Malinina, E. | bioRxiv Science

bioRxiv2023

Extracellular Matrix Regulates Neuronal Chloride Concentration via K+-Cl--Cotransporter 2

The neuronal intracellular chloride concentration [Cl-]i is critical for {gamma}-aminobutyric acid type A (GABAA) receptor-mediated transmission. Degradation of the extracellular matrix (ECM) is associated with raised [Cl-]I but neither the mechanisms underlying this effect nor the consequences for GABA- mediated transmission are well understood. Hitherto it has been unclear how to reconcile the effect of the ECM on [Cl-]i with the established role of cation-chloride cotransporters in setting [Cl-]I. In the present work we clarify the role of the ECM in the control of neuronal [Cl-]i. By measuring [Cl-]i in central neurons from male rats we show that the ECM affects basal [Cl-]i as well as the rate of Cl- extrusion after a high load. The mechanism is not via impermeant anions but through regulation of K+-Cl--cotransporter 2 (KCC2). ECM degradation is accompanied by an N-type Ca2+-channel- and calpain-dependent reduction in the amount of KCC2 protein, increased basal [Cl-]i, reduced Cl- extrusion capacity as well as by reduced inhibitory, or even an excitatory, effect of intense GABAA- receptor mediated trans mission. This implies a previously unrecognized pathway for the control of neuronal [Cl-]i and excitability by the ECM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/527837v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1ee0d30org.highwire.dtl.DTLVardef@1a3eb1aorg.highwire.dtl.DTLVardef@a00b44org.highwire.dtl.DTLVardef@143b43f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2021

Visual stimulation with blue wavelength light drives V1 effectively eliminating stray light contamination during two-photon calcium imaging

BACKGROUNDBrain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators emission spectra, resulting in significant contamination of calcium signals. NEW METHODTo overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicators emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials-VEPs) and 3D acousto-optic two-photon(2P) population Ca2+ imaging in mouse primary visual cortex (V1). RESULTSVEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. COMPARISON WITH EXISTING METHODSMasking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding without affecting V1 physiological responsiveness. CONCLUSIONSOur results show that the selected blue light stimuli evoke physiological responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics. HighlightsO_LIWhite and blue light trigger VEPs with similar amplitudes and latencies in mouse V1 C_LIO_LIBlue-and white-responding neurons are two largely overlapping neuronal populations C_LIO_LIBlue and white evoke Ca2+ responses similar in magnitude and latency C_LIO_LIBlue and white evoke Ca2+ responses similar in orientation/direction selectivity C_LIO_LIBlue stimuli could be an alternative to white ones in behavior and opto-physiological tests C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/433182v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@66c0a2org.highwire.dtl.DTLVardef@1f0cce9org.highwire.dtl.DTLVardef@1ce97borg.highwire.dtl.DTLVardef@24a3f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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