Whole-Brain Mapping of Direct Inputs to and Axonal Projections from GABAergic Neurons in the Parafacial Zone

Experimental procedures for retrograde monosynaptic tracing to identify direct inputs to PZ GABAergic neurons. A Design of viral vectors for RV-mediated trans-synaptic retrograde tracing. B Schematic of the PZ injection procedure for AAV-CAG-DIO-TVA-EGFP, AAV-CAG-DIO-RvG, and RV-EnvA-ΔG-DsRed in VGAT-Cre mice. C Schematic showing unilateral injection site of virus in the PZ in VGAT-Cre mice. D Representative images showing RV-labeled neurons (red) in VGAT-Cre mice but not in wild-type mice (blue, nuclei stained with DAPI). Scale bar, 550 μm. E Representative immunostaining images of the PZ from VGAT-Cre mice (left-most) showing that virus co-expression was restricted to the PZ. Scale bar, 200 μm. Starter cells (yellow, expressing both EGFP and DsRed) indicated by arrows in enlarged view of the PZ (right). Scale bar, 20 μm. 7n, facial nerve or its root