A Viral Labelling Study of Spinal Trigeminal Nucleus Caudalis Projection Neurons Targeting the Parabrachial Nucleus

Projection neurons (PNs) in the Spinal Trigeminal Nucleus Caudalis (Sp5C) relay orofacial nociceptive information to higher brain regions such as the thalamus and the parabrachial nucleus (PBN). Our understanding of Sp5C PN organisation and function has advanced less than the parallel spinal cord output system despite their corresponding roles for transmission of nociceptive signals from the orofacial region and body respectively. Viral vectors are an established approach for studying circuit connectivity in the nervous system, but different serotypes are known to produce variable results across circuits. As such, we sought to validate the utility of two common viral serotypes in spinal PN research: retrograde adeno-associated virus serotype 2 (rgAAV) and adeno-associated virus serotype 9 (AAV9), for identifying and analysing Sp5C PNs that project to the PBN. Following unilateral injections of either viral serotype into the PBN, many Sp5C projection neurons were retrogradely labelled. For both serotypes, these injections labelled Sp5C PNs bilaterally with a strong bias to the ipsilateral Sp5C. Within Sp5C, similar levels of PN labelling were present in both superficial and deep regions, contrasting previous work in spinal PNs that showed greater labelling by AAV9 versus rgAAV. Comparisons of the age dependence of labelling showed greater retrograde labelling of Sp5C projection neurons when injections were made in young adult animals. Finally, we demonstrate successful Cre-dependent recombination to selectively express channelrhodopsin-2 in Sp5C projection neurons. Together, these experiments show that rgAAV and AAV9 produce strong Sp5C PN transduction and provide a basis for future study of the afferent and efferent functions of the Sp5C PN population in health and disease.