Abstracts

Rationale: Cortical astrocytes are robustly arborized cells that extend their complex architecture to neuronal synapses and vascular structures throughout the parenchyma. This enables astrocytes to maintain CNS homeostasis over a large domain, a function directly coupled with neuronal activity. Following neurotransmission, astrocytes clear various ions and osmolytes from the extracellular space (ECS). The increase in intracellular osmolarity drives water into the cells, swelling astrocytes and reciprocally shrinking ECS volume. In the context of seizures, this period is of critical importance, as it generates increased neuronal haptic interactions and transient glutamate spikes, lending to the hypersynchronous neuronal discharge that is characteristic of seizures.



To study the effect of generalized seizures on both astrocytic volume and vascular volume regulation, we employed an in vivo multiphoton approach in a generalized seizure model. We hypothesize that astrocytes swell prior to seizure onset, then undergo regulatory volume decrease following seizure termination.


Methods: 8-week-old M Aldh1l1-GFP+ mice undergo dual cranial window and bipolar twist electrode implantation surgeries. After a 2-week recovery period, head-fixed animals are habituated to imaging conditions in a MobileHome Cage. 2 hours prior to imaging, animals are pre-treated with sulforhodamine 101 (SR101; 80 mg/kg i.p.) to counterstain vasculature red. 30 minutes prior to imaging, animals are implanted with an intraperitoneal port used to deliver pentylenetetrazol (PTZ) during the imaging session. Finally, head-fixed animals are imaged using a multiphoton microscope (MPM), with concurrent EEG acquisition. Animals are treated with 80 mg/kg PTZ i.p. to induce generalized behavioral and electrographic seizures. Volume data is analyzed using NIS-Elements Microscope Imaging Software and EEG data was acquired and analyzed using AcqKnowledge Data Acquisition and Analysis Software.


Results: (A) There is a significant increase in astrocytic volume leading up to the seizure (before vs. during) that is maintained even after the termination of the seizure (before vs. after), however there is no significant immediate regulatory volume decrease following the seizure (during vs. after)



(B) Parallel to the astrocytic findings, there is a trend towards increased vasculature diameter leading up to the seizure (before vs. during) and after the seizure (before vs. after), however there is no decrease in vasculature diameter after the seizure (during vs. after)


Conclusions: Our findings suggest that following pharmacological stimulation, astrocytes swell prior to seizure onset. Thus, it is essential to study how astrocyte swelling may contribute to ictogenesis. For example, astrocyte swelling may activate volume-regulated anion channels (VRACs) which could contribute to glutamate release during ictogenesis. We hypothesize that the subsequent decrease in ECS compounds the high [glutamate]_ECS and increases neuronal ephaptic interactions, thereby precipitating ictal activity.


Funding: N/A