Figure 6. Simultaneous Two-Photon Population Imaging and Targeted Intracellular Recordings during Epileptic Seizure Spread.

(A) Illustration showing simultaneous LFP recording, targeted intracellular recording, and dual-color two-photon imaging of both tdTomato-labeled Pv and GCaMP6f-expressing pyramidal cells from both the visual and the somatosensory cortex.

(B) Simultaneous intracellular (type A) recording from Pv interneurons (Pv1 and Pv2, red) approximately 400 μm below the surface of the pial layer, and population Ca²⁺ imaging (green) across the cortical column. The nanopipette was unchanged between recordings.

(C) Population Ca²⁺ transients from pyramidal cells (top, 5 individual cells in gray, population mean in blue) corresponded well with 4-AP-induced electrographic seizures (middle, LFP in red). Note the abrupt depolarization block in the Pv1 intracellular recording (bottom, black trace), which coincides with a sharp rise in population Ca²⁺ activity during seizure propagation into the field of view.

(D) Six consecutive seizures while recording from Pv1 and Pv2 (bottom, left and right, respectively, type A) depict the trend shown in (C). For each Pv, three consecutive optical break-ins of epileptic activity are superimposed (top, left and right, average population Ca²⁺ transient). Both Pv interneurons show consistent, immediate cessation of firing upon optical break-in of seizure activity, consistent with entering depolarization block. The intracellular membrane dynamics returned to the same baseline after each seizure period. No current was injected during voltage recordings. AP waveforms were not distorted, but firing rates changed after ictal break-in.

(E) Compared to baseline, Pv average firing rates increased during pending electrographic seizures (2 animals, 5 Pv interneurons, 15 preictal periods each, and 10.27 ± 1.956 spikes/s during the 5 s before seizure onset) but ceased upon optical break-in of ictal activity during electrographic seizure occurrence (2 animals, 5 Pv interneurons, 15 ictal periods, and 0.26 ± 0.13 spikes/s during the 5 s after seizure onset) (p = 0.006).

The concomitant RMP shift in (D) suggests a depolarization block.