Intrahippocampal AAV Injection
All procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University. Adult Wistar Furth and Long Evans rats (200g-400g, > 10 weeks old, n = 40, (17 = female, 23 = male)) were anesthetized with isoflurane (5% for induction; 2.5% for maintenance and surgery) and positioned in a stereotaxic frame. The microinjection was positioned at AP -4.0 mm, ML 3.0 mm, DV -3.1 mm relative to Bregma 53. In the experimental group, Adeno-associated virus encoding for EPG under hDlx promoter (AAV9-hDlx-EPG-IRES-EGFP) was injected into the CA3 region of the right hippocampus. Two control groups were included: A sham group that received an injection of virus encoding only for GFP (AAV9-hDlx- EGFP), and a group that did not receive an injection at all.
Electrophysiology
Electrophysiology took place two to three weeks after transgene delivery. Rats were anesthetized with isoflurane (5% for induction; 2.5% for surgery; and 0.5% throughout the recording) and a constant infusion of Dexmedetomidine (0.5mg/kg/hr) subcutaneously. Isoflurane was administered at 0-5 % during all recordings to minimize its effect on neuronal activity 54,55.
To obtain Local Field Potentials (LFPs), a monopolar tungsten microelectrode (impedance: 200-300kΩ, FHC, Bowdoin, ME) was inserted into the right hippocampus CA3 region. Baseline recording was acquired for ten minutes. Following the baseline recording, kainic acid (KA) (0.5µg/0.1µl) was injected into the right hippocampus and ten minutes after KA injection, recordings were acquired for 1h. The signals were recorded with an Intan Recording System (Intan Technologies, Los Angeles, CA) and Spike2 (Cambridge Electronic Design, Cambridge, UK) at a sampling rate of 20kHz 56-59.
Immunohistochemistry
Rats that developed seizures were perfused transcardially with 1X Phosphate Buffered Saline (PBS) and 4% Paraformaldehyde (PFA). The brains were extracted and placed in 4% PFA overnight after which they were placed in varying concentrations of sucrose cryoprotection solution until they sank (10%, 20% and 30% consecutively). Frozen brains were sectioned at a thickness of 30µm using a Leica CM3050 S Cryostat (Leica Biosystems, Buffalo Grove, IL) and slices were placed in 4C in PBS. Antigen retrieval was achieved by heating the brain slices to 95C for 5 minutes in sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0). After cooling, slices were washed in PBST (0.1% Tween 20) 3 times and a blocking solution added (donkey serum, Sigma-Aldrich, St. Louis, MO) for 1 hour. Slices were then incubated with the primary antibodies (1:500, Anti-GAD67, ab97739, Abcam, Boston, MA and Anti-GFP, Jackson ImmunoResearch Labs, West Grove, PA) and rocked overnight at 4C. The following day, slices were washed in PBST 3 times after which they were incubated with the secondary antibodies (1:500, Donkey Anti-Rabbit conjugated with Alexa Fluor 647, and Donkey Anti-Chicken conjugated with Alexa Fluor 488, Jackson ImmunoResearch Labs, West Grove, PA), and rocked in the dark for 3h at 4C. Sections were subsequently washed three times with PBS (15min each) and mounting media (with DAPI) was applied. Images were acquired using the Biotek Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments, Inc., Winooski, VT) configured on an inverted microscope.
Analysis and Statistics
Recordings were analyzed using NeuroScore software (Data Sciences International, St. Paul, MN). Signals were high-pass filtered at 0.1Hz and seizure detection performed with the following parameters: threshold ratio: 1.2, maximal ratio: 20, minimum value: 100µV, minimal spike duration: 1ms, maximal spike duration: 500ms. Seizure events were defined as having at least 4 spikes, minimum interspike interval of 0.05s, maximum interspike interval of 0.6s, minimum train duration of 7.5s, and a train join interval of 1s. Analyses were validated by visual inspection of traces offline. D’Agostino & Pearson test was performed on all data sets to tests for normal distribution of data points. Further statistical tests were selected based on these results.