Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain.Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables realtime,in situ,and long-term imaging of the living brain.Here,we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability.This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging.Notably,we were able to record single-spine spontaneous Ca2+ activity in specific circuits.Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits.Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo,and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.