Fig. 2.

Activation of entorhinal CaMKIIα-positive neurons reduces the severity of hippocampal seizures. a) An AAV construct encoding Cre-dependent ChR2-eYFP (pAAV-EF1α-DIO-hChR2(H134R)-eYFP) was injected into the EC of CaMKIIα-Cre mice to generate CaMKIIα-ChR2^EC mice (abbreviated to as “CaMKIIα-ChR2”). b) Histological images showing that ChR2.0-eYFP (triangles) was expressed in the EC and in the hippocampus (fibers). Immunohistochemical staining confirmed that ChR2-eYFP was expressed in CaMKIIα-positive neurons in the EC. c) The electrodes that would allow recording during electrical or optogenetic stimulation in the EC. d) Representative peri-event rasters showed the response of neurons to the LFES or photo-stimulation (1 Hz). Top Left: a PN excited by LFES; Top right: an IN exited by LFES; Bottom left: a PN exited by blue light; Bottom right: an IN inhibited by blue light. A neuron that showed a higher firing rate (at least three times higher than the baseline) immediately after stimulation was defined as an “excited” neuron, while a neuron that stopped firing (≥ 30 ms duration) within approximately 20 ms after stimulation was defined as an “inhibited” neuron. e) Population data showing that blue light (473 nm, 1 Hz) excited most PNs in the EC in 4 CaMKIIα-ChR2^EC mice, and LFES excited both PNs and INs in 3 wild-type (WT) littermates. f) The schedule diagrams for the delivery of LFES and light (above) and the labels used for different groups (below). Black rectangle indicates kindling stimulation, the green rectangle indicates a pulse of LFES, the blue rectangle indicates a pulse of blue optical stimulation, and the yellow rectangle indicates a trail of yellow optical stimulation. Animals were grouped to match afterdischarge threshold (ADT), which was defined as the minimal intensity that produced an afterdischarge (≥ 5 s). g) Entorhinal light stimulation (473 nm, 1 Hz) and LFES retarded the development of behavioral stages of seizures and afterdischarge duration (ADD) during hippocampal kindling acquisition (two-way ANOVA for repeated measures followed by Least Significant Difference (LSD) post hoc test). Black rectangle indicates kindling stimulation while the blue rectangle indicates blue light stimulation. In kindling acquisition experiments, we used 10 stimulations a day for three days in normal mice. The CaMKIIα-ChR2 control received yellow light stimulation; and the WT-control received no photo-stimulation. h) Both entorhinal photo-stimulation and LFES lowered the seizure stage and shortened ADD in kindled mice (two-way ANOVA for repeated measures followed by LSD post hoc test). Black rectangle indicates kindling stimulation while the blue rectangle indicates blue light stimulation. Separate kindled mice were grouped to match generalized seizure threshold (GST), which was defined as the minimal current intensity required to elicit a generalized seizure. i) Representative EEGs. The black rectangle represents the kindling stimulation. Data are displayed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the WT-control, and ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to the CaMKIIα-ChR2 control.