Sex Specific Effects of Salt Loading in Oxytocin Neurons From The Supraoptic Nucleus

Abstract

Arginine Vasopressin (AVP) and oxytocin (OXY) contribute to body fluid balance homeostasis. Salt loading (2% NaCl for 7 days) increases both AVP and OXY release in rats. The chronic increase in AVP release is associated with a change in the sensitivity of AVP neurons in the supraoptic nucleus (SON) to GABA so that GABAA receptor activation becomes excitatory. It is not clear if a similar mechanism is associated with chronic OXY release in this model. Our hypothesis is that changes in chloride homeostasis associated with salt loading occur in OXY neurons. To test this hypothesis, we used a chloride imaging approach with a ratiometric chloride sensitive dye, ClopHensorN (Addgene #50758) combined with an AAV with an oxytocin specific promoter (pFBOT563, Addgene # 40864). Adult, intact, Sprague Dawley rats of both sexes were anesthetized with isoflurane (2-3%) and were bilaterally injected with the AAV2-pFBOT--ClophensorN virus directly into the SON. After a two-week recovery period, the animals were sacrificed and the brains were rapidly removed. The SON was dissected away from the brain and the cells were dissociated, plated on cover slips, and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging on an inverted microscope. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells and then allowed to wash off. Background fluorescence was subtracted. In cells from males, muscimol resulted in chloride influx in 50% of the OXY cells tested while chloride influx was observed in all OXY cells tested from females. The results suggest that salt loading may influence GABA responses of OXY neurons in males but not females.