Factors affecting alcohol self-administration : : learning, environmental and genetic influences on behaviour

Abstract: Alcoholism is a serious problem for society and is a very difficult disease to treat clinically. Often, the addict has periods of abstinence from alcohol followed by relapse to drinking and this cycle of use/non-use can repeat over many years. The aim of this thesis was to further explore some of the factors affecting alcohol self-administration in animals, namely 1) The role of alcohol-associated cues in alcohol self-administration, 2) The effects of early life stress (known to change alcohol preference) on the neurochemistry of an individual 3) To determine whether the amount of alcohol an individual consumes is genetically determined and/or involves certain brain regions, and 4) To compare individuals that do or do not acquire a lever press response to determine whether this is explained by genetic differences. The first paper in this thesis investigated the effect of cues associated with alcohol on the self-administration of alcohol in Wistar rats. When alcohol is repeatedly used, classical conditioning occurs since the environmental stimuli surrounding alcohol use (e.g. smell, bottle, pub signs, etc.) become paired with the pharmacological effect of alcohol. We interrupted the normal routine of transport (a series of cues paired with ethanol) to observe what affect this would have on subsequent alcohol self-admi n i strati on. When rats were subjected to a 5-minute delay, active lever press response increased by 26% and consumption of ethanol (via blood alcohol measurements) significantly increased. No change in self-administration was observed after 10 or 15-minute delay periods or if the 5-minute delay occurred in a novel environment. The effect was also persistent since the 5-minute delay still increased alcohol consumption after a 1 0-day break in training (the alcohol deprivation effect paradigm). Additionally, the anti-craving compound naltrexone blocked the effects of the 5-minute delay. Preliminary experiments indicated that the 5-minute delay did not affect breakpoint on a progressive ratio schedule but did increase the rate of self-administration. Individual response to the 5-minute delay was correlated with the response to saline injection, which indicates a role for the response to stress in the 5-minute delay effect. The second paper explored the issue of stress and alcoholism. Stress early in life can place an individual at a greater risk for developing mental disorders later in life. The animal model of maternal separation has been developed to study the neurobiology underlying these effects. It is known that male Wistar pups separated from their mother for 6 hours per day (MS360) consume more alcohol than pups separated for 15 minutes (MS15). We hypothesized that this observation could be a result of differences in the glutamate system of the brain. Therefore, we investigated mRNA expression of glutamate receptors using real-time RT-PCR in the prefrontal cortex (involved in many mental disorders) and hippocampus (susceptible to stress and critical in learning). No differences in mRNA expression were observed in the prefrontal cortex but many changes were found in the hippocampus. MS360 rats had lower expression of NMDA NR2B and AMPA G1uR1 and G1uR2 subunits when compared to MS15 animals. In addition, the glutamate transporter GLAST was increased in MS360 rats relative to MS15 and this suggests a compensation in the system due to the excess glutamate released under stress. There was no difference between MS15 and animal facility reared animals which indicated that the observed changes were specific to the maternal separation procedure. Based on the results of the first two papers, we decided to further investigate alcohol self-administration on an individual level. We hypothesized that the amount of alcohol consumed by an individual animal could be a result of its genetic background. Using the qPCR technique, we investigated mRNA expression of receptors from the major neurotransmitter systems to see if there was a correlation to the amount of alcohol the individual consumed. We chose the prefrontal cortex, hippocampus and amygdala because addiction research has historically focused on the ventral tegmental area and its projection to the nucleus accumbens. We trained rats to self-administer over a short period (only 3 days of ethanol) since we could estimate the individual amount of long-term ethanol consumed in the session by using the mean of ethanol consumed on Day 2 and 3. Then, after a 2 week washout period, brains were collected and mRNA expression was analyzed using qPCR. Consumption was positively correlated to the expression of 6 receptors in the prefrontal cortex and these included GABAA á5 (r=0.92), AMPA G1uR1 (r=0.89), 5-HT3A (r=0.88) and the a adrenoceptors (álA r=0.85, á1B r=0.88, á2A r=0.87). Additional correlations were observed in the hippocampus (álA NM13A NR2A, GR) and amygdala NMDA NR2A 5-HT2C). Therefore, the amount of ethanol an individual rat self-administers may be genetically determined and the prefrontal cortex plays a critical role in guiding this behaviour. As an extension of Paper 3, we have also observed that not all animals will acquire the lever press response for reward over our standard training protocol. Often, these Low Consumers (do not press the lever or press it infrequently) are excluded from future experiments. We decided to investigate whether there was a difference in gene expression between High Consumers (those that press the lever) and Low Consumers using qPCR in the prefrontal cortex and hippocampus. A significant difference was observed by ANOVA but no single gene could explain this difference so a principal component analysis was used to identify combinations of genes responsible for the difference. This identified 4 genes: 5HT2A and inGlu, in the hippocampus and AMPA G1uR1 and adrenergic á2A in the prefrontal cortex. To observe whether this difference affected other measurable behaviours, we tested the individual response to novelty in an open field. It was found that Low Consumers moved a significantly higher distance than High Consumers and. this was unlikely to be a result of anticipation or searching for the lever by the High Consumer group. Additional analysis of the Low Consumers identified a critical role of the hippocampus in the acquisition of the lever press response. Overall, Paper 3 and 4 illustrate that difficrences in genetic background can influence the behaviour of rats. Expression in the hippocampus may affect the ability of the individual to learn to selfadminister alcohol and the prefrontal cortex seems to determine the amount consumed. Both of these observations have clinical relevance since stress affects the hippocampus and dys