatograph run, using 40 ml aliquots of the filtrate per injection. The HPLC hardware consisted of M600A dual piston pump (Waters), refrigerated autoinjection unit (WISP 715, Waters), 680 gradient controller (Waters), reversed phase catecholamine HR-80 column (ESA, Bedford, MA), column heater (Waters, set at 26 8C), electrochemical detector (5100 Coulochem, ESA, gain set at 15 /100) and Waters Maxima 820 workstation for data acquisition and processing. The detector electrodes were set at following potentials: guard cell /0.42 V, preoxidation electrode /0.1 V and the working electrode /0.37 V. The mobile phase was pumped through the system at a flow rate of 1.5 ml/min. Linear gradient elution program was used such as to increase concentration of mobile phase B (70% methanol) from zero to 8% in 15 min. Mobile phase A consisted of 5% methanol, 400 mg/l heptane sulfonic acid, 80 mg/l EDTA, and 6.9 g/l monosodium phosphate; pH was adjusted to 3.0. 4.3. Data analyses In all parameters measured, the vehicle-injected animals were not statistically different from the NS group and both groups were polled for subsequent analyses. Monoamine content in various brain regions
was analyzed by one-way ANOVA, using Scheffe’s test for a post hoc analyses where significant main effects of depletion were noted. Ethanol intake per each stage of Experiment 1 was expressed as g ETOH/day/kg body mass and analyzed using one-way-ANOVA. Total consumption over all stages of protocol was also calculated and subjected to the same analysis. Fluid intake data from Experiment 2 were analyzed using repeated measures ANOVA model with a priori contrasts set to test for differences among the selected groups.
5. Results 5.1. Brain catecholamine and indoleamine levels Neurochemical content in various brain regions of experimental animals is shown in Table 2. The content in the control (vehicle-injected, C) group is given in ng/ mg protein; the content in the 6-OH-DA infused rats is shown as a percentage difference from the controls. The VTA-lesioned group was divided into two groups according to the extent of depletion in the ventral tegmental region: L-high designates the group of three animals with high average dopamine depletion in the VTA (72%), L-low group designates group with less extensive depletion ( /42%). The extent of the dopamine depletion in the L-high was significantly greater than that in the L-low group (post hoc Scheffe, P B/ 0.05). Other brain regions also showed significant dopamine depletion by the neurotoxin: caudate-putamen (96 and 63%) and amygdala (86 and 69%, in the high and low depletion group, respectively). Decreases in dopamine content were also noted in the hypothalamus and in the prefrontal cortex (significantly different from the conTable 2 Brain dopamine, norepinephrine and serotonin content in vehicleinjected controls (expressed as ng/mg protein9sem) and in vta lesioned rats (expressed as percent depleted compared to vehicleinjected controls) Dopamine Norepinephrine
Serotonin
Vehicle-injected controls VTA Ventral striatum
7.8290.39 5.9690.37 56.197.48 10.4191.61
14.9793.81 13.1491.82
‘Low’ depletion VTA Ventral striatum
42*,** 54**
15 53
28 51
‘High’ depletion VTA Ventral striatum
72*,** 93*
15 47
15 38
* ‘High’ depletion group ‘Low’ depletion group, P B 0.05. ** Significantly different from vehicle control, P B 0.05.
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trols only in the prefrontal region of the high depletion group). Both metabolites of dopamine, i.e. DOPAC and HVA, were also decreased in lesioned rats (P B/0.05 in all tissues with an exception of the hypothalamus in Lhigh group and also in the hypothalamus, prefrontal cortex and ventral striatum in the L-low group). Group mean serotonin levels were not significantly decreased in any of the regions examined. In two regions (caudate-putamen and amygdala), a trend toward increased serotonin levels was noted in the lesioned animals. HIAA levels in the amygdala and in the caudate putamen were also elevated above the levels found in the control rats. In all other tissues a decrease in HIAA was noted. With the exception of the prefrontal cortex, the decrease in HIAA levels was not statistically significant in either the low or high lesion group. Norepinephrine levels in the L-high group were not significantly different from the control rats. In the L-low group the decrease was more pronounced and it reached a significant level in the caudate-putamen, prefrontal cortex, and amygdala (post hoc Scheffe, P B/0.05). 5.2. Experiment 1: effect of bilateral 6-OH-dopamine lesions in the ventral tegmental area on the acquisition of ethanol consumption and on ethanol preference Average daily ethanol intake during stage 2 (5% ethanol/20% sucrose) through 10 (10% ethanol/5% sucrose) of the sucrose-fading paradigm is shown in Fig. 1. The daily intake varied with time in all three groups, however there were no significant differences noted among the three groups (6-OH-DA-high; 6-OH-
Fig. 1. Average daily intakes of ethanol in control and 6-OH-DA treated rats. Three groups are shown; black bars are the lesioned group designated ‘low depletion’; white bars are the lesioned group designated ‘high depletion’; controls are shown as the small black square connected by the lines. The intakes are recorded as grams of ethanol consumed per kilogram body mass. The abscissa shows the sequence of the different concentrations of ethanol and sucrose presented to the animals as part of the sucrose-fading paradigm (Table 1).
