ADENO-ASSOCIATED VIRUS CONSTRUCT ENABLES DIFFERENTIATION OF VASOPRESSIN AND OXYTOCIN NEUROPEPTIDE-EXPRESSING MAGNOCELLULAR NEURONS IN THE HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RAT

Adeno-associated viral (AAV) vectors are useful tools for transfecting specific cell populations through the use of cell-type specific promoters. Recently, promoters that are specific for either vasopressin (AVP) or oxytocin (OXT) magnocellular neurosecretory cells (MNCs) have been designed that can be used with AAVs to selectively drive gene expression in these cells. The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226 - 250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies. R56 HL62569. Purpose (a): The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). Methods (b): In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226-250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Results (c): Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Conclusions (d): Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies.