Fig. 4: Increased ACh levels during seizures.

a Experimental setup for imaging of electrically evoked seizures. Mice were injected with AAV viruses encoding the ACh sensor iAChSnFr and the red-shifted calcium sensor jRGECO, then implanted with a stimulation electrode in the perforant path and a recording electrode in dorsal hippocampus. Imaging was performed while mice were running on a treadmill. b Top, Representative images of iAChSnFr (green), jRGECO (magenta), and merged signals. Scale bar: 100 µm. Bottom, Representative local field potential (LFP), ACh and calcium sensor signal recorded during evoked seizures. Yellow line denotes start of the perforant path stimulation. c Increased ACh and calcium signal fluorescence (F) was observed during electrically evoked seizures. Paired two-sided t-test. Mean ± SEM, n = 6 seizures from 4 mice. *P = 0.0124, ****P < 0.0001. d Experimental setup for imaging of spontaneous seizures in intrahippocampal kainic acid (IHKA) mice. Mice received unilateral injection of kainic acid into the dorsal hippocampus. After at least 3 weeks for the development of chronic spontaneous seizures, mice received hippocampal injection of AAV viruses encoding the ACh sensor iAChSnFr and the red-shifted calcium sensor jRGECO in the hemisphere contralateral to kainic acid injection and implanted with a recording electrode in the dorsal hippocampus in the hemisphere ipsilateral to kainic acid injection. e Significant correlation between ACh and calcium signal changes during spontaneous seizures. Responses are average signal changes in the 10 s after the start of the seizure. Linear regression line is shown with 95% confidence bands. n = 5 mice, 15 imaging sessions, 112 seizure events. f Average calcium and ACh responses aligned to the start of seizure for events that resulted in significant rises in calcium in the contralateral hemisphere across multiple mice and imaging sessions. Mean ± SEM, n = 3 mice, 4 imaging sessions, 21 seizure events. Source data are provided as a Source Data file.