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DA-low and control) at any stage of the protocol (ANOVA for each stage separately). Overall ethanol intake calculated across all stages (36.39/3.4, 41.29/5.9 and 32.09/1.4 for the Control, L-low and L-high group, respectively) also was not significantly different among the three groups (F2, 11/1.01). In the two-bottle choice test, three control and four lesioned rats (two L-low and two L-high) tested as preferers. Among these ‘preferers’, the percent of the total fluid intake consumed from the ethanol-containing bottle was 60 /85% for the controls and 59 /78% for the lesioned rats, respectively. 5.3. Experiment 2: naloxone administration, ethanol and water consumption during the maintenance phase of ethanol drinking The ethanol intake data from the 20-min daily access to a 10% ethanol in 5% sucrose solution before, during and after naloxone treatment are given in Fig. 2. The data were analyzed by a repeated measure ANOVA with a priori contrasts set to compare drinking under effects of naloxone to the drinking where no treatment, or only saline, was given. Naloxone decreased drinking in all three treatment groups to about 45 /60% of the consumption measured prior to the start of the treatment (naloxone vs. none contrast: F1, 9/31.01, P B/0.001; interaction with the level of depletion: F2, 9 /1.00, NS). Discontinuation of the naloxone treatment resulted in a rapid resumption of drinking, with the slowest recovery of drinking noted in the L-low group. Mean levels of consumption during naloxone treatment and during the
Fig. 2. The effect of naloxone on ethanol intakes in control and VTAlesioned rats. Three groups are shown; black bars are controls; gray bars are the lo-lesioned group and white bars are the hi-lesioned group. Intake is recorded as grams of ethanol consumed per kilogram body mass and the treatments are designated on the abscissa. The decrease in drinking due to naloxone was statistically significant (repeated measures ANOVA, naloxone vs. none contrast, F1,9 /31.01, P B/ 0.001); interaction with level of depletion was not, (F2,9 /1.00, NS).
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Fig. 3. The effect of naloxone on water intakes in control and VTA-lesioned rats. Three groups are shown; black bars are controls; gray bars are the lo-lesioned group and white bars are the hi-lesioned group. Intake is recorded as grams of water consumed per kilogram body mass and the treatments are designated on the abscissa. No significant differences were noted among the groups.
2 days of post-naloxone saline injections, however, were not statistically different from each other (post-naloxone saline vs. naloxone: F1, 9 /1.79, NS, interaction: F2, 9 /2.12, NS), suggesting that the effect of naloxone did not wear off completely in the 2 days after termination of naloxone treatment. Total daily (24-h) water intake before, during, and after the naloxone experiment was also assessed in this experiment (Fig. 3). To correct for differences in body weight the water intake was calculated as ml/kg body mass. The data were analyzed by a repeated measure ANOVA. A priori contrasts were set to compare drinking under the effect of naloxone to the drinking where no treatment or only saline was given. Average water intake among the three groups was not significantly different from each other, although on average the L-high lesion rats drank the least. Water intake after naloxone injection was not different from that when no injection was given (naloxone and none contrast).
prior to the drink access than when given saline (repeated measures ANOVA, F1, 9 /7.49, P B/0.023, interaction between intake and treatment F2, 9 /0.24, NS). In fact, 4 control and 2 lesion rats (one from each group) did not drink at all when given naloxone, and the rest of animals drank less than 60% of intake measured when injected with saline. In contrast, the intake of the sweetened drink without ethanol (5% sucrose) was not significantly decreased by naloxone (repeated measures ANOVA, F1, 9 /0.31, NS). In most rats the intake was actually higher (4 control rats, 1 L-low and 2 L-high rats) or remained the same (2 L-low). Two control rats which drank less sucrose when given naloxone drank about 70% of that amount when saline was given. Only one rat (L-high) drank very little sucrose when given naloxone (6% of intake when saline given).
6. Discussion 5.4. Differential effect of naloxone on consumption of drinks with and without ethanol The results are illustrated in Fig. 4. The top part of Fig. 4 depicts consumption data from the bottle containing sweetened solution with ethanol. On the bottom are consumption data for the solution containing the same amount of sucrose, but no ethanol. The data are given as g solution per kg body mass. Note that consumption of the ethanol containing drink (10% ethanol /5% sucrose) was significantly lower when rats received naloxone
Experiment 1 demonstrated that an intact mesolimbic dopamine system is not required for the acquisition of ethanol drinking. These results are consistent with other studies in which 6-OH-DA lesions of NAc/ventral striatum were employed to study the effect of mesolimbic dopamine depletion on ethanol self administration [31,23,8]. Each study used a different self-administration paradigm but reached a similar conclusion. Rassnick et al. [31] studied the effect of dopamine depletion in male Wistar rats on the maintenance phase of the operant reinforced drinking and Lyness et al. [23] studied the
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Fig. 4. The effect of naloxone and saline on a two-bottle choice paradigm. One bottle contained 10% ethanol and 5% sucrose; the other bottle contained only sucrose (5%). On one day the animals were given naloxone; several days later the study was run again but only saline was given. In the top panel are the data of the three treatment groups on the ethanol-sucrose mix. Here, naloxone has a significant effect of decreasing the amount consumed (measured in grams of fluid consumed per kilogram body mass) (repeated measure ANOVA, F1,9 /7.49, P B/0.023. In the lower panel are the data when the solution was sucrose alone. There was no effect of naloxone in this data set (repeated measure ANOVA, F1,9 /0.31, NS).
