Philippe De Witte

Alcohol and withdrawal: From animal research to clinical issues

The withdrawal syndrome in alcohol-dependent patients appears to be a major stressful event whose intensity increases with repetition of detoxifications according to a kindling process. Disturbances in the balance between excitatory and inhibitory neural processes are reflected in a perturbed physical state while disturbances in the balance between positive and negative reinforcements are reflected in a perturbed mood state. Our purpose is to link the different behavioral outcomes occurring during withdrawal with specific biological brain mechanisms from the animal to the human being. Better understanding of the various biological mechanisms underlying withdrawal from alcohol will be the key to design and to apply appropriate pharmaceutical management, together with appropriate therapy aimed at inducing protracted abstinence.

Neuroprotective and abstinence-promoting effects of acamprosate: elucidating the mechanism of action.

Acamprosate is an abstinence-promoting drug widely used in the treatment of alcohol dependence but which has a mechanism of action that has remained obscure for many years. Recently, evidence has emerged that this drug may interact with excitatory glutamatergic neurotransmission in general and as an antagonist of the metabotropic glutamate receptor subtype 5 (mGluR5) in particular. These findings provide, for the first time, a satisfactory, unifying hypothesis that can bring together and explain the diverse neurochemical effects of acamprosate. Glutamic acid is involved in several aspects of alcohol dependence and withdrawal, many of which can be modified by acamprosate. For example, during chronic exposure to alcohol, the glutamatergic system becomes upregulated, leaving the brain exposed to excessive glutamatergic activity when alcohol is abruptly withdrawn. The surge in glutamic acid release that occurs following alcohol withdrawal can be attenuated by acamprosate. The elevated extracellular levels of glutamic acid observed in withdrawal, together with supersensitivity of NMDA receptors, may expose vulnerable neurons to excitotoxicity, possibly contributing to the neuronal loss sometimes observed in chronic alcohol dependence. In vitro studies suggest that the excitotoxicity produced by ethanol can effectively be blocked by acamprosate. Moreover, glutamatergic neurotransmission plays an important role in the acquisition of cue-elicited drinking behaviours, which again can be modulated by acamprosate. In conclusion, the glutamatergic hypothesis of the mechanism of action of acamprosate helps explain many of its effects in human alcohol dependence and points the way to potential new activities, such as neuroprotection, that merit exploration in the clinic.

Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat

The experiments described here were aimed at further validating adjuvant arthritis as an animal model of chronic pain. It was found that the relative oral intake of a 0.008 mg/ml solution of fentanyl was higher in arthritic than in normal control rats; this difference was predicted by the notion that the analgesic effect of a substance may reinforce its intake in animals exposed to pain, more so than in normal pain-free animals. It was also found that body weight decreases and that vocalizations of aggregated rats increase as a result of the challenge; these effects suggest that the vegetative signs and the behavioral irritability which are characteristic of chronic pain in humans, also occur in arthritic animals. The pain which thus seems to be associated with adjuvant arthritis was estimated to have its onset on days 10-11, to peak on days 18-21, and to terminate on days 35-40 after inoculation with Mycobacterium butyricum.

Central effects of acamprosate: part 1. Acamprosate blocks the glutamate increase in the nucleus accumbens microdialysate in ethanol withdrawn rats.

One of the known behavioral actions of acamprosate is to decrease hypermotility during alcohol withdrawal. However, the mechanism of this effect remains unclear. In this study, the concentrations of excitatory and inhibitory amino acids were assayed by the microdialysis technique with OPA/BME precolumn derivatization and electrochemical detection in the nucleus accumbens of male Wistar rats which were either alcoholized by ethanol inhalation or simultaneously alcoholized and treated orally by acamprosate (400 mg/kg/day) for 4 weeks. Without treatment, extracellular glutamate increased during the withdrawal phase, while other amino acids tested (aspartate, arginine, taurine, alanine and GABA) remained stable. In contrast, the alcoholized rats treated with acamprosate failed to present the increase in glutamate during ethanol withdrawal, while other amino acids tested also remained stable. The observed glutamate increase could be responsible for the hyperexcitability observed during episodes of ethanol withdrawal. These results suggest that acamprosate is able to reduce the ethanol withdrawal syndrome by reducing the concentration of glutamate in the nucleus accumbens.

