Refining methods to map the central projections of the supraoptic nucleus

Introduction | The supraoptic nucleus (SON) is an anterior hypothalamic nucleus known for its release of vasopressin and oxytocin through the posterior pituitary. Evidence exists for the extra-pituitary efferent projections of SON neurons, however comprehensive mapping of the destinations and functions of these has not been established. We attempted to comprehensively map the SON’s connectome by refining an AAV vector-based mapping approach. Methods | AAVs were microinjected at the SON for reporter expression via a pan-cellular promoter CBA, or by endogenous promoters for CaMKIIα, oxytocin and vasopressin (Lewis rats: n = 24). We validated the phenotype specificity of CaMKIIα expression in glutamatergic neurons in the SON and lateral preoptic area using immunohistochemical colocalisation with VGLUT2. We designed and built a novel vector with a vasopressin promoter (AVP-cre) packaged into an AAV1/2 vector and tested its specificity for transfecting vasopressin neurons. We also used conventional tracers (retrograde CTB) to confirm putative central projections of SON neurons as proof of concept. Results | The CBA and CaMKIIα vectors effectively transduced SON neurons but were not SON exclusive. The CaMKIIα characterisation resulted in 88.5 % VGLUT2 colocalisation in the SON and 86.3 % in the lateral preoptic area. The AVP-cre vector was not specific to vasopressin neurons in the SON or PVN and failed to be taken up by magnocellular neurons. Anterograde mapping using CBA and CaMKIIα allowed for subtractive mapping of the transduced populations’ efferent projections. CTB retrograde mapping identified a major projection from SON to the median preoptic nucleus in rat that suggests involvement of the SON in thermoregulatory functions, and potentially other physiological responses. Conclusions | Exclusive anterograde labelling of SON central projections remains a challenge. The creation of a vasopressin-specific vector for expression of cre-recombinase remains of significance and will enable a variety of functional manipulation and projection mapping techniques. The identification of a central projection to the median preoptic nucleus allows for new avenues in SON physiological research.