Abstract

Although the mechanisms putatively responsible for the anesthetic and anxiolytic effects of high concentrations of ethanol have been well described, the mechanisms responsible for the pleasant (motivational) and rewarding effects of moderate levels of ethanol, which lead a person or animal to repeat its consumption (reinforcing), have not been defined. Studies in the past decade showed that rats selectively bred as alcohol drinkers will spontaneously selfadminister acetaldehyde into the brain ventral tegmental area, indicating that acetaldehyde, a product of ethanol oxidation, is rewarding. Unlike the ability of ethanol to freely enter the brain, livergenerated acetaldehyde does not cross the blood-brain barrier since the endothelial cells of the barrier degrade it. Thus, to mediate the rewarding effects of ethanol, acetaldehyde must be generated intracerebrally. Catalase is the main enzyme that oxidizes ethanol to acetaldehyde in the brain. The injection into the brain ventral tegmental area of a lentiviral vector that inhibits the synthesis of catalase virtually abolishes (85-95%) the voluntary consumption of ethanol of rats selectively bred for their alcohol preference. In the brain, ethanol-derived acetaldehyde can condense with dopamine to generate salsolinol. Rats will self-administer preformed salsolinol into the ventral tegmental area, showing rewarding effects at concentrations that are three orders of magnitude lower than those of acetaldehyde. A recent mass spectrometric and nuclear magnetic resonance study showed that the condensation of acetaldehyde with dopamine forms four salsolinol stereo/regio analogs. It is unknown, however, (a) which of the four salsolinol analogs is the rewarding agent and, (b) whether a salsolinol analog formed endogenously following ethanol drinking mediates the rewarding effects of ingested ethanol and reinforces its intake. The systemic administration of ethanol has been shown to increase the release of dopamine in nucleus accumbens, a structure that contains the axonal terminals of the dopaminergic cell bodies in the ventral tegmental area. A similar effect is observed following the injection of lower concentrations of acetaldehyde or salsolinol (analogs). Thus, the increased dopaminergic tone generated by both ethanol and acetaldehyde may be due to the formation of endogenous salsolinol analogs. It was recently shown that commercial salsolinol analogs activate mu-opioid receptors in the ventral tegmental area, which inhibit GABA neurons, in turn resulting in a disinhibition of (GABA-inhibited) dopamine neurons, thus increasing the firing of the latter. An increased dopamine cell firing and an increased release of dopamine in nucleus accumbens is a pivotal feature for all drugs of abuse. The proposed studies aim at testing the hypothesis that in vivo an adduct of brain acetaldehyde condensed with dopamine is the molecule responsible for the rewarding and reinforcing effects of ethanol. Studies will (i) determine which of the four condensation analogs activates the release of dopamine in cultures of the ventral tegmental area in vitro and in the ipsilateral nucleus accumbens in vivo. (ii) identify, from phage random peptide libraries, individual phages which externally display peptides that bind to salsolinol analogs and, by nucleotide sequencing, deduce the amino acid sequence of the peptides, (iii) generate in the brain salsolinol (analog) binding peptide(s) by injecting into the cerebrospinal fluid (CSF) lentiviral vectors that driven by a choroid plexus promoter synthesize and release the salsolinol analog binding peptide(s) into the CSF and (iv) determine in rats whether the specific salsolinol analog binding peptides released into the CSF inhibit both the rewarding effects of ethanol assessed as conditioned place preference and the reinforcing effects assessed by voluntary ethanol intake. Overall, the proposed studies will determine the role of salsolinol analogs produced in the condensation of acetaldehyde and dopamine as mediators of the motivational (rewarding) and reinforcing effects of ethanol. The studies may suggest new strategies for the treatment of alcoholism.