acquisition phase of intravenous self-administration in male Sprague/Dawley rats. Similar to our paradigm, Fahlke et al. [8] measured voluntary consumption in the home cage using female Wistar rats with continuous exposure to ethanol solutions. Moreover, while dopamine depletion was chiefly restricted to the NAc and the olfactory tubercle (at least as documented in Rassnick et al. [31] and Fahlke et al. [8]), in our study a large depletion was also produced in the VTA, as this was the site for the toxin infusion. Ikemoto et al. [17] studied the effects of 6-OHDA-lesions of nuc. accumbens on the acquisition and maintenance of ethanol consumption in the alcohol-preferring P-line of rats. In this strain, which has been bred for ethanol preference, nuc. accumbens lesions had no effect on ethanol drinking in animals that had previously established a pattern of drinking 10%
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ethanol solutions. However, when the lesions were made prior to any experience with ethanol, the lesioned rats failed to acquire the levels of ethanol drinking seen in sham-lesioned control animals. The results of these studies suggest that the intact mesolimbic system is not critical for self-administration of ethanol in animals that have already established a pattern of ethanol consumption. In addition, the intact mesolimbic dopamine system may not be critical for acquiring these behaviors unless the animal has been bred selectively for ethanol preference. In partial disagreement with these conclusions, however, are the findings of Quarfordt et al. [29], who reported that consumption of ethanol in an unrestricted 24-h three-bottle choice paradigm was increased in rats that sustained the lesions in the body of the NAc or in the tuberculum olfactorium. However, the consumption of ethanol was not altered in animals whose lesions were shown to be located ventral or medial to the shell of the NAc. This suggests that the function of dopaminergic terminals and/or other neurotransmitters within the NAc as they relate to the control of ethanol selfadministration are specified anatomically. Other anatomical and functional evidence to support this view has been presented elsewhere [14,15,2,46]. Moreover, evidence also exists to show that 6-OH-DA-induced depletion may affect other coexisting components within the NAc differentially [51,55]. Another possible explanation for the lack of the effect of mesolimbic dopamine depletion on the drinking pattern in our study may be the effect of ‘sparing’ and ‘functional recovery’, phenomena that have been described after 6-OH-DA lesions to the striatum (e.g. [43,57,56,3]). Recent microdialysis studies on recovery from 6-OH-DA lesions demonstrated that a very substantial depletion must be achieved in order to observe effects. For example in the striatum, a depletion of over 95% is necessary, otherwise a sufficiently large residual capacity will remain to support dopamine responses to drugs such as amphetamine [38,4] and thus potentially to ethanol also. Nevertheless it remains possible that the intact dopamine system is not the key element in ethanol reinforcement. Dopamine depletion has similarly been shown to be without effect on maintenance of apomorphine [34] or heroin [28] administration. In contrast, similar levels of depletion resulted in attenuation of amphetamine [23,7] or cocaine [35,37,36] self-administration. These results suggest that dopamine’s role in self-administration of drugs may not be the same for all drugs. Further evidence that there may be pathways other than the mesolimbic dopamine pathway involved in mediating the reward aspect of ethanol ingestion comes from a recent developmental study. Yoshimoto et al. [54] lesioned 3-day-old rat pups with 6-OH dopamine and tested for alcohol drinking at 14 weeks of age.
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Voluntary ethanol drinking (10% w/w with water) compared to water was assessed over 2 weeks with both bottles present at all times. Yoshimoto et al. [54] reported that the neonatally lesioned rats drank more ethanol under these conditions than controls. Their interpretation was that the early developmental stage of the lesion resulted in a neuroplastic response whereby serotonin fibers that project to NAc took on a prominent role in establishment of ethanol drinking and preference. A similar conclusion was reached by Cowan and Lawrence in their large review of opioid-dopamine interactions on ethanol consumption [5]. Cowan and Lawrence conclude that there are both dopaminedependant and dopamine-independent mechanisms involved in ethanol consumption. In the studies reported here, serotonin was measured to test whether there was an indication of serotonin fibers expressing ‘opportunism’ following the 6-OH-DA lesion. However, the serotonin levels (Table 2) paralleled those of dopamine providing no evidence of taking a prominent role. The results of Experiment 2 demonstrate that naloxone is effective in blocking ethanol consumption and that its effects are specific to ethanol. Naloxone decreased established ethanol drinking by 50% but had no effect on water consumption or on consumption of a sucrose solution without ethanol. In addition, naloxone’s effect is not dependent on an intact mesolimbic dopamine system since similar results were seen in lesioned and control animals. One of the earliest described effects of naltrexone on ethanol self-administration was by Altshuler et al. [1] in rhesus monkeys. Many subsequent studies have been carried out, primarily in rats; these are reviewed by Hubbell and Reid [16]. We published some preliminary results of the effect of naloxone in 6-OH-DA treated rats in 1993 [48]. However, we have seen no other reports of opiate antagonists on ethanol self-administration in animals with 6-OH-DA-induced lesions. It may not be surprising that naloxone inhibited consumption nearly identically in lesioned and intact animals since the 6OH-DA lesion had no effect itself on acquisition and maintenance of ethanol ingestion. On the other hand, opiate receptors are believed to reside on pre-synaptic dopamine terminals in the basal ganglia and this may help explain naloxone’s block of amphetamine action [44]. Similarly, interactions of the mesolimbic dopamine system and the endogenous opioid system have also been reported to influencing locomotor behavior [42]. Further, the recently described role of the mesolimbic dopamine system in reinstating extinguished ethanolseeking behavior [19] would lead one to suggest that depletion of 80 /90% of the nuc. accumbens dopamine, as in our study, should have some effect. Nevertheless, our results demonstrate that regardless of the level of
depletion achieved, naloxone had equivalent effects on voluntary ethanol ingestion in lesioned and control animals. It may be that the mesolimbic dopamine system responds to saliency in environmental stimuli, as Redgrave et al. [32] has postulated, but is not necessary for acquisition, maintenance or naloxone-induced inhibition of voluntary self-administration of ethanol in animals that are not bred for alcohol preference.
Acknowledgements Authors of the paper are greatly indebted to Dr Joseph Burleson for his help with data analyses and to Dr Deckel for his thoughtful review of the data and their interpretation.
