Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading




Farmer, George
Balapattabi, Kirthikaa
Little, Joel
Bachelor, Martha
Cunningham, J. Thomas


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Purpose 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, Cl ion causing increase in [Cl]i in SON AVP neurons. In this study, we combined virally mediated ClopHensorN, a relatively new ratiometric Cl imaging technique with capillary based Simple Wes to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons. Methods Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2-0VP1-ClopHensorN. The ClopHensorN (Addgene Plasmid #50758) was packaged in an AAV2 vector with an AVP promotor (Addgene Plasmid #40868). 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. Protein Simple Wes (12-230kDa matrix) 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. Results Muscimol application to SL SONs either significantly increased Cl efflux (p Conclusion Salt loading increases [Cl]i in SON AVP neurons through TrKB-KCC2 mechanism.