Final Report Summary - FAST (Fiber optic activation of serotonergic terminals during olfactory discrimination) Serotonin (5-HT) is a major neuromodulator which plays important roles in various brain functions and disorders, notably depression. Although it is clear that interfering with 5-HT through psychoactive drugs has strong emotional and behavioral effects (related to mood, aggression, anxiety and fear), the essential role of 5-HT-releasing neurons remains poorly understood. Previous attempts to interfere with the 5-HT system were carried using electrical and pharmacological interventions, both bearing intrinsic limitations. We proposed to overcome these limitations using "optogenetics", a recent approach for controlling neuronal activity with high neurochemical specificity and temporal resolution. Although we faced imponderables related to the extreme novelty of this approach, we are now able to use this technique to investigate the role of 5-HT in rodent behavior. These experiments are ongoing and should provide more definitive insights into the role of 5-HT in the very near future. Our approach consists in expressing channelrhodopsin-2 (ChR2), a light-gated channel, in 5-HT-releasing neurons in order to activate them using light. To deliver ChR2 to 5-HT neurons in rats, we designed a custom viral tool based on a 5-HT specific promoter (Fig. 1). In mice, we used another strategy called DiO (Doublefloxed inverted ORF) which was developed by another laboratory (Fig. 1) and is not available yet in rats. Specific expression of ChR2 in 5-HT neurons was achieved with both systems. To validate the efficiency of our approach, we measured the activity evoked by blue light in ChR2-expressing 5-HT neurons in vitro and in vivo. In vitro, we assessed the light sensitivity of ChR2-expressing neurons by measuring the minimal amount of light necessary to induce action potentials (Fig. 2). We found that ChR2 neurons were more sensitive in mice than rats, probably because ChR2 appears to be more highly expressed using the DiO system than our custom virus. In vivo, we assessed the range of light intensities that can be used without provoking artificial (not related to ChR2) neuronal activity (Fig. 3). Although we obtained photoevoked responses in rats, we are more confident about the efficiency of the photostimulation in mice. The characterization of photoreponses in mice in vivo is ongoing. The brain regions containing 5-HT neurons are located relatively deep below the brain surface. Since blue light is highly absorbed by the tissue, it has to be delivered through an optical fiber implanted into the brain. Optimal photostimulation in behaving animals requires the use of miniature fiberoptic connectors and rotary joints which were tested and optimized during the course of the project (Fig. 4). With our optogenetic tools in hands, we can now test the main theories about the role of 5-HT in behavior. Our photostimulation in vivo should induce a "rush" of 5-HT in the brain. By observing the effect of this rush on various behaviors, we hope to establish causal relations between 5-HT and certain aspects of behavior. These experiments will be carried primarily on mice (which appear to provide the best conditions for efficient 5-HT photostimulation), but some effort will also be dedicated to improving the technique in rats, which remain a model of choice for a number of complex behavioral experiments. The work achieved so far has established a strong methodological ground for conducting optogenetic experiments on the rodent 5-HT system. Deciphering the roles of 5-HT in relation to behavior is expected to have a beneficial impact on our understanding of the etiology of a number of psychiatric disorders (anxiety and panic disorders, depression and schizophrenia). In a more biotechnological perspective, this work has contributed to the development of an "optogenetic savoir-faire" which is currently spreading rapidly a cross neuroscience laboratories and is raising the state-of-the-art in neurobiological research.