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Research report
The acquisition and maintenance of voluntary ethanol drinking in the rat: effects of dopaminergic lesions and naloxone William J. Shoemaker *, Eva Vavrousek-Jakuba, Cynthia D. Arons, Fung-Cho Kwok Department of Psychiatry, School of Medicine, University of Connecticut Health Center, Farmington Avenue, Farmington, CT 06030, USA Received 26 April 2001; accepted 27 June 2002
Abstract Wistar male rats were microinfused bilaterally with 6-hydroxydopamine or vehicle into the ventral tegmental area. After recovery, ethanol drinking was established using a sucrose-fading paradigm, i.e. rats were given twice a day access to drinks containing increasing amounts of ethanol and decreasing amounts of sucrose. Mean daily intakes at each ethanol/sucrose concentration were similar irrespective of the level of dopamine depletion that, in some animals, reached 80 /90%. The percentage of rats testing as ethanol preferers in a two-bottle choice test also appeared similar in both the lesioned and control groups. After completing the sucrose-fading protocol, all rats were switched to one access per day during which they were presented with a drink containing 10% ethanol with 5% sucrose. Naloxone administration (15 min before the daily access period) decreased ethanol beverage consumption by about 50%, irrespective of the level of dopamine depletion. Total daily water intake was not altered by naloxone. In a two-bottle choice situation, naloxone suppressed intake of an ethanol drink (10% ethanol/5% sucrose), but not the intake of 5% sucrose alone. Thus, a lesion of the dopaminergic cell bodies that results in extensive depletion of dopamine in mesolimbic target regions produced no measurable effect on intake of the sweetened ethanol drinks during the acquisition phase of the sucrose-fading paradigm. Furthermore, during the maintenance phase of drinking, the marked effect of naloxone in inhibiting ethanol beverage ingestion (but not water ingestion or sucrose alone solutions) occurred despite extensive loss of dopaminergic innervation to telencephalic target regions. A preliminary account of these experiments appeared in an abstract form [48] and as an Internet publication [49]. (Supported by NIAAA grants P50-03510 and T32-0720). # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ventral tegmental area; Naloxone administration; Meso-limbic dopamine system
1. Introduction For many years, it has been suggested that the reinforcing properties of ethanol, which are thought to be central to its long-term abuse potential, are mediated in large part by the mesolimbic dopamine system [18,40]. This neural pathway, which consists of dopaminergic neurons in the ventral tegmental area (VTA) and its various targets, notably the nucleus accumbens (NAc), has been similarly implicated in the reinforcing properties of most other drugs of abuse [52,18,20,41]. For
* Corresponding author. Present address: Neurobiology Lab, Psychiatry Department, Room L-4111, University of Connecticut Health Center, Farmington, CT 06030-1410, USA. Tel.: /1-860-6793165; fax: /1-860-679-1296 E-mail address: [email protected] (W.J. Shoemaker).
example, acute administration of ethanol increases extracellular levels of dopamine in the NAc as determined by in vivo microdialysis, an action shared by acute exposure to opiates, cocaine, amphetamines, nicotine, and cannabinoids [6,53]. More than 10 years ago, Larry Reid and his associates demonstrated in a series of studies that morphine would increase alcohol drinking and that naloxone would decrease drinking in a rat model (see review [33]). There were subsequent reports [50,25] demonstrating efficacy of naltrexone in treating alcoholics. Shortly thereafter the FDA approved its use in treatment of alcoholism. Several reviews have detailed the relationship between ethanol and the opioid peptide system [9,10,45,47,12,5]. Recently, Rasmussen et al. [30] conducted a series of studies in Sprague/Dawley rats showing that physiologically meaningful blood ethanol levels acutely increased
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 2 9 0 - 5
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VTA and nuc. accumbens bendorphin levels within 30 min. By 3 h, they observed an increase in medial-basal hypothalamic POMC mRNA levels. These results are consistent with the in vitro work of Gianoulakis [11] showing that similar doses of ethanol produced secretion of bendorphin from mediobasal hypothalamic cultures. Similarly, Sarkar reported increased bendorphin secretion and POMC mRNA in cultured rat hypothalamic neurons following exposure to ethanol [26]. There is some question whether the opiate antagonists such as naloxone actually produce their effects on ethanol consumption by their action on opiate receptors directly or by their indirect effects on dopamine release. For example, endorphinergic-induced locomotor stimulation and reinforcement have been reported to be associated with an increase in dopamine release in the NAc [42]. The present study was designed to address some of the questions regarding the role of dopamine and the opiate system in the acquisition and maintenance of alcohol consumption. It is divided into two experiments. The purpose of Experiment 1 is to determine the effect of bilateral lesion in the VTA using 6-OH-dopamine (6OH-DA) on the acquisition of voluntary ethanol intake in a limited access paradigm. The purpose of Experiment 2 is to compare the effectiveness of naloxone administration on suppression of established ethanol drinking in both VTA-lesioned and in vehicle-treated control animals. Each experimental animal was a subject in both phases of the study.
2. Materials and methods
2.2. 6-OH-dopamine lesion surgery Surgery was performed under aseptic conditions. Rats were pretreated with the monoamine oxidase inhibitor, pargylline (50 mg/kg, i.p. Sigma Chemical Company, pargylline /HCl, 800 mg/16 ml saline) and with the noradrenaline uptake inhibitor desipramine (25 mg/kg, i.p., desipramine /HCl, Sigma Chemical Company, dissolved in saline, 400 mg/32 ml) 30 min prior to the administration of the anesthetic (sodium pentobarbital, The Butler Company, Columbus OH, 65 mg/kg body weight, i.p.). Anesthetized rats were positioned in the Kopf stereotaxic apparatus with the incisor bar adjusted to /3.3 mm from the intraaural line [27]. Craniotomy holes for the VTA placement were drilled bilaterally with the following coordinates (from bregma): 5.0 mm posterior and 9/0.9 mm lateral. The microinjections of the neurotoxin (6-OH-DA hydrobromide, Sigma Chemical Company, 6 mg/ml, fresh solution made daily, kept on ice until used) or of the same volume of the vehicle solution (Hank’s balanced salt solution containing 0.2% ascorbic acid, pH adjusted to 7.4, [21]) for control animals, were made using a CMA/12 microdialysis probe attached to CMA/100 pump (BAS, West Lafayette, IN; old probes from which the dialysis membrane was stripped were used) at a flow rate adjusted to 0.4 ml/min. The probe was lowered to the depth of 8.2 mm from bregma. The total volume of the solution delivered to each side of the brain was 1 ml (equal to 4 mg 6-OH-DA base). The infusion probe was left in place for an additional 2 min before removal to assure dispersion of the neurotoxin. Neurotoxin doses were the same as described by others [24]. Average weight at surgery was 3939/6.8 g. A ‘no surgery’ (NS) control group of 3 rats was also included in the experimental design. These animals were given all presurgery injections including the anesthetic, but had no craniotomy surgery.
