Studies have linked prenatal nicotine exposure to neurobehavioral defects, including attention deficit hyperactivity disorder, behavioral issues and depression. Nicotine binds highly to the nicotinic acetylcholine receptors (nAChRs) in the habenula, perturbing cholinergic signaling. The evolutionary conserved habenula contains the largest density of nAChRs and is known as the aversive brain due to its role in mediating stress, fear and anxiety. Our research aims to understand how cholinergic signaling modulates the activity and function of the habenula. For this aim, we have generated zebrafish mutants for the Vesicular Acetylcholine Transporter b (vachtb), expressed mainly in habenular neurons to specifically attenuate cholinergic signaling in the habenula. I performed volumetric calcium imaging using light-sheet microscopy on WT and mutant larvae. This allows the analysis of neuronal activity – both spontaneous and in response to aversive stimuli – in over 1000 neurons in the entire habenula with single cell resolution. Through clustering analysis, I confirmed previous results showing that the habenula consists of spatially segregated neurons with specific patterns of activity. In particular, I identified neuronal clusters that display anti-correlated activity in WT larvae, and show that the anticorrelated activity is reduced in mutants. This work will provide critical knowledge for understanding the role of cholinergic signaling during early development in modulating neuronal activity in a brain region mediating aversive emotions.