Exploiting C. elegans to provide insight into neural substrates of human alcohol dependence

Human alcohol (ethanol) consumption has a longstanding position within many societies despite the fact that its serious negative effects can sometimes outweigh its positive effects. Although alcohol has a very simple chemical structure it is a drug that has profound immediate effects on human behavior from relaxation and loss of social inhibition, through slurred speech and unsteady gait, to loss of consciousness and death. These changes in the pattern of behavior with increasing alcohol intake reflect its complex effects on the brain. There is also variation in the impact of alcohol on different people highlighting the fact that an individual's genetic background and their previous experience with alcohol both play an important role in how alcohol is experienced. Finally, it is often noted that ones' first drink does not taste good but continued drinking sometimes leads to a liking for alcohol which in the extreme can involve the development of dependence. Once dependence is established an individual's life can become dominated by a need to maintain a supply and avoid unpleasant symptoms that are experienced when alcohol is withdrawn. How does alcohol work? Like all drugs alcohol affects behaviour because it can interact with cells of the nervous system (including parts of the brain) causing cells to become more excited or inhibited. In effect alcohol has the ability to act like a skeleton key and open (excite) or close (inhibit) nerve cells. When alcohol acts in these ways it does so through special types of chemical structures, called proteins, that are found in cells and which can differ depending on the past experience and genetic structure of an individual. This means that ethanol can have quite different effects in different people and within the same person at different times. However, it seems to be an impossible task to be clear about the exact details of alcohol effects in higher animals because any observed effect represents the sum action of millions of nerve cells. This complexity means it is important to find simple ways of studying the effect of ethanol on nerves in a much simpler brain. In our proposal we want to use a model brain which is made of only 20 nerves in which we can sequentially remove the various proteins which ethanol works on to modify nerve function. This is done by using a small part of the brain from a simple worm C. elegans. This simplified model brain can then be used to tease apart how nerve cells might subtly change as a result of genetic factors and as a result of alcohol exposure. Importantly, this worm also shows some remarkably 'sophisticated' behaviours / it can move, explore its environment, find sources of food etc. Thus, as well as trying to understand the way alcohol works in an extremely simple nervous system we can also try to understand behaviours that are important in human alcohol dependence. For example, just as continued consumption of alcohol leads to changes in human behaviour when alcohol is withdrawn, we have also observed changes in the behaviour of C. elegans, when alcohol is withdrawn. Part of this project is aimed at trying to determine which genes are involved in alcohol withdrawal responses. Importantly, many of the genes that have been identified in different kinds of research into alcohol also exist in C.elegans. For this reason we can use this research to understand the way alcohol acts to control, and cause dysfunction, in human behavior.

We will combine the genetic tractability of C. elegans with robust functional assays of a defined microcircuit to provide an integrative analysis of the role of multiple effectors in the response to ethanol. We will use an established method, the electropharyngeogram (EPG), which records activity of the pharynx via a suction pipette applied to the mouth. The activity of the pharynx is regulated by an anatomically and neurochemically well-defined 20 neurone microcircuit which is sensitive to ethanol across the range of concentrations relevant to human intoxication, sedation and anaesthesia. The EPG provides information on the frequency of activity, the duration of excitation-contraction cycles which are neuronally regulated, and the activity of specific excitatory and inhibitory motorneurones. We intend to develop and apply automatic signal processing to EPGs. This will facilitate analysis of large data sets which we predict will tease out subtle effects of ethanol on the circuit. Furthermore, this will maximise the utility of the data sets by providing information on the frequency and relative timing of peaks that are the well-defined signatures of neural events. We will use this as the basis for defining the concentration-dependent response of the microcircuit to ethanol in wild type animals and a wide-range of mutants for putative ethanol effectors. We will build on this to develop EPG and other behavioural paradigms for ethanol tolerance by subjecting animals to episodic ethanol exposure prior to analysis. The behavioural assays are designed to be clinically and theoretically relevant to dependence. We will use these assays to test the hypothesis that acute sensitivity to ethanol is a predictor of tolerance, a key feature thought to underpin human alcohol dependency. In this way we will cross genetic variation with variation in environmental ethanol exposure in order to create a model of gene-environment interaction and ethanol dependence in C. elegans. Cofunded with MRC under 'Neurobiological Basis of Mental Health and Beahaviour'.