Biobehavioural Pharmacology: Research Annual Report 2003

Section Heads: Drs. A.D. Le & Denise Tomkins

The research goals of the Biobehavioural Pharmacology Section are: to understand the underlying behavioural and neurobiological mechanisms that initiate and maintain alcohol dependence; and to use this understanding to explore therapeutic agents for treating alcohol dependence. The majority of our research focuses on issues related to alcohol's reinforcing ability and relapse to alcohol drinking behaviour, with an emphasis on the role of stress in relapse. We continue to explore the role of specific central neurochemical systems in regulating these behavioural processes, in addition to examining the possible role of genetic factors involved in problem drinking and concurrent problems with other substances, such as nicotine.

Neurobiological Mechanisms of Stress-Induced Relapse to Substance Use

Our research has focused on the mechanisms underlying relapse to alcohol or other drug use caused by stress. Our working hypothesis has been that exposure to stress produces behavioural disinhibition (loss of control), which leads to relapse to substance use.

We have found that, in rats, the median raphe nucleus plays a critical role in stress-induced relapse to alcohol use. This brain area sends projections containing the neurotransmitter serotonin throughout the limbic system (a brain system that underlies emotion and learning).

While serotonin originating from the median raphe nucleus is important in stress-induced relapse, we found that another neurotransmitter, GABA, also has a critical role in relapse to alcohol. Injection of the drug muscimol, an agonist of GABAA receptors, into the median raphe caused reinstatement of alcohol-seeking. We also found that injecting bicucculine, a blocker of GABAA receptors, into the medial septum (a limbic brain structure involved in behavioural inhibition) also modestly reinstated alcohol-seeking. This is further evidence that the GABA system is involved in relapse. Changes in the activity of a direct or indirect GABA-containing projection between the median raphe and the medial septum may underlie these effects.

Our preliminary data show that direct injection of muscimol into the median raphe also activates c-fos, a marker of activated neurons, in various brain structures in a way that is similar to that induced by exposure to footshock stress. We believe that the observed effect on relapse to alcohol induced by injection of muscimol into the median raphe is due to its impairment of inhibitory control rather than through effects on incentive mechanisms, as the results of conditioned place preference testing did not indicate that injections of muscimol into the median raphe were rewarding.

Interaction between Alcohol and Nicotine

We have also furthered our research into the co-abuse of alcohol and tobacco. We have previously shown that repeated exposure to nicotine can enhance alcohol self-administration in rats.

Using our animal model of relapse, we have found that treatment with nicotine can also promote relapse to alcohol, by producing alcohol-seeking in animals whose alcohol self-administration has been extinguished. In animals that have previously experienced both alcohol and nicotine, injection of nicotine potently reinstated alcohol-seeking.

The results from these studies have strong implications for the treatment of concurrent alcohol and tobacco addiction.

5-HT Receptor Subtypes and Alcohol Reinforcement Processes

Studies in humans and animals suggest that the central neurotransmitter, 5-HT , is associated with problem alcohol use and dependence. We continue our work to assess how modulating activity at various 5-HT receptor subtypes affects alcohol self-administration behaviour. Through this work, we hope to better understand the neurobiological mechanisms underlying excessive alcohol consumption.

One receptor of particular interest is the 5-HT1B receptor. Human studies suggest that a locus predisposing people to antisocial alcoholism is linked to the 5-HT 1B receptor gene.

Previously, we clearly demonstrated that 5-HT 1B receptors play an important role in regulating alcohol intake in our animal models. Over the past year, we have extended these findings by exploring the behavioural basis of this phenomenon, as well as the specific brain areas that regulate it. Our data suggest that 5-HT 1B receptors are important for regulating both the initial drive to obtain alcohol as well as its consumption, particularly in highly motivated animals. Furthermore, we have now confirmed that two brain areas, the amygdala and the ventral tegmental area, are important in mediating these effects. Interestingly, activation of 5-HT 1B receptors within the ventral tegmental area leads to decreased alcohol intake, while in the amygdala, the same manipulation leads to increased alcohol intake. This difference in action shows that the regulatory effect of 5-HT 1B receptors within the brain is site-specific.

