Virally Mediated ClopHensorN Chloride Imaging in the Supraoptic Vasopressin Neurons

Abstract

A number of physiological manipulations in rats, including 2% salt loading (SL), alter chloride homeostasis effecting GABAA mediated inhibition in vasopressin (VP) neurons in the supraoptic nucleus (SON). Brain Derived Neurotrophic Factor (BDNF) and tyrosine receptor kinase B (TrkB) contribute to changes in chloride homeostasis in SON associated with SL. However, it can be difficult to measure these changes using traditional patch clamp electrophysiology. In this study, we used ClopHensorN (Addgene Plasmid #50758 from Colin Akerman), a relatively new technique for ratiometric intracellular chloride [Cl]i imaging to measure changes in the responses of acutely dissociated VP SON neurons to the GABAA receptor agonist muscimol following a 7‐day 2% SL protocol. ClopHensorN was packaged in an AAV2 vector with a VP specific promotor (Addgene Plasmid #40868 from Harold Gainer). Adult male Sprague Dawley rats (250–300 g b w) were anesthetized with isoflurane and bilaterally injected in the SON (300 nl) with rAAV2‐0VP1‐ClopHensorN. After two weeks of recovery, 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 (100 mg/kg IP) and decapitated. The brains were removed and the SONs isolated for mechanical and chemical dissociation. The cells were plated on coverslips that were placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. Some brains (injected with a VP reporter, AAV2‐p2.OVPI‐eGFP) were sectioned in a vibratome and live 300 um sections containing the SONs were used for electrophysiological recording in order to confirm the ClopHensorN findings. ClopHensorN positive cells were tested for changes in 445/556nm fluorescence ratio indicating decreases or increase in [Cl]i after focal application of muscimol (100uM). To confirm the function of ClopHensorN, transfected cells were dialyzed with micropipettes containing either a high [Cl] (130 mM CsCl) or 0 [Cl] (140 mM KGlu). Dialysis with CsCl produced a decrease and KGlu produced an increase in ClopHensorN fluorescence consistent with Cl− influx and efflux, respectively. In experiments with muscimol on SON neurons from SL rats, 6 out of 10 cells showed significant increases in chloride efflux while 4 out of 10 had no change in chloride flux. TrKB antagonist incubation significantly blocked muscimol induced chloride efflux in SL rats. We also saw significant increase in [Cl]i in 5 of 9 cells from euhydrated rats with muscimol application and this was unaffected by TrkB antagonism. Using loose patch voltage clamp recordings, neurons from euhydrated rats (6/6) were inhibited by muscimol (100 uM). Neurons from SL rats were either unresponsive to muscimol (5/7) or responded with a rapid and robust increase in activity (2/7) followed by a period inactivity (characteristic of depolarization block). Results with ClopHensorN are consistent with electrophysiology recordings suggesting that it is a viable alternative to electrophysiological recordings. The ClopHensorN's ability measure chloride ion dynamics could make it an important tool.Support or Funding InformationSupported by R01 HL119458This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.