Neuro-inflammation induced in the hippocampus of 'binge drinking' rats may be mediated by elevated extracellular glutamate content

The neuropathological and immune changes induced in the brain by ‘binge drinking’ have been investigated in a rat model. Evidence of neuro‐inflammation was identified in the ‘binge drinking’ rat model of alcohol abuse after 3 weeks of administration of 2 or 3 g/kg ethanol (EtOH), three times per day for two consecutive days, followed by 5 days of abstinence: Firstly, alveolar macrophages, isolated from these animals, showed significant increases in inducible nitric oxide synthase, as assayed by nitrite release, both before and after lipopolysaccaharide stimulation. Secondly, significant numbers of activated microglia were present in the dentate gyrus region of the hippocampus of the ‘binge drinking’ model, after major histocompatibility complex class II staining, by comparison with the control. Microdialysis studies in the ventral hippocampus identified a significant increase in the basal extracellular concentration of glutamate, in both the 2 and 3 g/kg administered ‘binge drinking’ rats. In contrast, no changes in the hippocampal extracellular concentrations, of GABA and taurine, or the dopamine and serotonin metabolites were observed under basal conditions. A further dose of EtOH induced a significant decrease in the concentrations of both 3,4‐dihydroxyphenylacetic acid and 5‐hydroxyindoleacetic acid, whereas glutamate, taurine and GABA levels were unaffected. There was no evidence that EtOH preference was initiated by the ‘binge drinking’ regimen. Our results suggest that the possible toxicity associated with ‘binge drinking’ maybe directed by the elevated glutamate levels, which in turn, activate phagocytic cells to release their inflammatory cytokines and chemokines, ultimately leading to neuro‐inflammation.

Taurine increases in the nucleus accumbens microdialysate after acute ethanol administration to naive and chronically alcoholised rats

The extracellular changes of amino acids (glutamate, taurine and GABA) in the nucleus accumbens of freely moving rats were estimated using the microdialysis technique following acute and chronic ethanol injections (1, 2, and 3 g/kg body weight). Compared to baseline values, taurine increased by 154% +/- 73%, 142% +/- 40% and 162% +/- 75% 20 min after the acute injection of respectively, 1, 2, and 3 g/kg body weight ethanol, while 40 min after ethanol injection, taurine had increased by 124% +/- 36%, 146% +/- 54% and 168% +/- 98%. No changes in either glutamate or GABA were detected at any time points assayed. In the rats which had received chronic ethanol administration prior to a further acute ethanol injection (1, 2, and 3 g/kg body weight), taurine increased by 138% +/- 73%. 144% +/- 39% and 180% +/- 85% 20 min after the ethanol injection at 40 min post ethanol injection taurine had increased by 134% +/- 44%, 160% +/- 56% and 158% +/- 45%, compared to the basal baseline value. No significant changes were observed in either glutamate or GABA microdialysate content in these chronic studies. The biological role played by taurine after acute ethanol injection in the nucleus accumbens remains unclear but may be associated with a yet, undefined mechanism, in reducing the cytotoxicity of ethanol.

Excitatory and inhibitory amino acid changes during repeated episodes of ethanol withdrawal: an in vivo microdialysis study.

Changes in amino acid levels in the hippocampus during repeated ethanol withdrawal were studied. Wistar rats were made ethanol-dependent by 4-week vapour inhalation. After this first cycle of chronic ethanol treatment, rats underwent repeated and alternate cycles of 24 h of withdrawal followed by 1 week of chronic ethanol treatment. The microdialysis technique was used together with high-performance liquid chromatography and electrochemical detection to separate and quantify different amino acids such as aspartate, glutamate, arginine, taurine, alanine and gamma-aminobutyric acid. During the first cycle of ethanol withdrawal, increases in glutamate, taurine and alanine levels were observed. During the third withdrawal period, further increases were detected in aspartate, glutamate, taurine and alanine in the comparison with levels in the control group. However, the arginine level during the third withdrawal period decreased when compared to that after the first withdrawal period. The results of this study demonstrate that excitatory amino acid levels increased with subsequent withdrawal periods.

