Male C57 black mice (initial ages of 6–8 months, Charles River Laboratory, Quebec, Canada) were used in the present experiments. These animals were housed in a vivarium that was maintained at 22°C with 12-hour light and dark cycles. Food and water were available ad libitum. Stimulations and recording experiments were done between 12-5 pm. All experimental procedures described below have been reviewed and approved by the Animal Care Committee of the University Health Network in accordance with the guidelines of the Canadian Council on Animal Care.
All electrodes were made of polyamide-insulated stainless steel wires (outer diameter 200 μm, Plastics One, Ranoake, VA). Twisted bipolar wires were used for stimulation and recording, with their intracranial tips ~100 μm apart. The extracranial tips of the twisted wire assembly were soldered to the female ends of two connecting pins, with one wire bent into an L-shape to separate the connecting pins (Figure 1A). Care was taken to fully remove the insulation layer before soldering. A liquid solder (Soldering Liquid Flux, Certanium Alloys and Research Company, Cleveland OH, USA) or weak phosphoric acid was used to ensure good contact between the stainless steel wire and the connecting pin. A single wire was similarly soldered to a single pin for reference. The resistance of each constructed wire electrode was ≤1.0 Ω. After being soldered to the connecting pins, the bipolar wires were then cut to ~3 mm in length to target the desired hippocampal CA3 area (see below). The single monopolar wire was cut to ≤0.5 mm for epidural position of the reference electrode. These electrodes were cleaned with 75% alcohol and stored in a sterilized glass vial until use. The connecting pins were detached from standard IC sockets (Samtec series SS socket strips, SS-132-G2, Electrosonic, Toronto, Ontario, Canada). These pins are 7.5 mm long in total; the male end of the pin is 3.2 mm long. The outer diameter is 1.8 mm or 0.5 mm for female or male end of the pin respectively (Figure 1A). Measured after being dissected out from implanted animals (n = 3), the total weight of implanted electrode assembly, including electrode wires, plastic base and dental acrylic (see below), was 0.50-0.52 grams. As adult mice were used in the present experiments, the weight of implanted electrode assembly was ≤2% of animal body weight.
Surgery for electrode implantation
Surgical procedures were modified from those we previously described [14, 21]. Briefly, the animal was anaesthetized with 2% isoflurane and then placed on a stereotaxic frame. After skin incision and exposure of the skull surface, the tip of a mini drill bit (see below) was aimed to bregma via a micromanipulator. After determining the bregma position, the drill bit was moved up but its X-Y position was unchanged, and a thin plastic base then was glued onto the skull surface. After the glue had cured, three small holes were then drilled through the plastic base and the skull according to the stereotaxic coordinates of the hippocampal CA3 area (bregma −2.5 mm, lateral ±1.3 mm, and a depth of 3.2 mm ). The reference electrode was positioned at bregma 1 mm, lateral 2.0 mm and a depth of 0.7 mm. The electrode depths were adjusted to accommodate for the thickness (200 μm) of the plastic base.
The plastic base was cut from a curved part of plastic weighting boats (polystyrene antistatic weighting dishes, Fisher Scientific, cat#08-732-115). The weighting boats were 140x140 mm in length-width and 25 mm in depth, with thickness of ~200 μm. The plastic base was soft and could be gently pressed to accommodate curvature of the skull, and thus bound tightly with the skull surface after being glued. We used a cyanoacrylate-type glue (Insta-cure+, cure time 5–15 seconds, made in U.S.A., cat# BSI-106C; obtained from Canadian Hobbycraft, Concord, Ontario, Canada). A motorized drill (model FM3545, Foredom Electric, Bethel, CT, USA) and a mini drill bit (part 115603, Ball Mills Carbide, CircuitMedic, Haverhill, MA, USA) were used to drill small holes (≤0.5 mm) through the skull. These holes were large enough for inserting the electrodes, but small enough to prevent dental acrylic leakage into the brain (see below).
