Ethanol self-administration in freely feeding and drinking rats: effects of Ro15-4513 alone,and in combination with Ro15-1788 (flumazenil)

Recent work in our laboratory demonstrated that Ro15-4513, a partial inverse benzodiazepine (BDZ) agonist, decreases ethanol (ETOH) self-administration in rodents under fluid deprivation conditions. The present study further examined the effects of Ro15-4513 (2.5 and 5.0 mg/kg) alone and in combination with Ro15-1788, (flumazenil) (8.0 and 16.0 mg/kg), a BDZ receptor antagonist on ETOH self-administration in freely feeding and drinking rats. Animals were trained to consume ETOH (11% v/v) using a limited access procedure. Measurements were taken at 10- and 60-min intervals. Ro15-4513 (2.5 and 5.0 mg/kg) markedly attenuated ETOH consumption at both intervals. The antagonistic actions of Ro15-4513 were completely blocked by the higher dose of flumazenil at both intervals; the lower dose failed to antagonize the Ro15-4513-induced reduction of ETOH intake. When flumazenil was given alone, both doses reduced ETOH self-administration at 60 min; although the magnitude of the antagonism was comparable to that of Ro15-4513 only with the highest does of flumazenil (16.0 mg/kg). Neither Ro15-4513 nor flumazenil alone or in combination significantly altered water intake at any of the tested doses. Rats pretreated with Ro15-4513 showed a substantial reduction in blood ethanol concentration (BEC) compared with the Tween-80 vehicle condition at the 10-min interval. However, the BEC of animals given Ro15-4513 in combination with flumazenil were similar to rats given Tween-80 vehicle. The present study extends our previous research by demonstrating that Ro15-4513 and flumazenil attenuate ETOH self-administration in non-food or water deprived rats. These studies suggest that the suppressant effects of Ro15-4513 and flumazenil on ETOH self-administration are associated with actions at the BDZ site of the GABA_A receptor complex. These data are discussed in relation to the possible mechanism(s) by which Ro-15-4513 and flumazenil exert their antagonism on ETOH self-administration.Portions of this work were presented at the Annual Meeting of the Research Society on Alcoholism under the symposium entitled GABA/BDZ Receptor Update, Marco Island, FL, June 10, 1991.We would like to dedicate this paper to the late Dr. Richard G. Lister, a friend, teacher, colleague and exceptional scientist who contributed substantially to the area of alcohol abuse and alcoholism. Over the past years, Dr. Lister investigated the interactions of alcohol with inverse benzodiazepine agonists and benzodiazepine receptor antagonists. His seminal and more recent papers played an important role in delineating the GABA benzodiazepine systems in the neurobehavioral actions of alcohol. Richard, we will miss you.     

Bremazocine reduces unrestricted free-choice ethanol self-administration in rats without affecting sucrose preference

It has been postulated that opioid systems in the brain may play a role in ethanol reinforcement. In this respect, μ- and δ-opioid receptors may mediate the rewarding effects whereas κ receptors are thought to mediate the aversive effects of opioids. Accordingly, long-acting benzomorphans such as bremazocine, that simultaneously act as μ and δ receptor antagonists and κ receptor agonists may be particularly effective in reducing ethanol self-administration. Therefore, we studied the effect of bremazocine on oral ethanol self-administration in rats using a paradigm [unrestricted free-choice drinking of 10% (v/v) ethanol], previously shown to cause long-term neuroadaptations in the nucleus accumbens and caudate putamen. Bremazocine (0.1 mg/kg, once daily for five consecutive days) reduced ethanol drinking by about 50% during the active period of the animals, whereas the intake of sucrose (3–10% w/v) was affected neither in naive nor in ethanol-experienced rats. This effect of bremazocine appeared not to be secondary to its acute sedative effect or the slight increase in total fluid consumption. Unlike bremazocine, the selective κ-opioid receptor agonist U50,488H (10 mg/kg, once daily) inhibited ethanol drinking only during the first of 5 treatment days and the opioid receptor antagonist naltrexone (0.3–10 mg/kg, once daily) only caused a modest (about 20%) suppression of ethanol drinking during the first hours after drug injection. Thus, bremazocine appears to be far more potent than the clinically applied drug naltrexone in this respect. Our data further support the role of opioid receptors in ethanol reinforcement and indicate that long-acting mixed-action opioids such as bremazocine may be useful as adjuvants for the clinical management of ethanol addiction. Received: 1 July 1998/Final version: 3 September 1998     

Exposure to nicotine enhances acquisition of ethanol drinking by laboratory rats in a limited access paradigm

