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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2018 Aug 17;39(11):2286–2294. doi: 10.1177/0271678X18795365

Reduction of spike generation frequency by cooling in brain slices from rats and from patients with epilepsy

Sadahiro Nomura 1,2,*,✉, Hiroyuki Kida 3,*, Yuya Hirayama 1, Hirochika Imoto 1,2, Takao Inoue 1, Hiroshi Moriyama 1, Dai Mitsushima 3, Michiyasu Suzuki 1
PMCID: PMC6827110  PMID: 30117752

Abstract

This study aimed to understand the mechanism by which brain cooling terminates epileptic discharge. Cortical slices were prepared from rat brains (n = 19) and samples from patients with intractable epilepsy that had undergone temporal lobectomy (n = 7). We performed whole cell current clamp recordings at approximately physiological brain temperature (35℃) and at cooler temperatures (25℃ and 15℃). The firing threshold in human neurons was lower at 25℃ (−32.6 mV) than at 35℃ (−27.0 mV). The resting potential and spike frequency were similar at 25℃ and 35℃. Cooling from 25℃ to 15℃ did not change the firing threshold, but the resting potential increased from −65.5 to −54.0 mV and the waveform broadened from 1.85 to 6.55 ms, due to delayed repolarization. These changes enhanced the initial spike appearance and reduced spike frequency; moreover, spike frequency was insensitive to increased levels of current injections. Similar results were obtained in rat brain studies. We concluded that the reduction in spike frequency at 15℃, due to delayed repolarization, might be a key mechanism by which brain cooling terminates epileptic discharge. On the other hand, spike frequency was not influenced by the reduced firing threshold or the elevated resting potential caused by cooling.

Keywords: Brain temperature, current clamp, depolarization, epilepsy, resting potential

Introduction

Brain cooling has been implemented for the last 50 years as an effective method for terminating epileptic discharges.1,2 Temperatures around 15℃ were found to suppress brain metabolism.3 Metabolic suppression improved the ischemic state, represented by lactate production, but it only mildly reduced cerebral blood flow.3 Brain cooling also inhibited neurotransmitter release,4 which terminated neuronal excitation5–11 and seizure propagation through the epileptogenic network. Brain cooling also protected the brain from secondary injury.12 Although 15℃ seems to be a very low temperature, it was found to be safe in neurofunctional11 and histopathological7 investigations. This safety permitted the development of a focal brain cooling device for intracranial implantation in patients with intractable epilepsy that involved unresectable foci. We previously collected biological evidence3–15 for the cooling application in a clinical study. We found that brain cooling caused changes in the electrophysiological properties of neuronal membranes that could lead to the termination of epileptic discharges.

Penicillin-induced epileptic discharges were associated with increased focal brain temperature,14 which could induce childhood febrile seizures.16–18 Studies in a febrile seizure model showed that the febrile state could increase the resting membrane potential, decrease the threshold potential, and increase the firing frequency, compared to the non-febrile state.19 One hypothesis suggested that, at high and low temperatures, neuronal cellular properties might shift to hyperactive and hypoactive states, respectively.

Here, we used the current clamp technique to investigate the effects of cooling on neuronal membrane properties and the action potential. Brain slices were prepared from the lateral temporal cortex samples acquired during a temporal lobectomy for patients with medically intractable epilepsy. To our knowledge, this study was the first to demonstrate the effects of cool temperatures on the cellular physiological properties of cortical neurons in patients with epilepsy. Specimens were prepared at a mild low temperature (room temperature, 25℃); at a low cool temperature (15℃), to mimic focal brain cooling treatments, and at a warm temperature (35℃), to approximate the physiological brain temperature. We chose 35℃, because cell viability is difficult to maintain in experimental chambers at 37℃. We performed the same measurements in normal brain slices prepared from Sprague-Dawley rats to compensate for the limited number of human samples.

