Astrocyte-originated connection mapping of presynaptic neurons using the rabies virus tracer

Astrocytes communicate bidirectionally with neurons by forming close contacts to synapses called tripartite synapses. Hundreds of thousands of synapses are embedded in a territory of single astrocytes, however, how astrocytes are functionally built into the neuronal circuits remains undetermined. To address this, we adopted the rabies virus (RV) for retrograde tracing of neuron-to-astrocyte connections in the mouse cerebral cortex. To sparsely and specifically transduce astrocytes with RV, a transposon vector expressing TVA, RV glycoprotein, and tdTomato under an astrocyte-specific promoter Gfa2 was introduced by electroporation at postnatal day (P) 0. Then, EnvA-pseudotyped and glycoprotein-deleted RV (EnvA-RV∆G-GFP) was injected at P30–50, and the brains were collected after five days of transduction. Neurons expressing only GFP were found around astrocytes expressing both tdTomato and GFP, demonstrating that EnvA-RV∆G-GFP primarily infected astrocytes and then spread to neurons. Secondarily infected neurons were also found in distant cortical regions, indicating that astrocytes receive long-range cortico-cortical projections as well as local connections. Topological analysis further revealed layer-specific neuron-to-astrocyte projections: layer I astrocytes receive horizontally widespread projection from infragranular layers, layer II/III astrocytes receive columnar inputs, and layers V and VI astrocytes mainly receive interlaminar projections. Moreover, astrocytic calcium response patterns evoked by local electrical stimulations to neurons coincided with the RV-revealed wiring diagram. Taken together, RV tracing enabled intra- and inter-regional mapping of neuron-to-astrocyte connections, thus is a potential tool for dissecting the astrocyte-neuron circuit organization.