The collection of data on the amygdala is particularly intriguing, as very few reported pharmacological manipulations have increased alcohol intake in animal models. Our findings may suggest that the amygdala exerts an important modifying influence on alcohol consumption under normal circumstances. Because this brain area has been linked with mediating the conditioned effects of psychoactive drugs that may elicit craving in humans, it is interesting that 5-HT 1B receptors also modify motivation to obtain alcohol in our animal models.

The 5-HT 2C receptor is also of interest to us. We previously reported a role for 5-HT 2C receptors in tonically regulating alcohol intake.

Recently, we have been trying to find how other members of the 5-HT 2 receptor family may modify the motivation to consume alcohol, by comparing our previous findings with those elicited by 5-HT 2A and 5-HT 2B/2C manipulations. Our data demonstrate that 5-HT 2B/2C receptors do alter alcohol intake; however, this alteration only occurs when the receptor is activated, not inhibited, and is most likely due to non-specific effects. Taken together, these findings suggest that, in this family of receptors, only the 5-HT 2C receptor constantly modulates alcohol intake.

We are continuing this line of research in the hope of better understanding the neural circuitry that helps regulate drinking behaviour.

GABAA Receptor Subunits, Drinking Behaviour and Voluntary Intake

Compelling evidence suggests that central GABAergic systems play an important role in regulating alcohol's effects, particularly those effects mediated via the GABAA receptor.

We continue to investigate regional differences in the expression of the GABAA receptor subunits. These differences have been demonstrated in the brains of high-alcohol preferring rats, and humans with drinking problems; therefore, they may represent one of the neurobiological factors underlying problem alcohol use.

The data generated thus far demonstrate that regional differences in GABAA receptor expression and subunit conformation affect drinking behaviour. We have found that these differences also affect the binding profile of some pharma-cological agents that interact with this receptor complex, including muscimol, flunitrazepam and diazepam, but not others, such as zolpidem. There appears to be a complex interaction between inherent alcohol preference, alcohol drinking history and the binding ligand employed.

Currently, we are analysing and interpreting the extensive database generated over the last year. Our data will provide important insights not only into the genetic and non-genetic GABAA receptor influences on alcohol preference and consumption, but also into potential interactions with, and/or influences over, other clinically used pharmacological agents that interact with this receptor complex, such as the benzodiazepines.

Sex Differences in Susceptibility to Alcohol-Induced Cognitive Deficits

To develop more effective treatment and prevention strategies for problem alcohol use in women, we must conduct basic research on the differences in alcohol's effects on brain function in men and women.
Clinical evidence suggests that women are more vulnerable to some of the negative effects of alcohol than men. While tests of psychomotor performance have consistently reported that men and women are equally impaired by alcohol ingestion, women appear to be more sensitive to the cognitive deficits induced by long-term alcohol exposure, particularly on tasks demanding divided attention or delayed recall.

Over the past year, we have continued to explore the long-term effects of alcohol exposure on cognitive function and behaviour, with specific emphasis on potential differences in susceptibility between males and females.

To support the clinical relevance of our experimental approach, we showed that, similar to humans, chronic alcohol exposure caused equal impairment of female and male rats on measures of psychomotor performance. As predicted from the human literature, female rats showed greater impairments on measures of delayed recall compared to their male counterparts when chronically exposed and then withdrawn from alcohol. Alcohol-exposed female rats also demonstrated a blunted response for obtaining a sweetened food reward compared to males. One possible interpretation of this finding is that alcohol exposure has elicited a dysphoric state in the females but not the males. We are interested in this difference because, clinically, women are more likely to present with concurrent alcohol problems and depression than are men. Currently we are examining the brain mechanisms that may potentially explain these sex differences.

In our work, we hope to unravel some of the gender differences in susceptibility to alcohol-induced cognitive impairments and uncover the role of GABAA receptors in these impairments. Ultimately, this research could help identify risk factors, protective factors and treatments for alcoholism that are specific to women.

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