Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains
Abstract number :
3.047
Submission category :
1. Basic Mechanisms / 1D. Mechanisms of Therapeutic Interventions
Year :
2021
Submission ID :
1825775
Source :
www.aesnet.org
Presentation date :
12/6/2021 12:00:00 PM
Published date :
Nov 22, 2021, 06:50 AM
Authors :
Negah Rahmati, PhD - Harvard Medical School and Massachusetts General Hospital; Kieran Normoyle, PhD - Neurology - Massachusetts General Hospital and Harvard Medical School; Joseph Glykys, MD, PhD - University of Iowa; Volodymyr Dzhala, PhD - Neurology - Harvard Medical School and Massachusetts General Hospital; Kyle Lillis, PhD - Neurology - Harvard Medical School and Massachusetts General Hospital; Kristopher Kahle, MD, PhD - Neurosurgery - Yale School of Medicine; Rehan Raiyyani, MS - Neurology - Harvard Medical School and Massachusetts General Hospital; Theju Jacob, PhD - Harvard Medical School and Massachusetts General Hospital; Kevin Staley, MD, PhD - Neurology - Harvard Medical School and Massachusetts General Hospital
Rationale: Differences in cytoplasmic chloride concentrations ([Cl-]i) of neurons lead to oppositely directed chloride flow, GABA signaling, and anticonvulsant effects. Variations in the concentration of Cl- in the cytoplasm of neurons is a predictable consequence of the uneven distribution of large, charged molecules such as actin, tubulin, and nucleic acid polymers. We tested the role of these immobile anions in the induction and stability of [Cl-]i microdomains in individual neurons, as well as and the non-uniform distribution of [Cl-]i at GABA synapses.
Methods: We determined the local [Cl-]i in murine hippocampus and cerebral cortex of both sexes by 3 different highly sensitive methods: 1. Two-photon imaging of SuperClomeleon, a ratiometric Cl--sensitive fluorophore consisting of two fluorescent proteins CFP and YFP, joined by a short polypeptide linker which allows FRET-based imaging. 2. Fluorescent Lifetime IMaging (FLIM) of a Cl--sensitive dye, MEQ (6-methoxy-N-ethylquinolinium iodide) delivered to the cytoplasm via whole-cell recording pipette. Unlike SuperClomeleon, MEQ is insensitive to pH, while for both fluorophores the Cl--sensitive signal is independent of the concentration of dye. 3. Electrophysiological measurements of the reversal potential of membrane currents elicited by local application of GABA and RuBi-GABA uncaging (EGABA).
Results: All three methods collectively demonstrate [Cl-]i microdomains in individual neurons in vitro and in vivo. In addition, there are highly significant correlations between [Cl-]i measured by SuperClomeleon imaging and MEQ FLIM with [Cl-]i calculated based on EGABA and Nernst equation. Our data demonstrate that [Cl-]i microdomains are highly stable over the course of hours and they persist after pharmacological inhibition of cation-chloride cotransporters, but progressively change after inhibiting the polymerization of the anionic biopolymer actin. We also found that these microdomains are not uniform at GABA synapses. This enabled each interneuron to impart a unique degree of charge accumulation, and consequent inhibition of neural activity. Some interneurons even generated postsynaptic responses in pyramidal cells that were not purely inhibitory.
Conclusions: Our findings open a new dimension of complexity for inhibitory GABA signaling, demonstrating new ways that the brain can store and process information. The findings also introduce novel targets for treatment of pathological states, including intractable epilepsy.
Funding: Please list any funding that was received in support of this abstract.: These studies were supported by NINDS grants R01NS040109 and K08-NS-091248.
Basic Mechanisms