Materials and methods

Preparation for electrophysiology

Animals

Male Sprague-Dawley rats (n = 19, 8 for cooling and 11 for warming experiments, Chiyoda Kaihatsu Co., Tokyo, Japan) were housed in individual plastic cages (40 × 25 × 25 cm) and maintained at a constant temperature of 23 ± 1℃ under a 12 h light/dark cycle, with water and food provided ad libitum. All experiments were performed according to the Guidelines for Animal Experimentation of the Yamaguchi University School of Medicine and conformed to Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines. The protocols were approved by the Institutional Animal Care and Use Committee of Yamaguchi University.

Rats were anesthetized with pentobarbital, and acute brain slices were prepared.20,21 Briefly, brains were rapidly perfused with ice-cold dissection buffer (25.0 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7.0 mM MgCl2, 25.0 mM glucose, 110.0 mM choline chloride, 3.10 mM pyruvic acid, and 11.6 mM ascorbic acid) and gassed with 5% CO2/95% O2.

Patients

This study included seven patients (four men, three women, aged 29–76 years, mean: 42.6 ± 15.7 years) with intractable temporal lobe epilepsy (Table 1). In six of the patients, epileptic foci were detected with subdural and depth electrode recordings, performed over two weeks, with video-electroencephalography (EEG). The seventh patient had meningioma and underwent a single-stage surgery. All patients met the surgical indication for temporal lobe resection, with or without hippocampectomy. During surgery, we acquired a columnar sample of cortical brain tissue from the middle temporal gyrus, 10 mm in diameter and 10 mm in height, for patch clamp analysis. Heat damage was avoided by acquiring samples before hemostatic procedures for coagulation. The sampling procedure was performed within 30 s to avoid ischemic injury. Four samples from patients with mesial temporal lobe epilepsy (MTLE) were histopathologically normal. Among these patients, three had hippocampal sclerosis and one had tentorial meningioma. The other three patients with lateral temporal lobe epilepsy had a histopathological diagnosis of focal cortical dysplasia (FCD) type I.22 All patients had taken Na+-channel antagonists as oral anticonvulsants until the morning of surgery.

Table 1.

Characteristics of patients with intractable epilepsy.

Case Age Sex Side Semiological lesion EEG lesion Pathology in sample Pathology in MRI Seizure type Seizure frequency Anticonvulsants
1 30 M R M. temporal H Normal Normal CPS, GTC weekly CBZ, LEV, LTG, TPM
2 40 F L M. temporal H Normal Cavernoma CPS Monthly CBZ, NZP
3 42 F R M. temporal H Normal Normal CPS Monthly CBZ, LTG
4 76 M R L. temporal C Normal meningioma CPS Monthly CBZ
5 29 M R L. temporal H + C FCD I Normal CPS, GTC Monthly CBZ, CLB, LEV, LTG
6 38 F R L. temporal C FCD I Normal CPS, GTC Monthly CBZ, LEV
7 43 M L L. temporal H + C FCD I HS CPS Monthly CBZ, LEV, VPA
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C: cortex; CPS: complex partial seizure; CBZ: carbamazepine; CLB: clobazam; FCD I: focal cortical dysplasia type I; GTC: generalized tonic convulsion; H: hippocampus; HS: hippocampal sclerosis; LEV: levetiracetam; LTG: lamotrigine; L. temporal: lateral temporal; M. temporal: mesial temporal; NZP: nitrazepam; VPA: valproate.

Tissue samples were immediately sliced, parallel to the axons, and prepared for whole-cell recordings, as previously described.20,21 Briefly, the tissue sections were rapidly placed in ice-cold dissection buffer (25.0 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7.0 mM MgCl2, 25.0 mM glucose, 110.0 mM choline chloride, 3.10 mM pyruvic acid, and 11.6 mM ascorbic acid) and gassed with 5% CO2/95% O2.

The study protocol was reviewed and approved by the Yamaguchi University Institutional Review Board, and conformed to the Helsinki Declaration. All patients and their families agreed to participate in our study after they were adequately informed of the aims, methods, anticipated benefits, potential risks, and discomfort associated with their involvement. All patients had the right to withdraw from the study at any time.

