499 Seizures in a Dish: Demonstrating and Analyzing Coordinated Network Activity in Human and Mouse Brain Slice with Calcium Imaging

Abstract

INTRODUCTION:Childhood epilepsy is a common and often devastating disease, and chronic seizures lead to a significant increase in premature mortality and developmental delay. Focal cortical dysplasia (FCD), a malformation of cortical development, is the primary cause of pharmacoresistant epilepsy in children undergoing resective surgery. There are no targeted medical or surgical therapies for FCD patients who have failed surgery or who are not surgical candidates.METHODS:Human tissue from patients with MCDs was resected during epilepsy surgery. Comparison tissue from CD1 mouse brains was also used. 400-µm brain slices were made with a vibratome. The slices were then incubated the slices in Fluo-8L, a low-affinity calcium-sensitive indicator for 40-180 minutes. Slices were imaged using an sCMOS imager at 15 fps on an upright microscope and standard patch-clamp rig with a custom-built temperature-controlled chamber with high aCSF flow rate (5-15mL per minute) to optimize tissue oxygenation. Network bursts are induced with a pro-ictal ACSF containing high potassium (8 mM) and 4-aminopyridine (100 micromolar).RESULTS:We developed a workflow for calcium imaging in human tissue as follows. Preliminary semi-manual analysis on each image stack was performed using ImageJ. Image stacks were motion corrected using the NoRMCorre algorithm and then processed using the calcium imaging analysis workflow (CaImAn) appropriate for 1-photon imaging. Hundreds of cells can be analyzed simultaneously. Ictal network activity may be induced with the pro-ictal ACSF, or with gabazine (1 mM, a GABA-A receptor antagonist, data not shown).CONCLUSION:We have demonstrated the feasibility of performing ex vivo calcium imaging to directly study the aberrant epileptic networks in human MCD tissue resected from children with intractable epilepsy. In future work, we will use this novel technique to uncover network anomalies underlying the epileptic networks in this disorder.