 Observations in humans suggest that the initial use of tobacco occurs in close temporal proximity to experimentation with alcohol. There have been relatively few research reports, however, examining possible interactions between these two agents. The present experiments examined the effect of nicotine exposure on the acquisition of ethanol drinking behavior in a limited access procedure. In experiment 1, rats were presented with 1-h access to ethanol solutions of increasing concentration for a period of 20 days. Subcutaneous injections of nicotine (0.6 or 1.2 mg/kg salt) or vehicle were administered 30 min prior to each ethanol presentation. Experiment 2 used a similar method, but rats were presented with water along with ethanol during the 1-h test session. Mecamylamine, a nicotinic receptor antagonist, was administered 30 min prior to the nicotine treatment. Nicotine was seen to produce a dose-dependent increase in ethanol drinking behavior which commenced at the 5% ethanol concentration and continued at 8% and again at 10%. In the second experiment, mecamylamine was observed to block completely the nicotine-induced increase in ethanol drinking behavior. The findings suggest that exposure to nicotine can facilitate the acquisition of ethanol drinking behavior in naive rats and that this effect is mediated by nicotine’s interaction at the nicotinic-cholinergic receptor. Received: 11 June 1998 / Final version: 1 October 1998     

Effects of opiate antagonist treatment on the alcohol deprivation effect in long-term ethanol-experienced rats

Rationale: Opiate antagonists are promising pharmacotherapeutic agents for the treatment of alcohol dependence, reducing craving and relapse rates in weaned alcoholics. However, preclinical findings indicate that they can also increase ethanol consumption and preference in animals with a strong liking for ethanol, depending on the dose and treatment regimen. Objective: The present study examined the effects of chronic, intermittent and acute opiate antagonist treatment on the alcohol deprivation effect (ADE) in long-term ethanol- experienced rats, which is an animal model of craving and relapse. Methods: Long-term ethanol-experienced rats were either implanted with mini-osmotic pumps delivering 0, 0.5 or 1 mg/kg per hour naloxone (chronic treatment) or received intermittent naltrexone injections (2×5 mg/kg per day SC). Effects of chronic and intermittent treatment on the ADE were studied in a four-bottle home cage drinking paradigm. In a second experiment, long-term ethanol-experienced rats trained in an operant ethanol self-administration paradigm received acute naltrexone treatment (0, 0.1, 1 or 10 mg/kg SC) before a 23-h session either during basal drinking or during the ADE. Results: Chronic naloxone treatment increased ethanol preference during the ADE. Intermittent naltrexone treatment at a dose comparable to the lower dose of chronic treatment moderately attenuated the ADE. Acute naltrexone treatment selectively reduced lever pressing for ethanol both during the ADE and during basal drinking only at the lowest dose, whereas higher doses also suppressed water intake. The ethanol-specific suppressant effect on the ADE was long lasting. Concerning basal drinking, however, naltrexone had a long lasting reductive effect only on lever pressing for water. Conclusions: A low dose of naltrexone and an intermittent treatment regimen seem to be necessary to maintain a specific reduction in ethanol intake in individuals with a high motivation to consume ethanol. These findings are consistent with the notion that, at low doses, opiate antagonists reduce the reward value of reinforcers. Received: 29 September 1998 / Final version: 15 March 1999     

Electrophysiological indices of acute effects of ethanol on involuntary attention shifting

Dose-related effects of ethanol (placebo, 0.30, and 0.60 g/kg) on behavioral and event-related brain potential (ERP) indices of involuntary attention shifting of audition were investigated. ERPs were recorded from 11 healthy social drinkers during a forced-choice reaction-time (RT) task. Subjects were presented with 100 and 200 ms tones (P = 0.50 for each) with a constant inter-stimulus interval (ISI) of 1 s. The task was to press either of two buttons, depending on the tone duration. The majority of the tones (“standards”) were of 700 Hz (P = 0.82). Occasionally, however, the frequency of the tones changed, deviating either slightly (750 Hz), moderately (900 Hz), or widely (1200 Hz; P = 0.06 for each) from the standard frequency. In accordance with previous findings, the task-irrelevant frequency deviations prolonged the RT. This RT prolongation was attenuated by alcohol with the 0.3 g/kg dose, thus suggesting less distraction by irrelevant stimulus deviations under the influence of ethanol. Furthermore, the P3a, reflecting involuntary attention shifting, was suppressed by alcohol even with the 0.3 g/kg dose. These findings demonstrate a detrimental effect of alcohol on involuntary attention shifting, evident with doses considerably smaller than previously described, and still juridically acceptable in road traffic in most countries. Received: 19 December 1997/Final version: 26 May 1998     

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC₅₀S for inhibition of NMDA-activated currents by ethanol were 129 mM ± 20 mM in hippocampal neurons and 126 ± 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC₅₀ of 174 ± 23 mM, which was not significantly different from the IC₅₀S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC₅₀ of 340 ± 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC₅₀, of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.     

