Labeling phrenic afferents with intrapleural AAV-PHP.S

Abstract

Background: Understanding the role of musculoskeletal afferents in health and disease relies on the ability to selectively label afferents. Traditional approaches involve using adeno-associated viral (AAV) tools to transduce afferents with intrathecal, intramuscular, or direct dorsal root ganglion (DRG) injections. However, these approaches are surgically invasive, have non-specific labeling, or do not target functional groups of afferents. For example, labeling phrenic afferents arising from the diaphragm muscle is challenging due to the presence of musculoskeletal and cutaneous afferents from the forelimb, neck, and shoulder in the C3-C5 DRGs.

New method: Using a new capsid variant of AAV9 with enhanced tropism toward afferents (AAV-PHP.S), we investigated if intrapleural injection of AAV-PHP.S transduces phrenic afferents in the cervical DRGs and spinal cord.

Results: In animals receiving AAV-PHP.S, we observed robust tdTomato labeling in the DRGs, dorsal roots, dorsal columns, and spinal projections throughout the spinal gray matter. We did not see the same pattern of afferent labeling when we transected the phrenic nerve prior to intrapleural injection, nor did we find any evidence for motor neuron labeling. Classification of labeled afferents suggests preferential labeling of large diameter proprioceptive neurons. Time-course experiments show tdTomato expression in DRG neurons plateaued by 2 weeks.

Comparison with existing methods: To our knowledge this is the first AAV-based method that preferentially targets phrenic afferents without also labeling phrenic motor neurons.

Conclusions: This approach labels phrenic afferents and may be used in combination with optogenetic or chemogenetic tools to advance our understanding of the functional role of phrenic afferents.

Keywords: AAV-PHP.S; Breathing; Intrapleural injection; Phrenic afferents; Respiratory; Retrograde labeling.