Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na⁺in Ischemia

Abstract

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na 1 indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na 1 signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na 1 influx, accompanied by considerable cell swelling. Both Na 1 loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na 1 transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na 1 loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.

Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex

“…Various pathways have been proposed and observed to account for the movement of water into and out of neurons, as neuronal size regulation is essential for their survival. Some of these pathways involve CCCs, VRACs, monocarboxylate transporters, potassium channels, Na + /H + exchangers, voltage-gated chloride channels, SLC26A11, TRPV4 ( Hellas and Andrew, 2021 ; Meyer et al, 2021 ; Pasantes-Morales, 2016 ; Rungta et al, 2015 ; Wilson and Mongin, 2018 ). Importantly, in neurons, the movement of ions is intimately linked to that of water.…”

“…Studies in juvenile and adult brain slices have shown that hypoxia induces astrocytic and neuronal swelling ( Hellas and Andrew, 2021 ; Meyer et al, 2021 ; Risher et al, 2009 ). Neuronal cytotoxic edema results from an influx of ions, primarily Na + and Cl − , accompanied by water, from the interstitial space into the intracellular compartment ( Glykys et al, 2017 ; Halstead and Geocadin, 2019 ).…”

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia

“…Prominent pathways for Na + influx into neurons are NMDA receptors, while in astrocytes, Na + ‐dependent glutamate transporters are predominant Na + loaders (Gerkau et al., 2018; Rungta et al., 2015; Song et al., 2020). In addition to these well‐established mechanisms, transient receptor potential vanilloid 4 (TRPV4) channels were recently shown to mediate Na + influx into neurons under ischemic conditions (Meyer, Gerkau et al., 2022).…”

“…TRPV4 channels are osmotically regulated unspecific cation channels that are activated in the ischemic penumbra and are expressed by neurons and astrocytes (Rakers et al., 2017; Yu et al., 2019). The contribution of TRPV4 to neuronal Na + loading in ischemic conditions (Meyer, Gerkau et al., 2022) suggests that their activation might also promote the loss of cellular ATP in ischemic conditions in both cell types. To address this question, we employed fluorescence‐based imaging of ATP with the nanosensor ATeam1.03 YEMK expressed in neurons or astrocytes in organotypic tissue slices of the mouse neocortex.…”

Astrocytic TRPV4 Channels and Their Role in Brain Ischemia

“…Astrocytic TRPV4 has been shown to be involved in a couple of Ca 2+ activated processes of the ischemic cascade–excitotoxicity [ 15 , 169 ], peri-infarct depolarization [ 170 ], oxidative stress [ 6 ] or astrocytic swelling [ 113 , 118 ].…”