We are using a newly established model to generate alcohol/ or drug-dependent rats or mice. Behavioural, genetic, pharmacological and neurochemical examinations on alcohol/ drug dependent rodents will help us to understand the neurobiological mechanisms of addictive behaviour. Studies with mutant mice (also derived from an ENU screen) and rats will help us to identify genes involved in the initiation of drug-seeking behaviour. In addition, an organbank with different alcohol-preferring rat lines has been established. The aim of the organ bank is the characterisation of molecular cascades involved in alcohol-derived diseases. In a comparative approach an organbank from alcohol dependent patients is used. Using massive microarray approaches and proteomic analysis from the material deriving from this organbank, we will be able to pin down molecular networks that trigger the pathological condition.
|Figure 1: We are in the course of establishing a central animal and human organbank for conducting genomics, transcriptomics and proteomics in liver, pancreas and brain tissue.|
The primary goals of our investigations are (i) the identification of risk alleles in the development of alcoholism and (ii) the characterisation of new anti-relapse compounds. Linking genetic and pharmacological findings we finally aim for of an individually adapted pharmacotherapy. In doing this, alcohol-dependent animals will be separated into different behavioural and neurobiological phenotypes (e.g. by the use of animal MRI) and will then be treated with a corresponding anti-relapse compound. Treatment responses will finally be correlated with specific genotypes and this pharmacogenetic information will then be translated into alcohol dependent patients.
In comparison, we are studying neuroplastic changes within the reward pathway induced by psychostimulants, opioids, nicotine, and cannabinoids and relate these changes to the observed behaviours such as behavioural sensitisation, conditioned place preference, intravenous self-administration and reinstatement of drug-seeking behaviour.
Currently, we are running 12 different research projects in DFG, BMBF and EU-funded networks. In the following section one project is described in more details.
One research project focuses on the involvement of clock genes in the modulation of drug-induced behaviours. A link between the neurobehavioural effects of drugs of abuse and period gene activity has been first established in Drosophila. Flies show behavioural sensitisation following repeated cocaine administration - a phenomenon that has been implicated in drug craving. Thus, they exposed the flies to volatilized free-base cocaine which produces a set of behaviours similar to those observed in rodents, including grooming and enhanced motor activity. Flies mutant in the period gene did not behaviourally sensitise after repeated exposure to cocaine whereas wild-type flies showed a strong sensitised response. This finding was further supported in mutant mice lacking functional Per genes.
Thus following repeated cocain injections, a sensitised behavioural response did not occur in Per1 mutant mice. In contrast, Per2 mutant mice exhibited a hyper-sensitised response to cocaine. Conditioned place preference experiments revealed similar results: Per1 mutant mice showed a complete lack of cocaine reward, whereas Per2 mutants displayed a strong cocaine-induced place preference. The role of the Per1 gene in the development of cocaine sensitisation has been confirmed in successive studies conducted in different rodents. Currently, these mice are tested for intravenous cocaine self-administration and reinstatement behaviour. In vivo microdialysis is performed in parallel in order to correlate extracellular dopamine and glutamate levels in the reward pathway with the observed behaviours.
|Figure 2: The interplay of drugs of abuse and clock genes. A dual role is described, in which clock gene activity determines the efficacy of psychoactive drugs and in which psychoactive drugs influence clock genes. Thus, the expression of these genes modulates the neurochemical state in the mesolimbic system and other brain areas, thereby facilitating or reducing the effects of drugs of abuse or antidepressants. However, these and other psychoactive drugs also influence the activity of clock genes. Once the activity of clock genes is altered (red), the neurochemical pathways that are modulated by these genes are subsequently affected as well as the physiological and behavioural functions driven by these pathways. Drugs of abuse induce neuroadaptations and thereby alter these biological outputs both directly and indirectly via their action on clock genes (green).|
Per1 and Per2 mutant mice have now also been studied in alcohol self-administration experiments. Using operant conditions, Per1 and wid type mice were trained to self-administer alcohol. Furthermore, extinction sessions were introduced, followed by reinstatement measures of ethanol-seeking behavior. In another set of animals, the mice were exposed to voluntary long-termn alcohol consumtion, endsued by a two-month deprivation phase, after whcih the alcohol deprivation effect - which is used a as measure of relapse- was examined. Mutant mice did not display a significantly divergent number of reinforced lever presses than wild type animals.
Furthermore, no significant differences between groups were obtained regarding reinstatement of ethanol-seeking behavior. Similar results wree obtained in the two bottle free choice paradigm. Specifically, no genotype differences concerning consumption and preference were observed over a broad range of different ethanol concentrations. Moreover, after the deprivation phase, both groups exhibited significant alcohol deprivation effects, yet no genotype differences. These data do not suggest a relationship between the circadian clock gene Per1 and ethanol reinforcement, -seeking and relaps behavior. In contrast, compared to wild type animals, Per2 mutant mice exhibit an enhanced alcohol intake and preference when pharmacologically relevant concentrations are offered.
Alterations in the brain reinforcement system of these mutant mice might drive an enhanced incentive motivation to consume more alcohol than control animals. The mesolimbic reinforcement system is modulated by various glutamatergic input pathways and in a series of experiments it was found that Per2 mutant mice have a hyper-glutamatergic state, especially in the nucleus accumbens. Regarding the large evidence given in the literature for an involvement of enhanced glutamate levels or alterations of the glutamatergic system in excessive alcohol consumption, one would expect a massive impact of the Per2 gene mutation on alcohol consumption via alterations within the glutamatergic system. This idea has been further confirmed by examining the effects of acamprosate in these mice. Acamprosate is used in the clinic for relapse prevention and it is suggested that acamprosate acts mainly on a hyper-glutamatergic state, yet having only little effect on a "normal" glutamatergic state. Therefore, acamprosate should be more effective in reducing alcohol consumption in Per2 mutant than in wild type mice. Indeed,following repeated acamprosate treatment, mutant mice showed decrease alcohol consumption along with a normalization of extracellular glutamate levels in the nucleus accumbens.
These new findings provied a clear link of the mous Per2 gene, the glutamatergic system, and excessive alcohol consumption. However, future animal research ought to address the question of whether the Per2 gene, and other clock genes, are also directly implicated in alcohol sensitivity, tolerance, withdrawel and most importantly in alcohol relaps behaviour. Most importantly, however, the link between Per2 and excessive alcohol consumption in mice could already been translated to humans. Thus association studies in different samples have demonstrated that specific genetic variations of the human PER2 gene are associated with high alcohol consumption.