Self-administration of the Analgesic Suprofen in Arthritic Rats - Evidence of Mycobacterium-butyricum-induced Arthritis As An Experimental-model of Chronic Pain

Abstract This study examined the hypothesis that arthritis induced by Mycobacterium butyricum in rats is associated with a condition of chronic pain. The procedures used in the present experiment were designed to promote the development of a learned taste preference for a solution of suprofen, an inhibitor of PG-biosynthesis, through a Pavlovian conditioning of the suprofen analgesia to the compounds taste. The data indicate that relative to normal control the adjuvant arthritis led to a significantly greater intake of a 0.5 mg/ml suprofen solution. The time-course of this increased suprofen intake resembled that of the inflammatory process in showing an initial marked rise during four weeks and a subsequent decline to a lower asymptote. The results suggest that Mycobacterium butyricum -induced arthritis may offer an experimental model of chronic pain in laboratory animals.

Effects of acamprosate on excitatory amino acids during multiple ethanol withdrawal periods.

BACKGROUND Our previous studies on the effects of acamprosate on enhanced locomotion during repeated withdrawals are now extended to the effects of acamprosate on excitatory amino acids in the hippocampus during repeated ethanol withdrawals. METHODS In this study, Wistar rats were made ethanol dependent by 4 weeks of vapor inhalation. After this first cycle of chronic ethanol treatment, rats underwent repeated and alternate cycles of 24 hr withdrawals and 1 week of chronic ethanol treatment. The microdialysis technique was used together with high-performance liquid chromatography and electrochemical detection to quantify different amino acids such as aspartate and glutamate. RESULTS An intraperitoneal administration of acamprosate (400 mg/kg) to naïve rats did not alter aspartate or glutamate levels compared with the saline groups. During the first cycle of ethanol withdrawal, the administration of acamprosate (400 mg/kg, intraperitoneally) 2 hr after the commencement of ethanol withdrawal decreased both aspartate and glutamate microdialysate levels when compared with their respective saline group. Acamprosate administration also significantly decreased glutamate levels during the third withdrawal compared with the saline group, whereas no changes were seen in aspartate levels. CONCLUSION The results of this work demonstrate that acamprosate reduced the excitatory amino acid glutamate increase observed during repeated ethanol withdrawal. These effects of acamprosate may provide a protective mechanism against neurotoxicity by reducing excitatory amino acids, particularly glutamate.

Conditioned stimulus preference after acetaldehyde but not ethanol injections.

Acetaldehyde, the first ethanol metabolite, has been suggested to mediate some of the behavioral effects of ethanol and particularly its reinforcing properties, although this later hypothesis remains extremely controversial. While several studies demonstrated the reinforcing effects of brain acetaldehyde, blood acetaldehyde accumulation is believed to be primarily aversive. In the present study, a conditioned reinforcement procedure has been used to investigate the reinforcing and/or aversive effects of intraperitoneal injections of both acetaldehyde and ethanol in Wistar rats. An olfactory stimulus was paired with daily injections of either ethanol (0, 0.25, 0.5, 1 and 2 g/kg) or acetaldehyde (0, 10, 20, 100 and 150 mg/kg). After eight conditioning sessions, all rats were tested for their stimulus preference or aversion. The results show that conditioning with small, 0.25 and 0.5 g/kg, ethanol doses induced neither preference nor aversion for the olfactory cue. In contrast, higher ethanol doses (1.0 and 2.0 g/kg) resulted in significant stimulus aversions. Acetaldehyde conditioning led to a biphasic stimulus preference, with a maximal preference around 20 mg/kg acetaldehyde. No evidence of aversive effects was found with increasing doses of acetaldehyde, even with concentrations close to the lethal limit. The present study clearly shows that systemic acetaldehyde injections induced significant stimulus preferences. This suggests that acetaldehyde may be, at least in part, responsible for the reinforcing effects of alcohol intake.