Micromanipulators were also used to individually insert the bipolar electrodes into bilateral hippocampal CA3 areas. After positioning and holding these electrodes with the micromanipulators, dental acrylic was overlaid onto the plastic base such that the bases of the connecting pins were covered by the dental acrylic (Figure 1B). Care should be taken to apply acrylic so as not to interfere with electrode positions and to contaminate connecting pins. We used a dental acrylic with hardening time of 6–9 minutes (Jet Tooth Shade, Reference No. 1404; Lang Dental Mfg. Co., Inc., Wheeling, IL, USA) to carefully cement the implanted electrodes. After the dental acrylic had hardened, the electrodes were released from the micromanipulators. The incised skin was then glued to the dental acrylic (Figure 1A,1B), which prevents infection in the implanted area. The animals were released from the stereotaxic frame and allowed to recover for at least one week before further experimentation.
Unilateral CA3 stimulation was conducted in all present experiments. Constant square-wave current pulses (duration of 0.5 ms, intensities of 10–150 μA) were generated from a Grass stimulator and delivered through an isolation unit (model S88H, Grass Medical Instruments, Warwick RI, USA). To establish an input–output plot for evoked CA3 field potentials, a single stimulation was applied every 30 seconds at intensities of 10–150 μA (10 μA increments; 5 consecutive responses at a given intensity). A standard kindling protocol [24, 25] was used. Animals were considered fully kindled after consecutive stage 5 seizures on 3 consecutive days.
Recordings and measurements
All recordings were from the CA3 area contralateral to the CA3 stimulation site. Signals were recorded via a 2-channel microelectrode AC amplifier (model 1800, A-M Systems, Carlsborg, WA, USA), with the input frequency band set in the range of 0.1-1000 Hz, and the amplification gain at 1000×. The signals were digitized at 5000 Hz (Digidata 1440A, Axon Instruments/Molecular Devices, Union City, CA, USA). Pclamp software (Version 9 or 10; Axon Instruments/Molecular Devices) was used for data acquisition, storage and analyses.
The amplitudes of evoked field potentials were measured from averaged traces (5 consecutive responses) at a given stimulation intensity. To measure hippocampal rhythmic activities associated with “active” and “inactive” behaviors, stable data segments of 5–10 seconds or 30–60 seconds were selected while the animals were moving/exploring or immobile/asleep. Spectral plots were generated from these data segments and peak frequencies were measured from the spectral plots for individual animals. To detect interictal-like spikes, individual animals were recorded for up to four hours before kindling was initiated. After animals were fully kindled, interictal spikes were recorded for 4–6 hours after the most recent ADs. A spike was only counted if its amplitude was large (≥2 times the amplitude of the background signal) and its waveform was similar to those previously described [26–29]. To minimize interference of movement-related artifacts, the rates of interictal spikes were measured in the periods (1–2 hours) while the animals were immobile/asleep. To measure AD durations, corresponding EEG data were treated with a 0.5 Hz high-pass (Bessel) filter to reduce slow drifts in EEG signals.
Animal’s behaviors were recorded with a high definition camera and analyzed by blinded experimenters. Behavioral seizures were scored using a modification of the Racine scale for the mouse : stage 0, no response or behavioral arrest; stage 1, chewing or head-nodding; stage 2, chewing and head nodding; stage 3, single or bilateral forelimb clonus; stage 4, bilateral forelimb clonus and rearing; stage 5, loss of righting reflex (falling).
Histological experiments were conducted as we previously described [15, 17]. Briefly, the animals were anesthetized with pentobarbital (70 mg/kg, i.p.) and trans-cardiacally infused with saline and then with 10% phosphate-buffered formalin solution before decapitation. Cryostat coronal sections 30 μm thick were obtained throughout the brain, stained with cresyl violet, and examined under a light microscope.
Statistical tests were performed with SPSS software (Version 20, SPSS Statistics, IBM). Data are presented as mean and standard error of mean (M ± SE) throughout the text and figures except where indicated.