2.1. Animals Three-month-old male Wistar rats were purchased from Harlan Industries, Indianapolis. All rats were housed in suspended metal cages (2 rats/cage) in the AAALAC-accredited Center for Laboratory Animal Care at the University of Connecticut Health Center. The animal room was maintained at 20 8C with a 12-h light/dark cycle (lights on 6 a.m.). Pelleted diet (Prolab Rat-Mouse-Hamster diet, Agway, Syracuse, NY) and water were available at all times. After lesion surgery all rats were returned to the same room, but were housed singly in polypropylene plastic cages with wood shavings for bedding. This housing arrangement was maintained throughout the remainder of the study. Each experimental animal was a subject in both Experiment 1 and 2. The overall length of time from surgery until sacrifice was 3 months.
3. Experiment 1 3.1. Acquisition of voluntary ethanol drinking in ‘twice-aday home cage limited access paradigm’ A modified version of ‘Samson’s sucrose fading procedure’ [13,39] was used to induce a stable pattern of ethanol drinking. Rats remained in their home cages during each access and had their regular water bottles and food available at all times. Experimental solutions were presented twice a day (at 10:00 h and at 14:00 h), for 20 min, 5 days per week. The drinks were offered at room temperature in small plastic bottles placed on wire cage tops. Intake was assessed from the difference in bottle weight before and after each access period (with corrections applied for drip). The individual bottles were
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stored overnight in a cold room and were cleansed and filled with fresh solutions at least once a week. The first experimental solution contained 20% sucrose with no ethanol added. Thereafter the composition of the experimental solution was changed so that it contained increasing amounts of ethanol and decreasing amounts of sucrose. The final solution contained 10% ethanol (v/ v) and 3% sucrose (w/v). Individual steps and the length of time at each step of the ‘sucrose-fading’ protocol are outlined in Table 1. The 6-OH-DA group, the vehicleinjected group and the NS controls received their first ethanol-containing drink 2 weeks after surgery. All solutions were made using 95% grain-based ethanol that was obtained as a generous gift from Heublein Company (Hartford, CT). Ethanol intakes were calculated as g ethanol per kg body weight (animals were weighed at weekly intervals). 3.2. Ethanol preference test Ethanol preference was assessed on day 4 of stage 10 (Table 1). Two solutions were offered to each animal simultaneously: (a). 10% ethanol in 5% sucrose and (b). 5% sucrose. The length of the access was 20 min. The test was given in the morning and a second time in the afternoon of the same day. The position of the experimental bottles was switched between each individual test session. At the start of the test, each cage was fitted with a freshly clean wire cage top. The top was placed in the opposite orientation to the usual placement (the trough for the food and water towards the back of the cage). This was done to eliminate any lingering odors and/or the placement-associated cues. Preference for ethanol was assessed by calculating the percent of ethanol containing drink consumed relative to the total volume drunk as an average of both sessions. Rats drinking 51% or more of their total fluid intake during that session
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from the bottle containing ethanol were coded as preferers.
4. Experiment 2
4.1. Maintenance of voluntary ethanol drinking and naloxone administration At the end of Experiment 1 all rats were switched to a ‘once-a-day’ limited access protocol. The drink contained 10% ethanol in 5% sucrose and it was given 5 times per week, for 20 min each morning. After 1 week on this protocol all rats were injected with saline (i.p., 0.25 ml) 15 min before access to the drink. Following 2 days of saline injections all rats received injections of naloxone for 4 consecutive days (Naloxone/HCl, Sigma Chemical Company, St. Louis, MO). Naloxone was dissolved in saline and it was given intraperitoneally, 15 min before the access to the drink, at a dose of 1 mg/kg body weight. The first round of naloxone treatment was followed by 2 days of saline injections. In addition to ethanol intake during the 20-min access, water consumption over 24 h was also measured. Four days after completion of the first naloxone trial, a second round of naloxone injections was started to test whether naloxone effect is specific to ethanol. On the second day of the new trial, the rats were given two bottles to drink from: 10% ethanol/5% sucrose and 5% sucrose alone. The intake from each bottle was recorded. Naloxone was then discontinued and 4 days later the same two-bottle choice paradigm was repeated, except that the rats were given saline injections instead of naloxone prior to the access of the two bottles.
Table 1 Ethanol presentation protocol: drink composition and length of time on the specified drink Drink stage
1 2 3 4 5 6 7 8 9 10
Drink composition % Ethanol (w/v)
% Sucrose (w/v)
0 5 5 5 5 5 5 10 10 10
20 20 15 10 7.5 5 3 5 3 5
Number drinks per day
Number of days
2 2 2 2 2 2 2 2 2 2
10 2 2 2 2 2 2 10 2 8
The protocol describes drinking experience of all animals prior to the start of naloxone experiment. Drinks were given at 10:00 h and at 14:00 h, 5 days a week, for 20 min during each access.
