摘要： 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 speci?c neuronal labeling enables real-time, 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 ?uorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label speci?c 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 speci?c 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 speci?c input circuits in vivo, and contribute to functional studies of de?ned neural circuits and the dissection of functional circuit connections.