More than 90% of slices heated to 40°C showed a "febrile-seizure like event," represented as initial epileptiform-like population bursts, followed by SDs. Figure 1A shows a typical "febrile seizure-like event" elicited by heating a hippocampal slice from 33.9 to 38.2°C. The epileptiform discharges demonstrated a frequency of 80 Hz (Fig. 1B), within the 30–100 Hz γ oscillation range. Summary of data from 24 slices showed that 71% (17/24) of hyperthermic epileptiform-like population spike activity fired at a frequency range between 30–50 Hz (Fig. 1C,D), with a mean oscillation frequency of 45.9 ± 3.0 (mean ± SE, n = 24).

Figure 2 shows the comparison between hyperthermic population spike activity and post-tetanic γ oscillations. In standard artificial cerebrospinal fluid (ACSF), a tetanic stimulation at 100 Hz, 100 μsec, 2 mA, for 200 ms (20 trains) elicited γ and β frequency population spikes, visible both with intracellular and extracellular recordings. In the same slice, heating to 38°C (2Ab) evoked population oscillation very similar to those of post-tetanic γ oscillations. Power spectrum analysis of the population of responses among all slices, showed similar frequency peak of hyperthermic and post-tetanic gamma oscillations (Fig. 2B). Among all slices, hyperthermic oscillations occurred at a frequency of 45.9 ± 3.0 Hz (mean ± SE, n = 24) and post-tetanic gamma oscillations at 47.1 ± 2.6 (mean ± SE, n = 34). These two frequencies do not significantly differ (Fig. 2C).

Results from this and our previous study showed that the SD always followed hyperthermic bursts 8, but tetanic stimulation induced only γ oscillations in the absence of SD (Fig. 2). Therefore, we investigated whether high-intensity tetanic stimulation could induce γ oscillations followed by SD. Fig. 3A illustrates a weak γ oscillation of field potential in response to tetanic stimulation at 2.25 mA. Tetanic stimulation at 3.25 mA induced a strong γ oscillation with a large field burst amplitude and long-lasting depolarization (Fig. 3B). In 6 of 7 tested slices, tetanic stimulation at 4.25 mA induced, not only prominent γ oscillations, but also SDs (Fig. 3C). These post-tetanic depolarizations had duration and time course very similar to those of hyperthermic SDs (Fig. 1A,2Ab).

We utilized the specific GABA_A receptor antagonist, bicuculline methiodide (BMI), to ascertain whether γ oscillations were dependent upon GABAergic mechanisms. The effect of BMI on the field potential in region CA1 is shown in Fig. 4. Bath-applied BMI (5 μM) changed the single evoked population spike to multiple spikes, in a reversible manner. In the same slice, tetanic stimulation delivered in control artificial cerebrospinal fluid (ACSF) induced typical γ oscillations, and these γ oscillations were abolished by addition of BMI to the perfusate. γ oscillations recovered partially by 80 minutes of wash in ACSF. Figure 4B shows another case in which high intensity of tetanic stimulation was applied to elicit γ oscillations and SDs. Addition of 20 μM BMI reversibly blocked γ oscillations, but not the SD.

In a second set of experiments using five slices (Fig. 5), we examined the effects of BMI on hyperthermic epileptiform-like population bursts. Bath-application of 10 μM BMI for 20 min abolished post-tetanic γ oscillations. After several minutes of exposure to BMI, slices demonstrated rhythmic spontaneous bursts of population spikes (Fig. 5B), indicating that GABA_A receptors have been blocked by BMI. Under these conditions, heating of the slice only evoked the SD without the initial population spikes (Fig. 5C).

Figure 6A shows the effect of the ionotropic glutamate receptor antagonist, kynurenic acid (Kyn-A, 7 mM), on γ oscillations. Kyn-A dramatically attenuated evoked synaptic transmission, but failed to prevent hyperthermia-induced γ oscillations and slow SDs, suggesting that excitatory synaptic transmission is not necessary for hyperthermic γ oscillations and SDs. In Fig. 6B, 30 mM QX-314 was incorporated into the recording electrode. The action potentials were blocked by intracellular QX-313 diffusion. Heating the slice to 37°C elicited a γ oscillation followed by a slow SD in the extracellular field recording. However, the intracellularly-recorded hyperthermic γ oscillation disappeared and was replaced by a smooth depolarization (Fig. 6B).