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4.2. Brain tissue analysis for catecholamine, indoleamine and protein levels Rats were sacrificed by decapitation following an anesthetic dose of sodium pentobarbital (65 mg/kg body weight). Brains were rapidly removed, placed on an icecold inverted Petri dish, cleansed of membranes, and dissected under a magnifying glass. Six regions of the brain were collected: ventral striatum (including the NAc and tuberculus olphactorium), caudate-putamen, hypothalamus, amygdala, medial prefrontal cortex, and VTA. Bilateral regions were pooled and all tissues immediately frozen on dry ice. All tissues were stored at /70 8C until assay. For assay, samples were thawed on ice, minced in 1 ml of 0.2 N perchloric acid, and sonicated for 1/1.5 min using Kontes sonicator fitted with a microprobe (Vineland, NJ; both power and tune dial set at 4). One hundred microliters (100 ml) of the homogenate was set aside for later protein analysis using BCA Protein Reagent kit from Pierce (Rockford, IL). The rest of the homogenate (900 ml) was processed as described by Lyness [22] by centrifugation (15 min at 10 000 /g ) followed by a filtration of the supernatant using 0.45 mm Millipore filters (Waters, Milford, MA). Supernatant filtrates were kept frozen at /70 8C until injected directly without further purification into the highpressure liquid chromatography system (HPLC). Norepinephrine, dopamine, homovanilic acid, dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindolacetic acid were detected in a single chromatograph run, using 40 ml aliquots of the filtrate per injection. The HPLC hardware consisted of M600A dual piston pump (Waters), refrigerated autoinjection unit (WISP 715, Waters), 680 gradient controller (Waters), reversed phase catecholamine HR-80 column (ESA, Bedford, MA), column heater (Waters, set at 26 8C), electrochemical detector (5100 Coulochem, ESA, gain set at 15 /100) and Waters Maxima 820 workstation for data acquisition and processing. The detector electrodes were set at following potentials: guard cell /0.42 V, preoxidation electrode /0.1 V and the working electrode /0.37 V. The mobile phase was pumped through the system at a flow rate of 1.5 ml/min. Linear gradient elution program was used such as to increase concentration of mobile phase B (70% methanol) from zero to 8% in 15 min. Mobile phase A consisted of 5% methanol, 400 mg/l heptane sulfonic acid, 80 mg/l EDTA, and 6.9 g/l monosodium phosphate; pH was adjusted to 3.0. 4.3. Data analyses In all parameters measured, the vehicle-injected animals were not statistically different from the NS group and both groups were polled for subsequent analyses. Monoamine content in various brain regions
was analyzed by one-way ANOVA, using Scheffe’s test for a post hoc analyses where significant main effects of depletion were noted. Ethanol intake per each stage of Experiment 1 was expressed as g ETOH/day/kg body mass and analyzed using one-way-ANOVA. Total consumption over all stages of protocol was also calculated and subjected to the same analysis. Fluid intake data from Experiment 2 were analyzed using repeated measures ANOVA model with a priori contrasts set to test for differences among the selected groups.
5. Results 5.1. Brain catecholamine and indoleamine levels Neurochemical content in various brain regions of experimental animals is shown in Table 2. The content in the control (vehicle-injected, C) group is given in ng/ mg protein; the content in the 6-OH-DA infused rats is shown as a percentage difference from the controls. The VTA-lesioned group was divided into two groups according to the extent of depletion in the ventral tegmental region: L-high designates the group of three animals with high average dopamine depletion in the VTA (72%), L-low group designates group with less extensive depletion ( /42%). The extent of the dopamine depletion in the L-high was significantly greater than that in the L-low group (post hoc Scheffe, P B/ 0.05). Other brain regions also showed significant dopamine depletion by the neurotoxin: caudate-putamen (96 and 63%) and amygdala (86 and 69%, in the high and low depletion group, respectively). Decreases in dopamine content were also noted in the hypothalamus and in the prefrontal cortex (significantly different from the conTable 2 Brain dopamine, norepinephrine and serotonin content in vehicleinjected controls (expressed as ng/mg protein9sem) and in vta lesioned rats (expressed as percent depleted compared to vehicleinjected controls) Dopamine Norepinephrine
Serotonin
Vehicle-injected controls VTA Ventral striatum
7.8290.39 5.9690.37 56.197.48 10.4191.61
14.9793.81 13.1491.82
‘Low’ depletion VTA Ventral striatum
42*,** 54**
15 53
28 51
‘High’ depletion VTA Ventral striatum
72*,** 93*
15 47
15 38
* ‘High’ depletion group ‘Low’ depletion group, P B 0.05. ** Significantly different from vehicle control, P B 0.05.
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trols only in the prefrontal region of the high depletion group). Both metabolites of dopamine, i.e. DOPAC and HVA, were also decreased in lesioned rats (P B/0.05 in all tissues with an exception of the hypothalamus in Lhigh group and also in the hypothalamus, prefrontal cortex and ventral striatum in the L-low group). Group mean serotonin levels were not significantly decreased in any of the regions examined. In two regions (caudate-putamen and amygdala), a trend toward increased serotonin levels was noted in the lesioned animals. HIAA levels in the amygdala and in the caudate putamen were also elevated above the levels found in the control rats. In all other tissues a decrease in HIAA was noted. With the exception of the prefrontal cortex, the decrease in HIAA levels was not statistically significant in either the low or high lesion group. Norepinephrine levels in the L-high group were not significantly different from the control rats. In the L-low group the decrease was more pronounced and it reached a significant level in the caudate-putamen, prefrontal cortex, and amygdala (post hoc Scheffe, P B/0.05). 5.2. Experiment 1: effect of bilateral 6-OH-dopamine lesions in the ventral tegmental area on the acquisition of ethanol consumption and on ethanol preference Average daily ethanol intake during stage 2 (5% ethanol/20% sucrose) through 10 (10% ethanol/5% sucrose) of the sucrose-fading paradigm is shown in Fig. 1. The daily intake varied with time in all three groups, however there were no significant differences noted among the three groups (6-OH-DA-high; 6-OH-
Fig. 1. Average daily intakes of ethanol in control and 6-OH-DA treated rats. Three groups are shown; black bars are the lesioned group designated ‘low depletion’; white bars are the lesioned group designated ‘high depletion’; controls are shown as the small black square connected by the lines. The intakes are recorded as grams of ethanol consumed per kilogram body mass. The abscissa shows the sequence of the different concentrations of ethanol and sucrose presented to the animals as part of the sucrose-fading paradigm (Table 1).
