Afferent and Efferent Connections of the Postinspiratory Complex (PiCo) Revealed by AAV and Monosynaptic Rabies Viral Tracing

ABSTRACT The control of the respiratory rhythm and airway motor activity is essential for life. Accumulating evidence indicates that the postinspiratory complex (PiCo) is crucial for generating behaviors that occur during the postinspiratory phase, including expiratory laryngeal activity and swallowing. Located in the ventromedial medulla, PiCo is defined by neurons co‐expressing two neurotransmitter markers (ChAT and Vglut2/Slc17a6). Here, we mapped the input–output connections of these neurons using viral tracers and intersectional viral‐genetic tools. PiCo neurons were specifically targeted by focal injection of a doubly conditional Cre‐ and FlpO‐dependent AAV8 viral marker (AAV8‐Con/Fon‐TVA‐mCherry) into the left PiCo of adult Chat Cre/wt : Vglut2 FlpO/wt mice, for anterograde axonal tracing. These experiments revealed projections to various brain regions, including the Cu, nucleus of the solitary tract (NTS), Amb, X, XII, Sp5, RMg, intermediate reticular nucleus (IRt), lateral reticular nucleus (LRt), pre‐Bötzinger complex (preBötC), contralateral PiCo, laterodorsal tegmental nucleus (LDTg), pedunculopontine tegmental nucleus (PPTg), periaqueductal gray matter (PAG), Kölliker–Fuse (KF), PB, and external cortex of the inferior colliculus (ECIC). A rabies virus (RV) retrograde transsynaptic approach was taken with EnvA‐pseudotyped G‐deleted (RV‐SAD‐G‐GFP) to similarly target PiCo neurons in Chat Cre/wt : Vglut2 FlpO/wt mice, following prior injections of helper AAVs (a mixture of AAV‐Ef1a‐Con/Fon oG and viral vector AAV8‐Con/Fon‐TVA‐mCherry). This combined approach revealed prominent synaptic inputs to PiCo neurons from NTS, IRt, and A1/C1. Although PiCo neurons project axons to the contralateral PiCo area, this approach did not detect direct contralateral connections. We suggest that PiCo serves as a critical integration site, projecting and receiving neuronal connections implicated in breathing, arousal, swallowing, and autonomic regulation.