Gamma oscillations are defined as coherent cortical oscillations at γ (30–100 Hz) band frequency frequencies in in vivo and in vitro models. In hippocampal slice preparations, γ oscillations can be triggered by high frequency tetanic stimuli, called post-tetanic γ oscillations 10111213. In addition to post-tetanic γ oscillations, the γ oscillations also can be elicited by metabotropic glutamate receptor agonists, carbacol, or free extracellular Mg²⁺1819. In this study, we report that γ oscillations can occur in a model of a clinical disorder, namely hyperthermia. Hyperthermic epileptiform-like population spikes occur within the band of γ frequencies (30–100 Hz). Post-tetanic γ oscillations can be mimicked by hyperthermic stimulation in the same slice. Tetanic stimulation at high intensity induces initial γ oscillations followed by a slow SD which resembles hyperthermic epileptiform-like population spikes followed by a slow SD. Finally, both post-tetanic γ oscillations and hyperthermic epileptiform-like population spikes were completely blocked by a GABA_A receptor antagonist, BMI. This experimental evidence suggests that the GABA_A receptor-governed γ oscillations underlie the hyperthermic population spikes in our in vitro model system.

Accumulating lines of evidence demonstrate that the genesis mechanisms for post-tetanic γ oscillations involve slow GABA_A receptor-mediated depolarization, extracellular K⁺ elevation and field effects 10111213. The present study showed that these three major factors also underlie hyperthermic γ oscillations. As shown in Fig. 1A and 2A, the hyperthermic γ oscillations always overlie the slow membrane depolarizations. For post-tetanic γ oscillations, the slow depolarizations were mediated by tetanic stimulation-induced GABAergic depolarizing action 20212223. This GABAergic depolarization is attributed to the tetanic stimulation-induced accumulation of intracellular chloride 24 and extracellular potassium 22. For hyperthermic γ oscillations, heating causes a Na⁺/K⁺ pump failure 8, in turn resulting in the accumulation of intracellular Na⁺ and Cl^-, as well as extracellular K⁺, finally causing a slow membrane depolarization. In our previous report, the extracellular K⁺ indeed elevated dramatically during hyperthermia-induced epileptiform-like population spikes and SDs 8. Field effects further contribute to post-tetanic γ oscillations 2526. High Frequency tetanic stimuli leads to cellular swelling 2728, increasing extracellular resistance. According to Ohm's law, this increases the voltage deflection recorded for a given current flowing through this extracellular resistance. Current generated by the active population travels through the resistances of the extracellular space and of the nearby inactive cells, therefore producing the extracellular population spikes and meanwhile depolarizing the inactive cells. In this manner, inactive cells are brought closer to threshold, enhancing their opportunity to firing together 293031. The extracellular resistance is the major determinant of field-effect strength 32. Since the extracellular volume fraction in CA1 stradium pyramidal is only 12% compared with approximately 18% in CA3 and granule cells 33, the CA1 pyramidal cells are particularly susceptible to field effects. The accumulation of extracellular K⁺ during hyperthermic SDs depolarizes membrane potential, which triggers initial γ oscillations. Cell swelling reduces the extracellular space, which triggers slow SDs 8, (Fig. 1A and 2A). In Fig. 6A, after blockade of synaptic transmission by kynurenic acid (7 mM), heating the slice still induced γ-oscillation followed by SD, suggesting that a non-synaptic mechanism (local field effect) may be involved in the generation of hyperthermic γ-oscillations and SDs. Figure 6B demonstrated that hyperthermic γ-oscillations disappear in the presence of an intracellularly delivered Na⁺ channel blocker (QX 314, 30 mM).

Epileptic activity can result from an imbalance between glutamatergic excitation and GABAergic inhibition. However, this simple balance model has been challenged by findings that GABAergic transmission remains effective in some epilepsy models, in epileptogenic human tissue 34353637383940, and by current findings of the excitatory effects of GABA 182223. Therefore, the GABA excitatory effects may work as a possible ictogenic mechanism under some special condition such as tetanic stimulation. Indeed, recently Köhling et al. reported that under epileptogenic condition (free Mg ²⁺ in ACSF), γ band oscillations arise from GABAergic depolarizations and that this activity may lead to the generation of ictal discharges 18. It has been reported that prolonged periods of γ oscillations are associated with temporal lobe seizures in in vivo rats 41. Furthermore, human EEG studies with subdural recording electodes showed that a γ band oscillation could be recorded at the start of typical seizure activity 42. In this respect, the hyperthermic γ oscillations may also play a critical role in generation of neuronal epileptiform activity. The extent to which the hyperthermic slice serves as a model of febrile seizures remains to be determined. If γ oscillations do prove to be important in clinical febrile seizures, then future work might profitably examine the therapeutic potential of mild disinhibition to disrupt inhibitory GABA-mediated synchrony at the start of ictal activity.