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Visualization of neuronal connectivity in NPARM PHOX2B mutants with a modified PACT protocol

NPARM PHOX2B mutations represent a subset of congenital central hypoventilation syndrome (CCHS). To understand the abnormal innervation of breathing column nuclei in CCHS, it is necessary to visualize hindbrain axonal projections and stereologically quantify differences in their connectivity. The task of visualizing neuronal connectivity typically relies on stereotactic injection of retrograde tracers; this method is often tedious, subject to inaccuracy and yields a low number of experimental subjects. When prepared for analyzation, the lipid molecules in the tissue inhibit the diffusion of immunofluorescent antibodies into tissue sections, thereby preventing entire visualization of target proteins in tissue. The present study proposes a twofold solution for the current methodology of neuronal connectivity visualization with the removal of lipid molecules from several transgenic, developmental tracer models to identify anatomical differences in the breathing column. Lipid molecules are removed from the tissue sections in a two phase process adapted from a Passive Clarity Technique (PACT) protocol. First, sections are cross‐linked and hybridized to hydrogel monomers. Next, the lipids are washed from the tissue bound to the monomers with a detergent [8% SDS]. The tissue sections are then stained with immunofluorescent antibodies, imaged using confocal microscopy and analyzed with stereology to quantify differences in nuclei innervation. This approach yields high quality images of the cell bodies and axons of Phox2b derived cells. This novel method of visualizing neuronal connectivity can be implemented in a diverse spread of studies to improve identification and morphology of neurons in several disease states.