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DA-low and control) at any stage of the protocol (ANOVA for each stage separately). Overall ethanol intake calculated across all stages (36.39/3.4, 41.29/5.9 and 32.09/1.4 for the Control, L-low and L-high group, respectively) also was not significantly different among the three groups (F2, 11/1.01). In the two-bottle choice test, three control and four lesioned rats (two L-low and two L-high) tested as preferers. Among these ‘preferers’, the percent of the total fluid intake consumed from the ethanol-containing bottle was 60 /85% for the controls and 59 /78% for the lesioned rats, respectively. 5.3. Experiment 2: naloxone administration, ethanol and water consumption during the maintenance phase of ethanol drinking The ethanol intake data from the 20-min daily access to a 10% ethanol in 5% sucrose solution before, during and after naloxone treatment are given in Fig. 2. The data were analyzed by a repeated measure ANOVA with a priori contrasts set to compare drinking under effects of naloxone to the drinking where no treatment, or only saline, was given. Naloxone decreased drinking in all three treatment groups to about 45 /60% of the consumption measured prior to the start of the treatment (naloxone vs. none contrast: F1, 9/31.01, P B/0.001; interaction with the level of depletion: F2, 9 /1.00, NS). Discontinuation of the naloxone treatment resulted in a rapid resumption of drinking, with the slowest recovery of drinking noted in the L-low group. Mean levels of consumption during naloxone treatment and during the
Fig. 2. The effect of naloxone on ethanol intakes in control and VTAlesioned rats. Three groups are shown; black bars are controls; gray bars are the lo-lesioned group and white bars are the hi-lesioned group. Intake is recorded as grams of ethanol consumed per kilogram body mass and the treatments are designated on the abscissa. The decrease in drinking due to naloxone was statistically significant (repeated measures ANOVA, naloxone vs. none contrast, F1,9 /31.01, P B/ 0.001); interaction with level of depletion was not, (F2,9 /1.00, NS).
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Fig. 3. The effect of naloxone on water intakes in control and VTA-lesioned rats. Three groups are shown; black bars are controls; gray bars are the lo-lesioned group and white bars are the hi-lesioned group. Intake is recorded as grams of water consumed per kilogram body mass and the treatments are designated on the abscissa. No significant differences were noted among the groups.
2 days of post-naloxone saline injections, however, were not statistically different from each other (post-naloxone saline vs. naloxone: F1, 9 /1.79, NS, interaction: F2, 9 /2.12, NS), suggesting that the effect of naloxone did not wear off completely in the 2 days after termination of naloxone treatment. Total daily (24-h) water intake before, during, and after the naloxone experiment was also assessed in this experiment (Fig. 3). To correct for differences in body weight the water intake was calculated as ml/kg body mass. The data were analyzed by a repeated measure ANOVA. A priori contrasts were set to compare drinking under the effect of naloxone to the drinking where no treatment or only saline was given. Average water intake among the three groups was not significantly different from each other, although on average the L-high lesion rats drank the least. Water intake after naloxone injection was not different from that when no injection was given (naloxone and none contrast).
prior to the drink access than when given saline (repeated measures ANOVA, F1, 9 /7.49, P B/0.023, interaction between intake and treatment F2, 9 /0.24, NS). In fact, 4 control and 2 lesion rats (one from each group) did not drink at all when given naloxone, and the rest of animals drank less than 60% of intake measured when injected with saline. In contrast, the intake of the sweetened drink without ethanol (5% sucrose) was not significantly decreased by naloxone (repeated measures ANOVA, F1, 9 /0.31, NS). In most rats the intake was actually higher (4 control rats, 1 L-low and 2 L-high rats) or remained the same (2 L-low). Two control rats which drank less sucrose when given naloxone drank about 70% of that amount when saline was given. Only one rat (L-high) drank very little sucrose when given naloxone (6% of intake when saline given).
6. Discussion 5.4. Differential effect of naloxone on consumption of drinks with and without ethanol The results are illustrated in Fig. 4. The top part of Fig. 4 depicts consumption data from the bottle containing sweetened solution with ethanol. On the bottom are consumption data for the solution containing the same amount of sucrose, but no ethanol. The data are given as g solution per kg body mass. Note that consumption of the ethanol containing drink (10% ethanol /5% sucrose) was significantly lower when rats received naloxone
Experiment 1 demonstrated that an intact mesolimbic dopamine system is not required for the acquisition of ethanol drinking. These results are consistent with other studies in which 6-OH-DA lesions of NAc/ventral striatum were employed to study the effect of mesolimbic dopamine depletion on ethanol self administration [31,23,8]. Each study used a different self-administration paradigm but reached a similar conclusion. Rassnick et al. [31] studied the effect of dopamine depletion in male Wistar rats on the maintenance phase of the operant reinforced drinking and Lyness et al. [23] studied the
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Fig. 4. The effect of naloxone and saline on a two-bottle choice paradigm. One bottle contained 10% ethanol and 5% sucrose; the other bottle contained only sucrose (5%). On one day the animals were given naloxone; several days later the study was run again but only saline was given. In the top panel are the data of the three treatment groups on the ethanol-sucrose mix. Here, naloxone has a significant effect of decreasing the amount consumed (measured in grams of fluid consumed per kilogram body mass) (repeated measure ANOVA, F1,9 /7.49, P B/0.023. In the lower panel are the data when the solution was sucrose alone. There was no effect of naloxone in this data set (repeated measure ANOVA, F1,9 /0.31, NS).