Marked individual variability in heart rate ‘normalization’ by ethanol

In order to test Kissin's (1974) concept of normalization by ethanol (deviant prealcohol parameter values becoming less deviant after alcohol) in nonalcoholics, data on unselected mice and nonalcoholic humans were analyzed. These data were on heart rates (HR) of 1055 HS mice and 24 young adults, measured before and after receiving a dose of ethanol (mice: 1.4g/kg, i.p.; humans: 1.3g/kg, oral). Both mice and humans, on the average, show marked normalization, initially low HR usually increasing after alcohol, and initially high HR usually decreasing. The correlation between (1) deviation in HR from the prealcohol mean and (2) change in HR after alcohol was-0.803 for mice and-0.538 for humans. There is very great individual variability, however, in the degree of this normalizing response, some individuals normalizing strongly and others not at all. Although first described in alcoholics, strong normalization by alcohol of several psychophysiological parameters is now known to occur in mice and seems likely to occur in some nonalcoholic humans. The possible relevance of these results to predisposition to alcoholism remains to be shown.     

Electrophysiological Response to Ethanol in P and NP Rats

Event-related potentials (ERPs) have been successfully used in human subjects to evaluate alcoholics as well as those at risk for the future development of alcoholism. In the present study, two lines of rats, those with a preference for ethanol consumption (P) and those not preferring (NP) to drink ethanol were studied using ERP-producing stimuli. Rats were implanted with electrodes in the frontal cortex and dorsal hippocampus (DHPC). A passive auditory "oddball" paradigm was used to record ERP responses following saline and two doses (0.5, 1.0 g/kg) of ethanol. P and NP rats differed under the saline condition in that P rats had smaller N1-like ERP components and larger P2 waves in both cortex and hippocampus. P and NP rats were also found to differ in response to ethanol administration. NP rats evidenced dose-dependent reductions in ERP component amplitudes such as the N1 recorded from cortical sites. P rats did not have such reductions in N1 amplitudes and in fact, displayed increased N1 amplitudes in hippocampal sites. These studies provide further electrophysiological evidence that rats with a genetically influenced preference for ethanol consumption differ from nonpreferring rats at baseline and have a less intense depressant or more stimulating response to ethanol challenge.     

Factors involved in the differential response to ethanol,barbital and pentobarbital in rats selectively bred for ethanol sensitivity

Two lines of rats, least affected (LA) and most affected (MA), had been selectively bred for their differential sensitivity to ethanol. Both males and females of the LA strain were observed to be less sensitive than their MA counterparts to the acute hypnotic and motor-impairing effects of ethanol. However, a lower ethanol metabolic rate of the MA males suggests that both CNS and metabolic factors contribute to their enhanced sensitivity to ethanol. By contrast, no differences were observed between the LA and MA males with respect to the hypnotic and subhypnotic effects of pentobarbital or to the clearance of this drug. MA females were more sensitive only to the hypnotic effects of pentobarbital, probably because of a smaller apparent volume of distribution. No strain difference was observed in the hypnotic effect or clearance of barbital. These observations suggest that, in spite of a differential sensitivity to ethanol, the LA and MA lines do not differ in their response to the barbiturates tested.     

Rats bred for ethanol sensitivity: Impairment of swimming by ethanol and pentobarbital

Rats selectively bred for disparate degrees of ethanol-induced depression of spontaneous locomotor activity (most affected = MA; least affected = LA) were trained on a swim task. Undrugged rats of the MA line swam significantly faster than rats of the LA line. Ethanol, 0.0–2.25 g/kg i.p., produced dose-dependent increases in swim time in rats of the 13th generation (F13). Averaged over trials, these increases were greater in LA than in MA rats and greater in males than in females, but there was no sex difference in peak impairment. Increases in swim time were uncorrelated with predrug performance. These findings were confirmed in younger F17 rats receiving 1.75 g EtOH/kg i.p. Although the lines differed in ethanol-induced impairment, F17 males of the two lines were not differentially impaired by pentobarbital (12.5–22.5 mg/kg, i.p.). The existence of task-dependent line differences in ethanol sensitivity emphasizes the nonunitary nature of ethanol-induced behavioral depression.