Electrophysiology

Coronal brain samples were cut into 350-µm slices with a Leica vibratome (VT-1200; Leica Biosystems, Nussloch, Germany) in dissection buffer. The slices were then transferred to a physiological solution (114.6 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM glucose, 4 mM MgCl2, and 4 mM CaCl2, pH 7.4) at 22–25℃, and gassed with 5% CO2 /95% O2, according to the method described by Boehm et al.23 Glass electrodes with resistances of 4–7 megaohm were manufactured with a horizontal puller (Model P97; Sutter Instrument, Novato, CA, USA) and filled with the appropriate solution. Whole-cell recordings were primarily performed in pyramidal neurons in epileptic lesions and recorded with an Axopatch-1D amplifier (Axon Instruments Inc., Union City, CA, USA). Recordings were digitized with a Digidata 1440 AD board (Axon), recorded at 5 kHz, and analyzed offline with pCLAMP 10 software (Axon).

Temperature analysis

Room temperature was maintained at approximately 25℃, and we monitored the temperature of the bath and chamber solutions with a thin thermocouple (IT-23, Physitemp Instruments Inc., Clifton, NJ, USA). The temperature in the chamber was maintained at 15℃, 25℃, or 35℃, by running the tube containing artificial cerebrospinal fluid through a temperature-controlled water bath (Figure 1(a)). For the moderate temperature (25℃), we used a warm water bath (27℃); for the warm temperature, we used a hot water bath (64℃); and for the cold temperature (15℃), we used a cold water bath (3℃), controlled with an immersion cooler (ECS-0SS, Tokyo Rikakikai Co., Ltd, Tokyo, Japan). We adjusted the flow rate of the pump (AC-2110, ATTO, Tokyo, Japan) to change the temperature within 2 min.

Figure 1.

Figure 1.

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Experimental set up. (a) The artificial cerebrospinal fluid (ACSF) solution (left) flowed continuously through a tube into the recording chamber (right). The ASCF temperature was adjusted by running the tube through a hot, warm, or cold bath (3℃, 27℃, or 64℃, respectively), to achieve the appropriate temperature upon mixing with the solution in the recording chamber. ASCF solution was continuously let out of the recording chamber to maintain the flow. A slice anchor was used to stabilize the brain slice in the recording chamber. (b) The cooling and warming protocol for the human study. Note that cooling was conducted prior to warming. In the rat study, the cooling and warming protocols were performed in separate rats (not shown).

We first tested the cooling (n = 8) and warming protocols (n = 11) on separate rat samples, and we adjusted the protocols to achieve consistent conditions. This calibration step allowed us to use as little of the human brain samples as possible (Figure 1(b)). Repeated cooling and warming may be harmful to neurons. Because the cooling protocol was considered most important, we began with the cooling protocol. Recordings were suspended until the temperature returned to 25℃ after the cooling or warming period. Data were excluded from the analysis when action potentials did not achieve the same level as observed before the cooling or warming period.

Current clamp recordings

For current clamp recordings, electrode pipettes were filled with 130 mM K-Gluconate, 5 mM KCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM Na-phosphocreatine, and 0.6 mM EGTA at pH 7.25. These electrodes were inserted into cortical layer II/III neurons. We counted the number of spikes that occurred during current injections stepped from −100 to +550 pA, that lasted 150–300 ms. We identified the minimal voltage that induced action potentials to determine the firing threshold for each neuron. The afterhyperpolarization (AHP) amplitude was evaluated by measuring the voltage at spike initiation and the lowest voltage during AHP. When the current injection failed to induce any spikes, we excluded both threshold and spike duration data from the analyses.

Data analysis

All results are expressed as the mean ± standard error of the mean (SEM). We used one-way factorial ANOVA, followed by a post-hoc analysis with Tukey’s honest significant difference (HSD) test, to compare means among multiple groups. We used the Mann–Whitney U test to evaluate the differences between groups. P values < 0.05 were considered statistically significant.

