Labeling phrenic afferents with intrapleural AAV-PHP.S.

Area of Science:

• Neuroscience
• Molecular Biology
• Physiology

Background:

• Selective labeling of musculoskeletal afferents is vital for studying health and disease.
• Current adeno-associated viral (AAV) methods for afferent labeling are invasive, non-specific, or fail to target functional groups.
• Labeling phrenic afferents from the diaphragm is particularly challenging due to confounding afferents in cervical dorsal root ganglia (DRGs).

Purpose of the Study:

• To investigate the efficacy of intrapleural injection of a novel AAV9 capsid variant (AAV-PHP.S) for selectively transducing phrenic afferents.
• To determine if this method labels phrenic afferents in the cervical DRGs and spinal cord.
• To establish a new, less invasive method for studying phrenic afferent function.

Main Methods:

• Utilized a new AAV9 capsid variant (AAV-PHP.S) with enhanced afferent tropism.
• Administered AAV-PHP.S via intrapleural injection in an animal model.
• Verified labeling specificity by transecting the phrenic nerve prior to injection and assessing for motor neuron labeling.

Main Results:

• Observed robust tdTomato labeling in DRGs, dorsal roots, dorsal columns, and spinal cord projections following intrapleural AAV-PHP.S injection.
• Confirmed that labeling was phrenic nerve-dependent and did not involve motor neurons.
• Identified preferential labeling of large-diameter proprioceptive neurons, with expression peaking by 2 weeks.

Conclusions:

• Intrapleural injection of AAV-PHP.S provides a novel and effective method for selectively labeling phrenic afferents.
• This technique avoids labeling motor neurons and offers improved specificity compared to traditional methods.
• The approach facilitates future studies combining genetic tools (optogenetics, chemogenetics) to elucidate phrenic afferent roles.