Spontaneous Bursting Slice from Post-Kainic Status Epilepticus (rat, in vitro)

Systemic kainic acid (KA)-induced status epilepticus (SE) in rats is an accepted model of temporal lobe epilepsy (TLE), where an initial insult results in SE and is followed by a sustained latent period that subsequently gives way to the development of recurrent spontaneous seizures. The medial entorhinal cortex-hippocampal (mEC-HC) slices obtained from these rats exhibit recurrent epileptiform discharges (REDs) that are pharmacoresistant to many traditional antiseizure drugs (West et al., 2018). The different sensitivities of REDs to these antiseizure drugs may reflect persistent molecular, cellular, and/or network‐level changes resulting from disease. These data serve as a foundation upon which future therapeutics may be differentiated and assessed for potentially translatable efficacy in patients with refractory epilepsy.

A modified low-dose kainic acid administration protocol (Hellier et al., 1998) is used to induce SE in adult male Sprague–Dawley rats weighing 150–200 g. Animals are injected intraperitoneally (IP) with an initial dose of 10 mg/kg KA (Tocris Bioscience, catalogue # 0222) to induce SE. If the animals do not develop a stage 4-5 seizure on the Racine scale within an hour, an additional dose of 5 mg/kg KA is injected every 30 minutes up to a maximum of 40 mg/kg total dose until a stage 4-5 seizure occurs. The Racine scale (Racine, 1972) defines wet dog shakes, facial/jaw clonus and head nodding as stage 1–2, forelimb clonus as stage 3, and rearing on hind legs and loss of balance as stages 4-5 seizures. The animals are observed, and seizures recorded for a minimum of 3.5 hours. Animals are required to display at least one stage 4-5 seizure every hour for 3.5 hours to be included in experiments. After 3.5 hours of behavioral monitoring, animals are given an injection of lactated Ringer’s solution subcutaneously to prevent dehydration and aid in recovery. Animals are then placed back in the holding facility in an individual cage with free access to food and water and allowed to recuperate for 2-3 weeks prior to in vitro experimentation.

For the in vitro, electrophysiology experiment rats are anesthetized using sodium pentobarbital (50 mg/kg, IP) and the brains are quickly removed and placed in ice cold, oxygenated 95% O2-5% CO2 sucrose artificial cerebrospinal fluid (ACSF) solution containing (in mM): sucrose (200), KCl (3.0), NaH₂PO₄ (1.4), MgSO₄ (2.0), NaHCO₃ (26.0), glucose (10.0), and CaCl₂ (2.0). The brains are sliced in to 350 µm thick horizontal sections in a ventral to dorsal orientation and incubated at room temperature for 1-2 hours prior to experiments in oxygenated ACSF containing NaCl (126 mM) instead of sucrose and 10 μM glycine (pH = 7.33–7.38; osmolarity = 290–310 mOsm). Slices are then transferred to a submersion chamber (SliceMate, Scientifica) and are continuously perfused with oxygenated ACSF at a rate of 2.0-2.5 ml/min and maintained at a constant temperature of 31 ± 1 °C. Occurrence of recurrent epileptiform discharges are facilitated with ACSF containing 6 mM K₊, 10 μM glycine, and 0.1 mM Mg₂₊.

Extracellular field excitatory postsynaptic potentials (fEPSPs) are recorded from eight brain slices simultaneously using a SliceMaster high-throughput brain slice recording system (Scientifica). Borosilicate glass electrodes (World Precision Instruments TW-150) are filled with ACSF and pulled to 1.5-2.5 MΩ resistance using a flaming brown micropipette electrode puller (Sutter Instrument Company Model P 97). A twisted nichrome/formvar coated wire stimulating electrode is used to evoke fEPSPs. The stimulating electrode is placed in the angular bundle fibers while the recording electrode is positioned in layer II of the mEC. Stimulation is evoked at a rate of one stimulation every 5 seconds using Slice-ISO stimulators (npi electronic GmbH), and fEPSPs are measured using Slice-2A amplifiers (npi electronic GmbH). The minimum amplitude criteria for evoked fEPSPs in the mEC is ≥0.2 mV. Spontaneous fEPSPs are recorded using a low pass 1 kHz filter, at a sampling rate of 10 kHz, and recorded in gap-free mode using a Digidata 1550 (Axon Instruments). Data is acquired using pCLAMP 10.4 and analyzed using CLAMPFIT 10.4 software (Axon Instruments). After 20 minutes of baseline recording of recurrent epileptiform discharges, investigational compounds are applied via bath exchange for 20 minutes followed by a 20-minute washout to assess reversibility.

Data are high-pass filtered before analysis (Bessel 8 pole filter, with a cutoff of 5 Hz). A threshold search method is used to identify spontaneous events (threshold set to no less than 2x baseline noise). These data are then subjected to burst analysis with a burst-delimiting interval of 1000-1500 msec. Burst duration, frequency, and amplitude are averaged and binned in 60-second increments to assess baseline stability of these measures; data from individual slices that exhibit unstable baselines or drift >20% over 20 minutes are excluded. Concentration-response curves are fit to data when the investigational compound is tested at a minimum of 4 concentrations, and half maximal effective concentration (EC50) is calculated (slope constrained to one, and initial baseline values are constrained to 100% and 0%, GraphPad Prism version 6).

For all electrophysiological experiments, stock solutions of investigational compounds are made fresh on the day of the experiment and frozen then reused for multiple experiments and applied via bath perfusion of the combined mEC brain slice. The working concentration of dimethyl sulfoxide (DMSO) is kept below 0.1% for each solution.