Results

Data for the cooling protocol were collected from 29 cells isolated from eight rats (average 3.63 ± 0.56 cells/rat, range: 2–7 cell/rat). A representative case is shown in Figure 2(a). The resting membrane potential was significantly increased at 15℃ compared to the precooling condition (precooling: −63.6 ± 1.6 mV, cooling: −54.0 ± 2.0 mV, recovery from cooling, −65.1 ± 1.5 mV, P < 0.001, one-way repeated-measures ANOVA, F(2,56) = 78.8; P < 0.001, Tukey’s HSD test). The cooling condition did not significantly affect the threshold (precooling: −29.6 ± 1.8 mV, cooling: −30.3 ± 1.9 mV, recovery from cooling: −29.1 ± 1.2 mV, P = 0.14, one-way repeated-measures ANOVA, F(2,46) = 2.00). Current injections of 400 pA and 500 pA were required to induce an action potential at 25℃. In contrast, injections of only 100 pA induced action potentials at 15℃. In addition, the spike duration was significantly extended at 15℃ (precooling: 1.36 ± 0.20 ms, cooling: 5.42 ± 0.21 ms, recovery from cooling: 1.37 ± 0.21 ms, P < 0.001, one-way repeated-measures ANOVA, F(2,46) = 77.4; P < 0.001, Tukey’s HSD test; Figure 2(b)). During the cooling condition, the number of spikes plateaued at 200 pA; in contrast, at 25℃, the number of spikes increased linearly with increasing levels of current injections (Figure 2(c)). A two-way repeated-measures ANOVA indicated that these effects were mainly due to cooling (F(2, 1069) = 13.53; P < 0.001) and the level of injected current (F(13,1069) = 81.50; P < 0.001), and a significant interaction was observed between the temperature and the current (F(26,1069) = 5.34; P < 0.001).

Figure 2.

Figure 2.

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Changes in neuronal activity with cooling in rat brain slices. (a) Representative examples of action potentials induced by current injections in rat brain slices for the cooling condition. Traces show each series of current injections performed before (precooling) during (cooling) and after cooling (recovery). (b) Mean resting membrane potential, firing threshold, and spike duration. Note that all these parameters recovered to the precooling levels. The number of neurons is shown at the base of each bar. (c) Mean input/output relationship during precooling (filled), cooling (blue), and recovery from cooling (open). *P < 0.05 vs. precooling. Error bars indicate ± SEM.

Data for the warming protocol were collected from 33 cells isolated from 11 rats (average 3.00 ± 0.27 cells/rat, range 1–4 cells/rat). A representative case is shown in Figure 3(a). The 35℃ warming produced no change in resting membrane potential compared to the prewarming condition (prewarming: −68.1 ± 0.9 mV, warming: −69.2 ± 1.4 mV, recovery from warming: −66.8 ± 1.9 mV, P = 0.24, one-way repeated-measures ANOVA, F(2,64) = 1.47). However, the warming strongly and significantly increased the threshold (prewarming: −29.6 ± 1.0 mV, warming: −20.0 ± 1.9 mV, recovery from warming: −27.2 ± 1.3 mV, P < 0.001, one-way repeated-measures ANOVA, F(2,40) = 16.7; P < 0.001, Tukey’s HSD test). Warming caused the spike duration to shorten significantly (prewarming: 1.44 ± 0.01 ms, warming: 0.89 ± 0.01 ms, recovery from warming: 1.67 ± 0.01 ms, P = 0.0006, one-way repeated-measures ANOVA, F(2,40) = 8.95; P = 0.027, Tukey’s HSD test; Figure 3(b)). The number of spikes induced by increasing current injections was less at 35℃ than at 25℃; however, the difference was not significant (Figure 3(c)). Two-way repeated-measures ANOVA indicated that these effects were mainly due to warming (F(2, 1112) = 40.75; P < 0.001) and the injected current (F(13,1112) = 35.93; P < 0.001), and a significant interaction was also observed between the temperature and the current (F(26,1112) = 2.71; P < 0.001).

Figure 3.

Figure 3.

