High Salt Loading Increases Brain Derived Neurotrophic Factor in Supraoptic Vasopressin Neurons

Abstract

Purpose: Salt loading (SL) upregulates Brain Derived Neurotrophic Factor (BDNF) and causes increased arginine vasopressin (AVP) release from supraoptic nucleus of the hypothalamus (SON). BDNF diminishes or reverses the GABAA inhibition in the SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrKB) phosphorylation. This creates a feed forward loop that drives AVP release. However, the source of BDNF is not known. Hypothesis: We hypothesize that SON is the source of BDNF contributing to increased AVP release in SL rats. Methods: Adult male Sprague Dawley rats were anesthetized with isoflurane (2-3%) and bilaterally injected in the SON (300 nl/side) with an AAV2 vector with a U6 promoter containing either shRNA against BDNF or a control construct with an mCherry reporter. The vectors were injected at a titer of 1.0 X 1013 GC/ml (Vector Biolabs). Two weeks after the stereotaxic injections, the rats were provided with either water or 2%NaCl to drink for 7 days. At the end of the protocol, rats were anesthetized with inactin (100 mg/kg IP) and brains were collected and flash frozen. Fresh frozen brains were prepared for Laser Capture Microdissection (LCM) by cutting 10μm thick coronal sections through the hypothalamus at the level of the SON. Using LCM, we verified the accuracy of the injections by visualizing the mCherry reporter and collected the SON to measure changes in the BDNF mRNA and AVP hnRNA expression using quantitative Real Time PCR. Subset of brains from each group were used for Western blot analysis of punch samples containing the SON. Rats that did not have successful virus injections in the SON were separately analyzed. Plasma osmolality, hematocrit, and AVP concentration were measured. Data were analyzed by one-way ANOVA with Bonferroni comparisons. Results:SL was associated with significant increases in BDNF mRNA and AVP hnRNA in SON (P Conclusion: The results indicate that BDNF produced in the SON contributes to increased AVP secretion during SL. Supported by R01 HL119458