Chronic drug intake will be studied using a intravenous self-administration (SA) protocol in C57/BL6 mice. After an initial acquisition period (first 5 days), where a FR1 schedule of reinforcement will be applied, the FR will be progressively increased to 5 and so maintained for the rest of the experiment. During the two months of SA training we will measure the intensity of three addiction like-behaviours twice: i) persistence in drug taking, ii) excessive motivation for the drug, and iii) resistance to punishment. Finally, as previously done for the rat, animals will be scored for each addiction-like behaviour independently. Animals will be then separated in four groups depending on the number of positive criteria met (0, 1, 2 or 3, respectively). If the score is in the 33% highest percentile of the distribution, the individual will be considered positive for that addiction-like criterion. This classification will be done for the three studies drugs, cocaine, morphine and nicotine.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuro- modulators. Neuroimaging of these mice will be performed using the pertinent radiolabelled com- pounds for the different receptors and the resulting data will be correlated with information on the glycolytic metabolism of the same mice brains by means of [18F]-FDG PET.

In order to understand the behavioural switch from normal drug-seeking to compulsive drug-seeking and from normal drug taking to compulsive drug taking the quinine and sucrose test will be done at different time points using adult male, adult female or adolescent male rats: Alcohol deprivation effect (ADE) parameters will be assessed. The transfer of the rat model to mice will be done. The rat model protocol will be exactly followed with male C57/BL6 mice and the same ADE parameters will be assessed.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuromodulators at 3 different time points and compared to alcohol-naïve and alcohol-experienced agematched control animals using. Neuroimaging of these mice will be performed using the pertinent radiolabelled compounds for the different receptors. The resulting data will be correlated with information on the glycolytic metabolism of the same rat brains by means of [18F]-FDG PET.

Food-seeking reinstatement in wild type C57/BL6 mice and in obese ob/ob mice and their wild-type littermates will be studied, taking into account age and gender differences, using an operant response maintained by food under a FR1 schedule of reinforcement. Extinction protocols in which the previously active lever has no consequences, associated or not to cue light or footshock, will be adoperated. Food-seeking relapse patterns in those animals will be studied when receiving non-contingent food pellets with or without stress or cue stimuli, and associated to motor activity.

Cocaine or nicotine self-administration reinstatement in wild type mice, taking into account age and gender differences, will be characterised using intra-jugular vein catheters and an operant response maintained under a FR1 schedule of reinforcement. Extinction protocols will be carried out as explained above and the reinstatement of drug behaviour, once achieved the acquisition criteria, will also be studied in mice receiving non-contingent injection of cocaine or nicotine.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuromodulators. Neuroimaging of these mice will be performed using the pertinent radiolabelled compounds for the different receptors and the resulting data will be correlated with information on the glycolytic metabolism of the same mice brains by means of [18F]-FDG PET.

The validation of the compulsive food seeking/taking model in rat will be carried out with two different anti-obesity compounds (e.g. sibutramine, rimonabant) to identify potential specific efficacy regarding different factors of eating control like energy demand, reward and compulsion. Four tests will be employed: i) anticipatory place preference, ii) restricted food access, iii) bitter-tasting Cafeteria Diet (CD), and iv) restricted CD access. The assessment of the influences of sex and age on both the development of compulsive eating disorder and anti-obesity treatment will be carried out using three tests: i) automated feeding recording, including unrestricted food access, one day of starvation and bitter-taste adulteration of CD; ii) limited access to CD; and iii) limited access to food, alternating between CD and standard chow. Meal pattern analysis will be done for i).

The analysis of the role of feedback signals for maintenance of eating and development of compulsive patterns will be done allowing the rat to eat at any time except from refractory periods (1, 3 or 10 min), that are inserted after a given time period of feeding. Meal pattern analysis will be always done.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuromodulators. Neuroimaging of these rats will be performed using the pertinent radiolabelled compounds for the different receptors and the resulting data will be correlated with information on the glycolytic metabolism of the same rat brains by means of [18F]-FDG PET.

The transfer of the rat model of compulsive food seeking/taking (WP4) to C57/BL6 mice will be done through: i) adapting the general schedule for behavioural testing diet-induced obese versus nonobese mice, ii) adapting a set of experimental interventions suitable for the detection of causal factors including compulsive components to mice, iii) adapting the meal pattern analysis of circadian recordings; and iv) comparing the results with a range of compulsive behaviours associated to eating disorder.

The validation of diet-induced obesity, as well as the influence of sex and age, for eating disorder and associated compulsive-like behaviour will be performed in mouse reference models.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuromodulators. Neuroimaging of these mice will be performed using the pertinent radiolabelled compounds for the different receptors and the resulting data will be correlated with information on the glycolytic metabolism of the same mice brains by means of [18F]-FDG PET.

The different animal models, backcrossed for at least 5 generations on a C57/BL6 genetic background, will be analyzed at behavioural levels using the mouse models of compulsive behaviours described in WP1 (modified conflict), WP2 (deprivation), WP3 (reinstatement) and WP5 (food seeking/taking). Significant gender differences in the results obtained may be studied using genetically modified mice strains for the androgenic receptor and/or for the estrogenic receptor.

