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- MATERIALS AND METHODS
Summary: Purpose: We sought to determine whether cooling brain tissue from 34 to 21°C could abolish tetany-induced neuronal network synchronization (gamma oscillations) without blocking normal synaptic transmission.
Methods: Intracellular and extracellular electrodes recorded activity in transverse hippocampal slices (450–500 μm) from Sprague–Dawley male rats, maintained in an air–fluid interface chamber. Gamma oscillations were evoked by afferent stimulation at 100 Hz for 200 ms. Baseline temperature in the recording chamber was 34°C, reduced to 21°C within 20 min.
Results: Suprathreshold tetanic stimuli evoked membrane potential oscillations in the 40-Hz frequency range (n = 21). Gamma oscillations induced by tetanic stimulation were blocked by bicuculline, a γ-aminobutyric acid (GABA)A-receptor antagonist. Cooling from 34 to 21°C reversibly abolished gamma oscillations in all slices tested. Short, low-frequency discharges persisted after cooling in six of 14 slices. Single-pulse–evoked potentials, however, were preserved after cooling in all cases. Latency between stimulus and onset of gamma oscillation was increased with cooling. Frequency of oscillation was correlated with chamber cooling temperature (r = 0.77). Tetanic stimulation at high intensity elicited not only gamma oscillation, but also epileptiform bursts. Cooling dramatically attenuated gamma oscillation and abolished epileptiform bursts in a reversible manner.
Conclusions: Tetany-induced neuronal network synchronization by GABAA-sensitive gamma oscillations is abolished reversibly by cooling to temperatures that do not block excitatory synaptic transmission. Cooling also suppresses transition from gamma oscillation to ictal bursting at higher stimulus intensities. These findings suggest that cooling may disrupt network synchrony necessary for epileptiform activity.
Focal cooling has been suggested as a treatment for epilepsy. In vivo experiments confirmed that cooling reversibly inactivates mammalian cortex (1–4). Clinical case reports have documented use of cooling in a variety of epileptic conditions (5–10). Recent studies in hippocampal slices and in whole rats showed that rapid cooling to 22.0°C can halt seizure activity induced by 4-aminopyridine (11,36). Rapid cooling also blocked single-pulsed–evoked potentials in dentate granule cell layer neurons.
Mechanisms of cooling as an antiepileptic therapy remain unknown. Tetany-induced discharges in the range of 30–120 Hz, also called gamma oscillations, resemble spontaneous oscillations seen under a variety of epileptic conditions (12). These oscillations reflect network synchronization mediated by γ-aminobutyric acid (GABA)A-ergic depolarizations. Gamma oscillations recently have been identified in vitro in slice models of epilepsy. They are seen in transition to epileptiform bursting, spontaneously and in response to single-pulse stimulation in low-Mg2+ conditions, after high-frequency tetanic stimulation, and in juvenile slices subjected to hyperthermia in a febrile seizure model(12,13). In this study, we provide evidence that cooling disrupts epileptiform activity, at least in part, by blocking gamma oscillations.
MATERIALS AND METHODS
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- MATERIALS AND METHODS
Transverse hippocampal slices (450–500 μm) were prepared from juvenile (postnatal days 14–30) and adult (>30 days old) Sprague–Dawley rats (n = 21). Rats were anesthetized with halothane and then decapitated. The brain was placed into an oxygenated Ringer's solution at 10°C, mounted on a vibratome, and 450-μm slices were cut. Slices were incubated at room temperature for ≥60 min, and then maintained in an interface chamber at 32–35°C. Artificial CSF (aCSF) containing (in mM): NaCl, 117; KCl, 5.4; NaHCO3, 26; MgSO4, 1.3; NaH2PO4, 1.2; CaCl2, 2.5; glucose, 10 was bubbled with 95% O2 and 5% CO2. Blunt glass micropipettes filled with NaCl were used to record field potential from CA1 and sharp glass micropipettes (70–120 MΩ) filled with potassium acetate were used for intracellular recordings. For single-pulse–evoked potentials, Schaffer collaterals were stimulated with a bipolar platinum wire electrode for 100 μs. Recordings were made from stratum pyramidale in CA1 by a field and an intracellular electrode. For tetany–induced discharges, stimulation was changed to a train of 100 Hz for 200 ms.
Recordings were made only after ≥60 min of incubation at room temperature, followed by ≥30 min at control temperature (32–34°C) in the interface chamber. Temperature was controlled by using a thermostat with feedback from a temperature sensor (Fine Science Tools, Inc., Foster City, CA, U.S.A.). Previous experiments used a sensor connected to a probe penetrating the slice. After demonstrating excellent temperature correlation with only a small time delay (data not shown), a less cumbersome probe positioned just under the interface chamber was used for these experiments. Cooling was aided by continuously exchanging water in the chamber water jacket.
Cooling experiments (n = 21) were initiated at 32–34°C. Cooling to ∼21°C was accomplished within 20 min. The water bath in the slice chamber was slowly recirculated with ice water and the aCSF replaced with aCSF precooled to 4°C. Recordings were made every 3 min during cooling. Single-pulse–evoked potentials were measured first, and then tetanic stimulus was delivered. Stimulus intensity was kept constant through the cooling and rewarming. Stimulus intensities just above threshold to produce gamma oscillations (usually 1.25–2.25 mA) were used in 14 slices. Higher intensities (150–200% of threshold) were used to elicit gamma oscillations, followed by ictal bursts in five slices.
Analysis was performed off-line by using spike-counting software (Origin pClamp). Initial frequency for an oscillation was defined as the spike frequency during the first 250 ms of the oscillation. Latency was defined as the time between the offset of the stimulus and the first spike of the oscillation. For single-pulse–evoked potentials, amplitude was measured in the following way: A line was drawn connecting the peaks of the two positive deflections in the population spike. The distance from the midpoint of this line to the trough of the negative deflection was measured as the amplitude. Half-time durations were measured by drawing a line perpendicular to the amplitude line at its midpoint. The duration between the two points where the potential tracing crossed this line was the half-time duration, a reflection of the width of the waveform.