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Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization

Lookup NU author(s): Dr Ryley Parrish, Connie Mackenzie-Gray Scott, Dr Alex Grundmann, Dr Faye McLeodORCiD, Neela Codadu, Dr Alexandru Calin, Dr Hannah Alfonsa, Professor Andrew Trevelyan



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Copyright © 2023 the authors.The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K+ concentration ([K+]extra) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K+]extra that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride cotransporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), both in vitro and in vivo This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally.SIGNIFICANCE STATEMENT Halorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K+ ions into the cells; (2) the resultant drop in extracellular [K+] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K+] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K+]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.

Publication metadata

Author(s): Parrish RR, MacKenzie-Gray Scott C, Jackson-Taylor T, Grundmann A, McLeod F, Codadu NK, Calin A, Alfonsa H, Wykes RC, Voipio J, Trevelyan AJ

Publication type: Article

Publication status: Published

Journal: Journal of Neuroscience

Year: 2023

Volume: 43

Issue: 5

Pages: 685-692

Print publication date: 01/02/2023

Online publication date: 09/12/2022

Acceptance date: 02/12/2022

Date deposited: 17/02/2023

ISSN (electronic): 1529-2401

Publisher: Society for Neuroscience


DOI: 10.1523/JNEUROSCI.1141-22.2022

ePrints DOI: 10.57711/e8j1-za73

PubMed id: 36639898


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Funder referenceFunder name
BB/P019854/1Biotechnology and Biological Sciences Research Council (BBSRC)
National Institute for Health and Care Research
MR/R005427/1Medical Research Council (MRC)