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Changes in neuronal activity with warming in rat brain slices. (a) Representative examples of action potentials induced by current injections in rat brain slices during the warming condition. The action potential traces show each series of current injections performed before (prewarming), during (warming), and after warming (recovery). (b) Mean resting membrane potential, firing threshold, and spike duration. The number of neurons is shown at the base of each bar. (c) Mean input/output relationship during prewarming (filled), warming (red), and recovery from warming (open). *P < 0.05 vs. prewarming. Error bars indicate ± SEM.

We recorded from 43 neurons in seven human slices (average 7.17 ± 1.25 cells/slice, range 3–10 cells/slice). Typical examples of the cooling and warming effects are shown in Figure 4(a). In cell 1, the spikes were strongly attenuated in both cooling and warming conditions. In cell 2, the spikes were inhibited, especially in the late phase of cooling. In cell 3, the spikes increased during warming. The firing patterns were monotonous during cooling and varied during warming conditions. We performed the cooling–warming protocol in 40 of the 43 neurons; the remaining three neurons were tested only under cooling conditions. Accordingly, the latter three neurons were excluded from the statistical analyses of warming conditions. Cooling to 15℃ significantly increased the resting potential (precooling: −65.5 ± 1.0 mV, cooling: −54.0 ± 1.5 mV, recovery from cooling: −60.7 ± 3.4 mV; warming: −66.4 ± 1.5 mV, recovery from warming: −60.1 ± 2.1 mV, P < 0.001, one-way repeated-measures ANOVA, F(4,162) = 5.73; P < 0.001, Tukey’s HSD test). Warming to 35℃ affected the threshold (precooling: −33.0 ± 1.9 mV, cooling: −33.7 ± 0.9 mV, recovery from cooling: −32.6 ± 1.5 mV; warming: −27.0 ± 0.9 mV, recovery from warming: −30.0 ± 1.0 mV, P = 0.0035, one-way repeated-measures ANOVA, F(4,146) = 4.11; P < 0.001, Tukey’s HSD test). Cooling to 15℃ also significantly extended the spike duration (precooling: 1.85 ± 0.01 ms, cooling: 6.55 ± 0.01 ms, recovery from cooling: 1.70 ± 0.001 ms; warming: 1.11 ± 0.002 ms, recovery from warming: 1.70 ± 0.001 ms, P < 0.001, one-way repeated-measures F(4,146) = 44.2; P < 0.001, Tukey’s HSD test; Figure 4(b)). The number of spikes at 15℃ increased with increasing levels of injected current, similar to the behavior observed at 25℃ and 35℃, up to 100 pA; then, the number of spikes plateaued, in contrast to the continued increasing number of spikes with increasing current levels observed at 25℃ and 35℃. Two-way repeated-measures ANOVA determined that these effects were mainly due to cooling (F(4, 2326) = 39.63; P < 0.001) and the level of injected current (F(13,1069) = 91.26; P < 0.001). Again, a significant interaction was observed between the temperature and the current (F(52,1069) = 3.55; P < 0.001) (Figure 4(c)).

Figure 4.

Figure 4.

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Changes in neuronal activity with cooling and warming in human brain slices. (a) Three representative examples of action potential traces induced by a series of current injections in human cells during cooling/warming conditions. (b) Mean resting membrane potential, firing threshold, and spike duration. The number of neurons is shown at the base of each bar. (c) Mean input/output relationship during precooling (filled), cooling (blue), recovery from cooling (gray), warming (red), and recovery from warming (open). *P < 0.05 vs. precooling. Error bars indicate ± SEM.

We compared parameters in the precooling state, at 25℃, to determine whether the intrinsic properties were different between the FCD cortex (three patients) and the histologically normal MTLE cortex (four patients). There were no significant differences in the resting potentials (MTLE: −65.5 ± 1.9 mV, FCD: −65.1 ± 5.5 mV, P = 1.000, Mann–Whitney U test), the spike thresholds (MTLE: −31.1 ± 2.9 mV, FCD: −44.4 ± 6.1 mV, P = 0.064, Mann–Whitney U test), or the spike durations (MTLE: 1.751 ± 0.125 ms, FCD: 2.271 ± 0.309 ms, P = 0.355, Mann–Whitney U test).

