Acute averse life events may prompt dynamic reorganization of neuronal networks, which eventually might lead to psychiatric disorders such as depression. The serotonergic (5HT) system in the dorsal raphe nucleus, a major system involved in mood control, is known to be susceptible to such events. The neurons in this nucleus project to distributed regions throughout the brain, in particular to regions in the limbic system, the hippocampus, the amygdala, and the prefrontal cortex. Mouse fMRI offers a unique opportunity to investigate non-invasively modifications of the neurotransmitter system at the whole brain level induced by pharmacological modulation, or stressful life events. In this study we used optogenetic methods to dynamically control 5HT neurons in the dorsal raphe nucleus assessing dynamic changes in cerebral blood volume (CBV) as fMRI readout. Electrophysiological recordings confirmed the neuronal nature of the response.ePet-cre transgenic mice expressing cre recombinase in 5HT neurons and wild-type controls were stereotactically injected in the dorsal raphe nucleus with adeno-associated viruses containing a sequence encoding for the channelorodopsin2 fused with enhanced yellow fluorescent protein (ChR2-eYFP). Two weeks later, a light-fiber implant was surgically inserted at the site of the dorsal raphe nucleus. Histological staining were performed to confirm the expression of ChR2-eYFP and colocalization with 5HT. Local field potential and multi-unit recordings were performed on brain slices and in vivo. CBV-fMRI measurements were performed on a 7T Bruker Pharmascan system with a transmit/receive surface coil using multi-shot gradient-echo EPI, TE 5.6ms, TR 1000ms, FA 90°, 2 segment, 0.315x0.273mm² in plane resolution. Freely breathing animals were anesthetized with isoflurane 0.5% and medetomidine 0.1mg/kg bolus and 0.2 mg/kg/h infusion s.c. Iron nanoparticles were injected i.v. prior to fMRI experiments. 6 blocks of 20s blue-light stimulation consisting of 5ms pulse at 20Hz and laser power set at 40mW were spaced 160s apart for the stimulation of the 5HT system (control group, n=17). In a group of mice (n=9) Fluoxetine (4.5 mg/kg i.v.) was administered before the fMRI scan to modulated serotonergic signaling. Finally, to mimic a stress response a group of animals (n=7) were held in a restrainer for 60 min before undergoing fMRI. GLM analysis was performed on motion corrected and high-pass filtered time series by modeling each stimulation individually. Second level analysis was performed to compare fluoxetine vs. control and restrain vs. control. Statistical maps were corrected with threshold-free cluster enhancement.Histological analysis confirms the expression of ChR2 confined in the dorsal raphe nucleus (Fig 1A), and in projection areas such as in the prefrontal cortex (Fig 1B&C). Electrophysiological recordings indicate increased spiking rate in dorsal raphe nucleus upon blue light stimulation (Fig 2A), but decreased spiking rate (Fig 2B) and local field potential power (Fig 2C) in sensory cortex. Blue light stimulation elicited an increase in CBV in the dorsal raphe, and a corresponding decrease in the projection area, including isocortex, caudate putamen, and hippocampi (Fig 3 A&B). No CBV changes have been observed in wild-type controls, which could not integrate the ChR2-eYFP coding sequence (Fig 3A). Statistical comparisons of fluoxetine vs. control conditions indicate an increase in response amplitude elicited following fluoxetine injection across the projection areas (Fig 4A), while restraint prior to fMRI session lead to the opposite effect in these regions (Fig 4B).The tight control of local neuronal networks represents a crucial element in mood-regulation in the healthy and disordered brain. The ability to follow the changes in the circuits involved in mood disorders non-invasively and over long period of times is attractive to understand the pathological alterations induced by stressful events, and to understand the effects of mood-altering pharmacological intervention on specific neurotransmitter systems. Optogenetic modification of 5HT neurons allows for a dynamical control of selected neurons, which in turn elicit a robust neuronal and hemodynamic response. The response observed correspond to the expected serotonin projection areas. We could not find indication of unspecific effects as reported in earlier reports as indicated by the lack of response in control animals. The CBV-fMRI response was susceptible to both pharmacological and behavioural modulation. Indeed, the amplitude of the CBV response in the projection areas was increased following fluoxetine administration, consistent with elevated 5HT transmission induced by the 5HT reuptake inhibitors. Restraint leads to an opposite effect, a decrease in CBV response, which indicates a reduction in 5HT transmission following stressful events. Opto-fMRI allows for a sensitive read-out to follow specific circuits involved brain disorders, and may thus allow a mechanistic understanding the effect of pharmacological action on these circuits.