Browsing by Subject "supraoptic nucleus"
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Item ADENO-ASSOCIATED VIRUS CONSTRUCT ENABLES DIFFERENTIATION OF VASOPRESSIN AND OXYTOCIN NEUROPEPTIDE-EXPRESSING MAGNOCELLULAR NEURONS IN THE HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RAT(2014-03) Knapp, Blayne A.; Little, Joel; Cunningham, TomAdeno-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.Item Water Deprivation Evokes Changes in Glutamate Neurotransmission in Magnocellular Neurosecretory Cells of the Hypothalamic Supraoptic Nucleus(2015-05-01) Knapp, Blayne A.; Cunningham, J. Thomas; Mifflin, Steve W.; Schreihofer, Ann M.The regulation of vasopressin (AVP) is critical to maintaining body fluid homeostasis, and the activity of magnocellular neurosecretory cells (MNCs) correlates to the amount of hormone secreted into circulation. The balance of excitatory and inhibitory inputs are important elements of activity, however the mechanisms leading to changes in AVP regulation are not fully understood. Water deprivation (WD), a physiological challenge, was used to examine changes in excitatory neurotransmission in MNCs, measured using patch-clamp electrophysiology and ratiometric calcium imaging. An adeno-associated virus construct containing an AVP gene promoter and enhanced green fluorescent protein (EGFP) reporter allowed us to distinguish vasopressin from oxytocin MNCs. In EGFP-labeled cells (GFP+ MNCs), 48-hour WD treatment resulted in significantly greater mini-excitatory postsynaptic current (mEPSC) amplitude as compared to euhydrated animals. GFP+ MNCs exhibited greater calcium mobilization than GFP- MNCs independent of treatment group, and we observed less cytosolic calcium mobilization with 48H WD treatment.