Discussion

We investigated the intrinsic electrophysiological properties of neurons in brain slices. Changes in these properties were previously shown to contribute to hyperexcitability in a number of epilepsy models.24–26 Ca2+-dependent bursting24 and a marked increase in the amplitude of the spike after depolarization, particularly in low threshold bursts,25 appeared to contribute to the epileptogenicity of the hippocampus in the pilocarpine model of temporal lobe epilepsy. In addition, a reduced seizure threshold was found in a mouse model of Rett syndrome.27 We analyzed the neuronal properties of lateral cortices derived from three patients with FCD type I and four patients with MTLE. We found that the neurons in these patients had similar firing thresholds. FCD type I is characterized as the abnormal arrangement of normal neurons and MTLE samples were histologically normal; consequently, we did not expect epileptogenesis to arise from an abnormality in an intrinsic neuronal property, but by an abnormality at the network level.

We found that neuronal electrophysiological properties were affected by temperature. When we sorted the data according to temperature, i.e. 35℃, 25℃, and 15℃, the changes were serially augmented. The appearance of the first action potential to a small current injection was accelerated by lowering the temperature from 35℃ to 25℃, which reduced the threshold, and by lowering the temperature from 25℃ 15℃, which increased the resting potential. The resting potential increase during hypothermia was due to the reduced activities of the Na+-K+ pump and the temperature-sensitive, two-pore domain K+ channels (TREK-2 and TRAAK).28–30 However, these effects did not change the number of spikes. The number of spikes was determined by the width of the action potential, as observed when the temperature was lowered to 25℃ and 15℃ (Figures 2 and 4). Moreover, the spikes at 35℃ were sharper than those at 25℃; this finding suggested that the number of spikes at 35℃ could have been larger than the number at 25℃, if we could have maintained the neurons in superb condition at 35℃. However, superb conditions are unrealistic in vitro, because they require sufficient oxygen and energy supplies, as observed in vivo.

Thompson et al.31 reported that cooling to 30℃ from 37℃ decreased the number of action potentials in guinea pig hippocampal neurons. Their results indicated that activation of Ca2+-dependent K+ channels induced spike frequency adaptation by increasing the depth and duration of the AHP.32–35 However, in our study, at 15℃, there was no typical AHP form. The decrease in spike frequency we observed at 15℃ was not achieved by a decrease in AHP, but rather by a broadening of the action potential waveform. This broadening represented a potential mechanism for the interruption of epileptic discharges. Prolonged inactivation of the K+-induced Na+ channel was shown to cause similar spike broadening and retardation.36 Further investigation is needed to investigate the ion channels responsible for these temperature-dependent changes.

The antiepileptic mechanism of focal brain cooling is different from that of anticonvulsant Na+ channel blockers. Carbamazepine, topiramate,37 phenytoin,38 valproic acid,39,40 lamotrigine,41 and lacosamide42 bind to the Na+ channel inactivation gate to modulate the voltage-dependent Na+ current,43–45 and they do not affect membrane properties.46–48 The present study results suggested that the number of spikes decreased due to a broadening of spike duration and that the effect was unresponsive to increased current injection. Therefore, the broadening of spike durations could represent the mechanism by which brain cooling terminates epileptic discharge. This effect is likely to be additive to the effect of Na+ channel blockers in controlling epilepsy.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by Grants-in-Aid for Scientific Research B (S.N.) and Scientific Research C (H.K.).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

Sadahiro Nomura and Hiroyuki Kida contributed equally to this article.

Sadahiro Nomura operated on the patients and prepared the manuscript.

Hiroyuki Kida performed the experiments, analyzed data, and prepared the manuscript, including the figures.

Yuya Hirayama, Takao Inoue, and Hiroshi Moriyama performed the experiments.

Hirochika Imoto managed and operated on the patients.

Dai Mitsushima and Michiyasu Suzuki were responsible for the research design and supervision.

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