The Role Of POA^Oprk1 Neurons In Regulating Fluid Homeostasis

Objective:To investigate the main functions of preoptic area(POA)Oprk1 neurons(POA^Oprk1+)in the rat hypothalamus and specific mechanisms of regulating fluid metabolism.Contents:In Oprk1-Cre transgenic rats,we use stereotaxic methods to inject adeno-associated virus expressing chemical genetic receptors in the preoptic area.By intraperitoneal injection of CNO we activate POA^Oprk1+,resolve functions of these neurons in the preoptic area and to understand the circuit mechanisms of fluid homeostasis.Methods:First,we generated Oprk1-cre rats and used 7 week old male rats for basal metabolic cage experiments.We record several physiological and metabolic indexes,such as drinking,urine output and urine ion concentration.Based on these results,we study only metabolically normal rats,and randomly divide them into experiment and control groups.Second,we injected virus encoding Gq-DREADD in the experimental group and virus encoding fluorescent protein in the control group.After virus injection,we returned the rats back to their home cage.After virus completely expressed,we did further metabolic cage experiments,recorded physiological and metabolic variables and monitored blood Glucose.After completing behavioral experiments,we perfused the rats with paraformaldehyde and collected samples,such as plasma,brain,liver,kidney and pancreas,then cut brain sections with a freezing microtome to observe the location of virus injection in combination with antibody staining.We used paraffin slicing machine to cut other tissues and used hematoxylin eosin staining to observe cell morphology.Finally,we investigate which neuron types and downstream nuclei are activated via anterograde virus tracer labeling and c-Fos antibody staining.Results:After virus entirely expression,intraperitoneal injection of CNO activate POA^Oprk1+neurons successfully transfected with the virus.We found the experiment rats drink more water,urine more,decrease food ingestion and lose weight,andpotassium,sodium,and chloride ion concentration in the urine were all decrease while total amount of ions output without changing,and in plasma,sodium,chloride ion concentration decreased,urine and plasma osmotic pressure decreased,too.We monitored blood Glucose increase,but insulin and Glucagon was not changed significantly.Renal glomerulus,pancreatic islet cells have obvious abnormal morphology.However,there were no significant changes in control group in water intake,urine output and so on.We further investigated the mechanism of above phenomenon,we found the supraoptic nucleus and paraventricular nucleus that were the downstream nuclei by doing anterograde virus injection.And specific antibody staining suggested that oxytocin and vasopressin play an important role in the change.Conclusion:Activation of POA^Oprk1+neurons through chemical genetics technology,in Gq rats,there were drinking more,eating less,urining more and losing more weight.Also concentration of ions in blood and urine,osmotic pressure were changed.Meantime oxytocin and vasopressin were secreted more by endocrine cells in supraoptic and paraventricular nuclei.The results illustrated POA^Oprk1+neurons can be regulated by oxytocin and vasopressin synthesis and release of the regulation of homeostasis in rats.