The brain controls various physiological functions through a complex network of neural circuits. To elucidate the function of individual neural circuits, it is necessary to perform both the anatomical mapping and functional manipulation of these circuits in living animals. Our understanding of the neural mechanisms underlying physiological functions has recently been advanced by technological innovations in optical approaches to manipulate neural activity and viral vector systems for in vivo gene delivery. The application of optogenetic tools to neurons in the brain facilitates the selective and reversible manipulation of neuronal activity with millisecond precision using light-sensitive proteins. Regarding gene transfer into specific neural circuits, the lentiviral system for highly efficient retrograde gene transfer (HiRet)Highly efficient retrograde gene transfer (HiRet) offers the advantages of neural circuit selectivity and stable transgene expression. The combined approach of optogenetic tools and HiRet vectors allows the cell type-specific manipulation of neuronal activity and animal behavior with high spatial and temporal precision. I herein describe a protocol for manipulating the activity of neural circuits involved in the control of thirst and salt appetite in freely moving animals as an example of the optogenetic approach using HiRet vectors. This application will be widely utilized to characterize individual neural circuits underlying physiological functions.