Astrocyte Swelling

Excitability of the CNS is exquisitely sensitive to the tissue volume and, consequently, the size of the extracellular space (ECS). Nearly a century of work has established that acute reductions in plasma osmolality cause accumulation of water in the CNS and swelling of neural tissue, along with cognitive impairment and, in severe cases, seizures or death. In vitro work (brain tissue slices) has demonstrated that hypoosmolar conditions constrict the ECS and enhance ephaptic (electrical) interactions among neurons, driving them toward seizure and augmenting seizure activity produced by other established models. Similarly, an acute increase in extracellular osmolality can abolish seizures in progress. The close temporal association between seizures and constriction of the ECS is not limited to direct changes in osmolality, and has been observed in different seizure models by several different groups, including ours. Moreover, we have shown that such changes in ECS volume precede seizure development. 

A reduction in ECS volume reflects an increase in cellular volume, and it has become increasingly apparent that astrocytes may account for the majority of cellular edema preceding a seizure. Astrocytes are highly permeable to water (partially due to their selective expression of the water channel aquaporin-4, or AQP4), and are thought to mediate the bulk of CNS water transport. This central role renders astrocytes particularly vulnerable to hypoosmolar conditions. Astrocytic uptake of K+, a crucial component of neuronal activity, is also accompanied by inward water fluxes and induces neuronal activity-dependent tissue swelling, which may be particularly high during the lead up to a seizure. Interestingly, swollen astrocytes have in some cases been reported to recover their original volume by releasing osmolytes and water through a number of channels, including the glutamate-permeable “volume-regulated anion channel” or VRAC (more recently identified as LRRC8). Glutamate released in such a manner would increase the chances of nonsynaptic NMDA receptor activation on neurons, (particularly if the ECS is already constricted).

In vivo 2 photon image showing perivascular astrocytes (eGFP) in contact with penetrating arterioles (SR101) in a section of somatosensory cortex.

To date, there have been no studies which examine astrocyte swelling and/or swelling-induced glutamate release directly as a contributor to seizure development. The main goal of our collaboration with Dr. Todd Fiacco is to fill this void. Through a combination of in vivo (our laboratory) and in vitro (Fiacco laboratory) techniques, we are addressing two main questions: (1) What role does astrocyte swelling play in seizure generation?; (2) What role does astrocyte volume-activated glutamate release play in seizure generation? Current approaches in our laboratory include 2 photon imaging/quantification of astrocyte volume before and during seizures, and electroencephalogram (EEG) recording of seizure threshold and duration after direct hippocampal infusion of VRAC antagonists or anisoosmolar solutions. These approaches are complemented by a number of slice physiology techniques employed by the Fiacco lab including optogenetic astrocyte swelling, patch clamp-mediated astrocyte swelling or loading of excess glutamate, and patch clamp recordings of neuronal excitability.

Max intensity z-projection through 50 µm of somatosensory cortex in vivo, with astrocytes labeled by eGFP. Occasional blood vessels are visible as outlines formed by astrocyte endfeet.