acquisition phase of intravenous self-administration in male Sprague/Dawley rats. Similar to our paradigm, Fahlke et al. [8] measured voluntary consumption in the home cage using female Wistar rats with continuous exposure to ethanol solutions. Moreover, while dopamine depletion was chiefly restricted to the NAc and the olfactory tubercle (at least as documented in Rassnick et al. [31] and Fahlke et al. [8]), in our study a large depletion was also produced in the VTA, as this was the site for the toxin infusion. Ikemoto et al. [17] studied the effects of 6-OHDA-lesions of nuc. accumbens on the acquisition and maintenance of ethanol consumption in the alcohol-preferring P-line of rats. In this strain, which has been bred for ethanol preference, nuc. accumbens lesions had no effect on ethanol drinking in animals that had previously established a pattern of drinking 10%
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ethanol solutions. However, when the lesions were made prior to any experience with ethanol, the lesioned rats failed to acquire the levels of ethanol drinking seen in sham-lesioned control animals. The results of these studies suggest that the intact mesolimbic system is not critical for self-administration of ethanol in animals that have already established a pattern of ethanol consumption. In addition, the intact mesolimbic dopamine system may not be critical for acquiring these behaviors unless the animal has been bred selectively for ethanol preference. In partial disagreement with these conclusions, however, are the findings of Quarfordt et al. [29], who reported that consumption of ethanol in an unrestricted 24-h three-bottle choice paradigm was increased in rats that sustained the lesions in the body of the NAc or in the tuberculum olfactorium. However, the consumption of ethanol was not altered in animals whose lesions were shown to be located ventral or medial to the shell of the NAc. This suggests that the function of dopaminergic terminals and/or other neurotransmitters within the NAc as they relate to the control of ethanol selfadministration are specified anatomically. Other anatomical and functional evidence to support this view has been presented elsewhere [14,15,2,46]. Moreover, evidence also exists to show that 6-OH-DA-induced depletion may affect other coexisting components within the NAc differentially [51,55]. Another possible explanation for the lack of the effect of mesolimbic dopamine depletion on the drinking pattern in our study may be the effect of ‘sparing’ and ‘functional recovery’, phenomena that have been described after 6-OH-DA lesions to the striatum (e.g. [43,57,56,3]). Recent microdialysis studies on recovery from 6-OH-DA lesions demonstrated that a very substantial depletion must be achieved in order to observe effects. For example in the striatum, a depletion of over 95% is necessary, otherwise a sufficiently large residual capacity will remain to support dopamine responses to drugs such as amphetamine [38,4] and thus potentially to ethanol also. Nevertheless it remains possible that the intact dopamine system is not the key element in ethanol reinforcement. Dopamine depletion has similarly been shown to be without effect on maintenance of apomorphine [34] or heroin [28] administration. In contrast, similar levels of depletion resulted in attenuation of amphetamine [23,7] or cocaine [35,37,36] self-administration. These results suggest that dopamine’s role in self-administration of drugs may not be the same for all drugs. Further evidence that there may be pathways other than the mesolimbic dopamine pathway involved in mediating the reward aspect of ethanol ingestion comes from a recent developmental study. Yoshimoto et al. [54] lesioned 3-day-old rat pups with 6-OH dopamine and tested for alcohol drinking at 14 weeks of age.
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Voluntary ethanol drinking (10% w/w with water) compared to water was assessed over 2 weeks with both bottles present at all times. Yoshimoto et al. [54] reported that the neonatally lesioned rats drank more ethanol under these conditions than controls. Their interpretation was that the early developmental stage of the lesion resulted in a neuroplastic response whereby serotonin fibers that project to NAc took on a prominent role in establishment of ethanol drinking and preference. A similar conclusion was reached by Cowan and Lawrence in their large review of opioid-dopamine interactions on ethanol consumption [5]. Cowan and Lawrence conclude that there are both dopaminedependant and dopamine-independent mechanisms involved in ethanol consumption. In the studies reported here, serotonin was measured to test whether there was an indication of serotonin fibers expressing ‘opportunism’ following the 6-OH-DA lesion. However, the serotonin levels (Table 2) paralleled those of dopamine providing no evidence of taking a prominent role. The results of Experiment 2 demonstrate that naloxone is effective in blocking ethanol consumption and that its effects are specific to ethanol. Naloxone decreased established ethanol drinking by 50% but had no effect on water consumption or on consumption of a sucrose solution without ethanol. In addition, naloxone’s effect is not dependent on an intact mesolimbic dopamine system since similar results were seen in lesioned and control animals. One of the earliest described effects of naltrexone on ethanol self-administration was by Altshuler et al. [1] in rhesus monkeys. Many subsequent studies have been carried out, primarily in rats; these are reviewed by Hubbell and Reid [16]. We published some preliminary results of the effect of naloxone in 6-OH-DA treated rats in 1993 [48]. However, we have seen no other reports of opiate antagonists on ethanol self-administration in animals with 6-OH-DA-induced lesions. It may not be surprising that naloxone inhibited consumption nearly identically in lesioned and intact animals since the 6OH-DA lesion had no effect itself on acquisition and maintenance of ethanol ingestion. On the other hand, opiate receptors are believed to reside on pre-synaptic dopamine terminals in the basal ganglia and this may help explain naloxone’s block of amphetamine action [44]. Similarly, interactions of the mesolimbic dopamine system and the endogenous opioid system have also been reported to influencing locomotor behavior [42]. Further, the recently described role of the mesolimbic dopamine system in reinstating extinguished ethanolseeking behavior [19] would lead one to suggest that depletion of 80 /90% of the nuc. accumbens dopamine, as in our study, should have some effect. Nevertheless, our results demonstrate that regardless of the level of
depletion achieved, naloxone had equivalent effects on voluntary ethanol ingestion in lesioned and control animals. It may be that the mesolimbic dopamine system responds to saliency in environmental stimuli, as Redgrave et al. [32] has postulated, but is not necessary for acquisition, maintenance or naloxone-induced inhibition of voluntary self-administration of ethanol in animals that are not bred for alcohol preference.
Acknowledgements Authors of the paper are greatly indebted to Dr Joseph Burleson for his help with data analyses and to Dr Deckel for his thoughtful review of the data and their interpretation.
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