Molecular analysis of selected brain areas will be carried out for IEG genes, clock genes and neuromodulators. Neuroimaging of these rats will be performed using the pertinent radiolabelled compounds for the different receptors and the resulting data will be correlated with information on the glycolytic metabolism of the same rat brains by means of [18F]-FDG PET.

An overall comparison of the complete phenotypical characterisation of the different strains of genetically modified mice targeting the glucocorticoid receptor (GR) gene with wild-type mice, and an assessment of the physiological role of the different receptors studied in the four animal models of compulsive behaviour studied will be carried out in view of pre-clinical screening of potentially therapeutic compounds for treating compulsion.

The experimental protocols for the synthesis of three of the radiolabelled compounds to be used in the project are not yet available and will be set up in this WP. The radiolabelled compounds [11C]- Carfentanil and [11C]-N-methyl-SR 141716A, with the common radioisotope 11-Carbon, will be generated from [11C]CH3I and the desmethylated forms of the corresponding compounds under the experimental conditions described (see WP7). The radiolabelled compound [11C]-GR103545 will be synthesized from [11C]CH3OH and desmethylcarbonyl GR103545 (see WP7). HPLC purification, sterile filtering and quality control will be carried out in all cases. The methodologies for the synthesis of the other two radiolabelled compounds needed in this project, namely, [11C]-Raclopride and [18F]- FDG, are already available and will be used as described.

Validation of the pharmacological profile of [11C]-N-methyl-SR 141716A will be done by examining the distribution of the CB1 receptor in brains and peripheral tissues of wild-type and CB1 knockout mice. This validation is mandatory as this radiolabelled compound has never been synthesized. A method for the analysis of mouse brain PET images applying partial volume correction will be set up using highresolution magnetic resonance (MR) images and/or from an MR Atlas of a few animals. Full validation of the method will be carried out by means of autoradiography.

Production of the five radiolabelled compounds for PET studies, [11C]Carfentanil for µ-opioid receptors, [11C]-N-methyl-SR 141716A for CB1 receptors, [11C]-GR103545 for F-opioid receptors, [11C]- Raclopride for D2 receptors, and [18F]-FDG for the correlation of the resulting data with information n the glycolic metabolism of the same mice or rat brains will be timely carried out using methods in force.

The Consortium will disseminate the results obtained by peer-reviewed journal publications, communications to scientific congresses and technical reports to the scientific community, always acknowledging the support by the EC. To ensure maximum exposure of the results of our studies we will establish and open access page on the Consortium website that will provide scientific information on the progress of the research in terms understandable by the general public. In addition, non-special lists summaries of the project objectives and outcomes for promotion purposes by the EC and for diffusion in enterprise or commercial exhibitions will be made available. The ultimate objective of the present project is to provide information that can be used by the participants or other research groups and pharmaceutical companies seeking new animal models, as well as to develop novel drugs or other therapies for the treatment of compulsive disorders and other related psychiatric diseases.

The potential of increasing the competitiveness of the participating SMEs against competition inside and, particularly, outside Europe will be further stressed by the careful and enterprise-oriented set-up of exploitation plans of those outcomes of the project retained to be innovative. Therefore, all partners will be involved and one of the SMEs (P8) will be the WP leader. Those plans will be discussed within the consortium (preliminary plan), with the advice of external experts and stakeholders, before being released (definitive plan). Deliverables susceptible to be object of exploitation plans may be: the four validated animal models of compulsive disorders transferred to mice (modified conflict, deprivation, reinstatement, food seeking/taking), weight transducer technology, software development in food/drinking devices control, the pharmacologically-validated food seeking/taking model in rats using anti-obesity compounds, the methodology for the synthesis and the pharmacological validation of the new radiolabelled compound [11C]-N-methyl-SR 141716A, complete experimental protocols for mice PET in neuropharmacology, parallel behavioural, molecular and neurochemical data of genetically modified mice for glucocorticoid receptors, and molecular markers for compulsion in the four animal models mentioned.

The Consortium will allocate a steering group (SG) formed by P1, P2, P3 and P6 with the role of assisting the co-ordinating person in the assessment of all scientific, technical, financial, legal, ethical and gender aspects of the project. Accordingly, P1 will be responsible of the project management tasks, P2 will oversee ethical and gender issues, P3 will advise on scientific and training issues and P6 will take care of quality assurance, links with SMEs and intellectual property rights (IPR) issues. The SG will also hold a tight relationship with P8 (leader of WP8), thus keeping a particular attention on and an active role in the development and completion of the dissemination and commercial exploitation plans. All these tasks are described in the corresponding sections (6, 7, 8 and 9).

The steering group will meet regularly according to the planned schedule and maintain continuous contacts by e-mail and telephone in order to assure the correct development of the planned activities.

A particular care will be given to the preparation and submission of contract and technical deliverables. When necessary, the steering group and, in particular, P3 will take care of the evaluation of those deliverables prior to their submission, eventually with the collaboration of other participants in the consortium and/or with the advice of external experts. In this context, the steering group will maintain frequent communication with the different team and activity leaders to monitor the progress of the work, identify possible problems and find suitable solutions.

Meetings and workshops, either internal or open training events, organised within the project will be also supervised by the SG. The timely exchange of junior students between partners for training and education purposes will also be supervised and, whenever needed, instigated by the SG.