Ethanol as a prodrug: brain metabolism of ethanol mediates its reinforcing effects

Abstract

Background: While the molecular entity responsible for the rewarding effects of virtually all drugs of abuse is known, that for ethanol remains uncertain. Some lines of evidence suggest that the rewarding effects of alcohol are mediated not by ethanol per se but by acetaldehyde generated by catalase in the brain. However, the lack of specific inhibitors of catalase has not allowed strong conclusions to be drawn about its role on the rewarding properties of ethanol. The present studies determined the effect on voluntary alcohol consumption of two gene vectors, one designed to inhibit catalase synthesis and one designed to synthesize alcohol dehydrogenase (ADH), to respectively inhibit or increase brain acetaldehyde synthesis.

Methods: The lentiviral vectors, which incorporate the genes they carry into the cell genome, were (i) one encoding a shRNA anticatalase synthesis and (ii) one encoding alcohol dehydrogenase (rADH1). These were stereotaxically microinjected into the brain ventral tegmental area (VTA) of Wistar-derived rats bred for generations for their high alcohol preference (UChB), which were allowed access to an ethanol solution and water.

Results: Microinjection into the VTA of the lentiviral vector encoding the anticatalase shRNA virtually abolished (-94% p < 0.001) the voluntary consumption of alcohol by the rats. Conversely, injection into the VTA of the lentiviral vector coding for ADH greatly stimulated (2 to 3 fold p < 0.001) their voluntary ethanol consumption.

Conclusions: The study strongly suggests that to generate reward and reinforcement, ethanol must be metabolized into acetaldehyde in the brain. Data suggest novel targets for interventions aimed at reducing chronic alcohol intake.

Figure 1. Anticatalase-Lentiviral vector administration into the ventral tegmental area reduces voluntary alcohol intake in rats bred as alcohol drinkers

(A) Rats significantly (p<0.001) reduced their alcohol intake when injected with a lentiviral vector coding for a shRNA against catalase (Anticatalase-Lenti) into the VTA (n=10) compared to that after the injection of an empty lentiviral vector (control-Lenti) (n=10). Animals were allowed free availability of 10% (v/v) ethanol and water four days after the administration of the lentiviral vectors (B). No differences in body weight were observed along the experiment when Anticatalase-treated rats were compared to controls, indicating a good general health of the animals. Abscissa: days of ethanol availability. Deviations shown are S.E.M.

Figure 2. Anticatalase-Lentiviral vector administration into the ventral tegmental area

(A) Inhibition by anticatalase-lentiviral vector of dopamine efflux into the microdialysis fluid of nucleus accumbens (shell) induced by the systemic administration of ethanol (1g/kg i.p.). (B) Anticatalase-lentiviral vector does not affect dopamine efflux into the microdialysis fluid of nucleus accumbens (shell) induced by D-amphetamine (0.1mM) addition to the microdialysis fluid. (C) Anticatalase-lentiviral vector does not affect dopamine efflux into the microdialysis fluid of nucleus accumbens (shell) induced by KCl (100 mM) addition to the microdialysis fluid (n=5 to 7). Respective control values (100%) ranged 0.57 to 0.82 nM dopamine and were not influenced by the viral vectors.

Figure 3. Immunocytochemistry against tyrosine hydroxylase (TH) of a coronal mesencephalic section

Rats were injected with a 1μl solution containing anticatalase-Lenti-shRNA (8×10⁴ virus/μl) into the ventral tegmental area (coordinates: B-5.2; L-0.8; V-7.2), two months after the stereotaxic injection. Open arrow heads show the tract left by the injection cannula. VTA, ventral tegmental area; SNc, substantia nigra, pars compact; SNr, substantia nigra, pars reticulata; FR, fasciculus retroflexus; LM, medial lemniscus. Scale bar= 500μm. Inset: Magnification of the area within the rectangle, showing normal morphology of TH-positive cells in the vicinity of the tip of the injection cannula; scale bar= 20μm.

Figure 4. Induction of alcohol dehydrogenase synthesis increases voluntary alcohol intake in rats bred as alcohol drinkers

(A) Rats significantly (p<0.001) increase their alcohol intake when injected with a lentiviral vector encoding alcohol dehydrogenase (ADH) (Lenti-ADH) into the ventral tegmental area (n=7), compared to that observed after treatment with an empty lentiviral vector (control-Lenti) (n=7) (A). Four days after the injection of the lentiviral vectors, animals were allowed free availability of 5% (v/v) ethanol and water. (B) No differences were observed in body weight along the experiment when Lenti-ADH-treated rats were compared versus control-empty-Lenti virus-treated rats. Abscissa: days of ethanol availability. Deviations shown are S.E.M.