Protocol for labeling epileptic-status-related neuronal ensembles in mouse hippocampal kindling model

Abstract

Epileptic networks are characterized by two states, seizures or more prolonged interictal periods. Here, we present the procedure for labeling seizure-activated and interictal-activated neuronal ensembles in mouse hippocampal kindling model using an enhanced-synaptic-activity-responsive element. We describe the seizure model establishment, tamoxifen induction, electrical stimulation, and calcium signal recording of labeled ensembles. This protocol has demonstrated dissociated calcium activities in the two ensembles during focal seizure dynamics and can be applied to other animal models of epilepsy. For complete details on the use and execution of this protocol, please refer to Lai et al. (2022).¹.

Keywords: Behavior; Neuroscience.

Graphical abstract

Figure 1

Syringe equipped with glass micropipette A 1 μL syringe filled with paraffin oil is connected with a glass micropipette filled with paraffin oil via the hot melt glue and mounted on a syringe pump holder.

Figure 2

Hand-made double-stranded spiral electrode An electrode made of stainless-steel wire coated with Teflon, twisted into a double-stranded spiral with the tip divided into a u-shape and peeled off its insulating layer.

Figure 3

Process of making a female connector A female connector is made from the double-row female header according to the following steps: (1) cut to each piece with three pairs of electrode pins; (2) tilt each electrode pin outward; (3) make the two adjacent electrode pins on the same side contact each other; (4) apply silicone sealant to the bottom of the electrode pins.

Figure 4

Surgical supplies Supplies required for AAV injection, electrode and fiber optic cannula implantation surgery.

Figure 5

The main experimental steps of AAV injection, electrode and fiber optic cannula implantation (A) Fix the head of the mouse in the stereotaxic apparatus. (B) Determine the coordinate of the piriform cortex (AP: +0.5 mm, ML: -2.8 mm, DV: -5.4 mm) for the AAV injection site and drill a hole with a skull drill. (C) Determine the coordinate of the CA3 (AP: -2.9 mm, ML: -3.0 mm, DV: -3.2 mm) for the electrode site and drill a hole with a skull drill; then drill 4 more holes at the anterior bregma, posterior lambda, and the left and right sides of the skull suture separately with a skull drill; finally, screw in skull screws in each hole for dental cement fixation. (D) Wrap copper wire around the anterior bregma screw and posterior lambda screw for cortical EEG recording and grounding reference, respectively. (E) Slowly insert the electrode into the pre-drilled hole. (F) Fix the electrode and screw it with a small amount of dental cement (core fixation). (G) Bend the exposed part of the electrode near the cement by 90 degrees. (H) Fix the bend with a small amount of dental cement. (I) Insert the fiber optic cannula into the AAV injection site, and the implantation depth should be 0.2 mm shallower than the AAV injection depth. (J) Unwind the double-stranded spiral electrode and strip the insulation from the tip of the wires (∼1cm). (K) Connect the electrode and the copper wire at the anterior and posterior screws to electrode pins of the female connector with solder. (L) Apply dental cement at the skull openings with electrode wires and the space between the skull and the bottom side of the female connector.

Figure 6

Expected outcomes (A) Representative image from coronal slices showing restricted GCaMP6s expression of the APir’s IP-Ens with a trace of a fiber optic cannula above (scale bar, 200 μm). (B) Average fluorescence value of the IP-Ens during FS from 4 mice with 3 repeated trials each. (C) Heatmap of fluorescence corresponding to Figure 1B. (D) Group data of trials showing quantification of the average ΔF/F value during base states and FS corresponding to Figure 1B and 1C. Paired t-test, p = 0.0830. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (E) Representative image from coronal slices showing restricted GCaMP6s expression of the APir’s FS-Ens with a trace of a fiber optic cannula above (scale bar, 200 μm). (F) Average fluorescence value of the FS-Ens during focal seizures from 4 mice with 3 repeated trials each. (G) Heatmap of fluorescence corresponding to Figure 1F. (H) Group data of trials showing quantification of the average ΔF/F value during base states and FS corresponding to Figure 1F and 1G. Paired t test, p = 0.0392. Please find further details and results in our original paper.