Abstracts

Rationale: Following synaptic transmission events, astrocytes uptake glutamate from the extracellular space (ECS), a phenomenon coupled to the transmembrane flux of Na⁺, K⁺, H⁺, and Cl^-. The increase in intracellular osmolarity drives water into the cell causing astrocytes to swell, reciprocally causing ECS volume to decrease. The period of critical importance in seizures occurs when the ECS shrinks, generating transient glutamate spikes and increasing neuronal haptic interactions, leading to the hypersynchronous neuronal discharge characteristic of seizures. To study the effect of astrocytic volume regulation on the generation of seizures, we employed an in vivo multiphoton approach using a generalized seizure model.

Hypothesis: Astrocytes swell prior to seizure onset, then undergo regulatory volume decrease following seizure termination.

Methods: Eight- to 10-week-old M Aldh1l1-GFP+ mice underwent dual cranial window installation and bipolar twist electrode implantation surgeries. After a 2-week recovery period, head-fixed animals were habituated to imaging conditions. 4 hours prior to imaging, animals were pre-treated with sulforhodamine 101 (SR101; 80 mg/kg i.p.) to stain vasculature and perivascular astrocytes red. Finally, animals were imaged using a multiphoton microscope (MPM), with concurrent EEG acquisition. Animals were treated with pentylenetetrazol (PTZ, 75 mg/kg i.p.) to induce generalized seizures. Volume data were attained and analyzed using NIS-Elements, and EEG data were acquired and analyzed using AcqKnowledge.

Results: We found that: (1) cortical astrocytes undergo significant volume increase prior to seizure onset, (2) astrocytes remain swollen throughout the seizure, and (3) astrocytes exhibit some volume decrease following seizure termination; however, they do not return to baseline volume during the post-ictal phase.

Conclusions: These findings suggest that in the context of hyperexcitation, astrocytes assume a state of pathological volume regulation.

Proposed mechanism: PTZ, a GABA(A) receptor antagonist, tips the excitatory/ inhibitory balance of the CNS towards glutamatergic neurotransmission. Astrocytes remove excess glutamate and ions from the peri-synaptic space, and this transmembrane flux is osmotically coupled to water, causing astroglia to swell past physiological levels. The subsequent decrease in ECS compounds the high [glutamate]_ECS and increases neuronal haptic interactions, thereby precipitating ictal activity. These results suggest that targeting astrocyte swelling mechanisms may be a promising approach to attenuate seizures in vivo.

Funding: Not applicable