1 Academician Workstation of Animal Disease Control and Nutrition Immunity in Henan Province, Henan Joint International Research Laboratory of Veterinary Biologics Research and Application, Anyang Institute of Technology, Anyang 455000, China

2 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

3 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

4 Biomedical Center, Huazhong Agricultural University, Wuhan 430070, China

5 Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

Abstract 

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 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 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.

Keywords

In vivo Ca2+ imaging; Cranial window; Rabies virus; Dendrite; Primary visual cortex; Corticothalamic projection; Neural circuit tracing

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