Authors :
Presenting Author: Sandhya Sriram, BS – University of California, Riverside
Paul Pham, BS – University of California, Riverside
Mahsa Miri, BS – University of California, Riverside
Wayne Dewing, BS – University of California, Riverside
Devin Binder, PhD – University of California, Riverside
Todd Fiacco, PhD – University of California, Riverside
Rationale:
Astrocyte volume changes feature in a variety of physiological processes but are especially impacted in disease states. Small increases in extracellular potassium concentrations or decreases in extracellular osmolarity that trigger astrocyte swelling are sufficient to generate seizure-like activity in intact hippocampal tissue. We have demonstrated that astrocyte swelling constricts the extracellular space (ECS), increasing the effective glutamate concentration both within and outside of the synapse which has a unique effect on neuronal excitability involving slow transmission but not point-to-point synaptic transmission (Walch et al., 2022). Astrocytes rely on several channels and transporters to adapt to changing environments, but the volume regulated anion channel (VRAC) is of particular interest due to its potential role in regulatory volume changes, as well as in the release of glutamate during astrocyte swelling. Though pharmacological inhibition of VRAC has been shown to mitigate tissue damage in an in vivo ischemia model (Yang et al., 2019), the direct contribution of astrocytic VRAC to ECS constriction prior to seizure activity has not been thoroughly investigated.
Methods:
Using a conditional knockout in a mouse model to ablate VRAC selectively in astrocytes (VRAC cKO), we set out to measure the impact of VRAC loss in long-term astrocyte volume regulation. This was done through real-time volume imaging of SR101-labeled astrocytes in live hippocampal slices obtained from adult mice as various ionic or osmotic challenges were applied to evoke cell swelling. We then sought to determine the consequences of astrocyte VRAC cKO on acute volume-driven increases in neuronal activity in the form of both rapid and slow excitatory currents that are characteristic of epileptiform activity. These currents were measured using voltage clamp recordings from CA1 pyramidal neurons in hippocampal slices.
Results:
We observed that, over long-term exposure to elevated extracellular potassium, VRAC cKO astrocytes showed marginally reduced swelling compared to controls. Over long-term application of 40% hypoosmolar ACSF, VRAC-deficient astrocytes display increased swelling compared to controls. This difference in responses is likely due to the different mechanisms driving water intake in each model, and future experiments will be aimed at elucidating the specific contributions of VRAC to these mechanisms. Over the short term, VRAC cKO astrocytes display the same or similar volume change as controls in response to elevated potassium, and in recovery. We also found that, for both control and VRAC cKO, neuronal activity is elevated during periods of astrocyte swelling, but VRAC cKO has differential effects on slow, high amplitude events during astrocyte volume reduction.
Conclusions:
We show that the effects of VRAC cKO are highly context dependent, based on both the time course and method of evoking astrocyte swelling. Future experiments will examine VRAC’s role in astrocyte shrinking and neuronal and network excitability, as well as potential interactions between VRAC and other volume-related channels and transporters expressed by astrocytes.
Funding: NIH NINDS 1R01NS136434
NIH NINDS 1R01NS137603