Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading

Abstract

Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl− co‐transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride ion causing increase in [Cl]i in SON AVP neurons. In this study, we have combined virally mediated ClopHensorN, a relatively new ratiometric chloride imaging technique with capillary based Simple Western technology to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons.Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2‐0VP1‐ClopHensorN. The ClopHensorN (Addgene Plasmid #50758 from Colin Akerman) was packaged in an AAV2 vector with a AVP specific promotor (Addgene Plasmid #40868 from Harold Gainer). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß‐Actin protein expression. The standard matrix (12–230kDa) in Protein Simple Wes was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi‐squared test and one‐way ANOVA with Bonferroni comparisons.Muscimol application to SL SONs either significantly increased chloride efflux (p<0.05;13/20) or did not change chloride flux (7/20). TrkB antagonist (AnA,50uM) significantly blocked the chloride effluxes. SONs from euhydrated rats showed muscimol induced chloride influx (p<0.05;11/16). KCC2 antagonist (VU 0240551,10uM) significantly blocked the chloride influxes in euhydrated rats. SON AVP neurons that responded to pharmacological inhibitors during chloride imaging were viable and expressed KCC2 co‐transporter and ß‐Actin. Neurons that did not respond during chloride imaging did not had KCC2 and ß‐Actin protein expression. Salt loading increases [Cl]i in SON AVP neurons through TrKB‐KCC2 dependent mechanism.Support or Funding InformationSupported by R01 HL119458 and 18PRE34060035This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.