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

High salt loading (SL) upregulates Brain Derived Neurotrophic Factor (BDNF) and causes increased arginine vasopressin (AVP) release from the supraoptic nucleus of the hypothalamus (SON). BDNF can have profound effects on GABA A mediated inhibition through several mechanisms, including the regulation of chloride transporters through tyrosine receptor kinase B (TrkB) activation. Increase in intracellular chloride, [Cl]i can diminish or reverse the inhibitory effects of GABA A on vasopressin neurons creating a feed forward loop that drives AVP release. However, the specific source of BDNF is yet to be elucidated. In this study, we used adeno‐associated viral vectors with shRNA against BDNF to test the hypothesis that the salt loading produces BDNF release from the SON, which in turn contributes to AVP release in SL rats. Adult male Sprague Dawley rats (250–300 g b w) were anesthetized with isoflurane (2–3%) and bilaterally injected in the SON (300 nl/side) with an AAV2 vector with a CMV promoter containing either shRNA against BDNF or a control construct with an mCherry reporter. The vectors were injected at a titer of 1.0 × 10 13 GC/ml (Vector Biolabs, Malvern, PA). 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 using 2‐ Methyl butyrate. Eleven to fifteen fresh frozen brains from each treatment groups 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 injection sites 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 by 2 −ΔΔCt method. Nine to twelve 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. Blood samples were collected to measure plasma osmolality, hematocrit, and circulating vasopressin concentration by ELISA (Phoenix Pharmaceuticals, Inc.). Data were analyzed by one‐way ANOVA with Bonferroni pairwise comparisons. SL was associated with significant increases in BDNF mRNA and AVP hnRNA in SON (P < 0.05, n = 6–7). SL also significantly increased phosphorylation of TrkB in SON (P < 0.05, n = 6–9) and increased plasma AVP (P < 0.05, n = 5–6). SON injections of shBDNF blocked the increases in BDNF mRNA, AVP hnRNA, and TrkB phosphorylation (all P < 0.05) in SON of SL rats. BDNF knockdown in the SON prevented the increases in plasma AVP associated with SL (P< 0.05). Injections of shBDNF outside of the SON did not significantly affect the responses to SL. The results indicate that BDNF produced in the SON is necessary for increased vasopressin secretion during high salt loading. Support or Funding Information Supported by R01 HL119458 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .