Cellular Mechanisms of Desynchronizing Effects of Hypothermia in an In Vitro Epilepsy Model
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- Motamedi, G.K., Gonzalez-Sulser, A., Dzakpasu, R. et al. Neurotherapeutics (2012) 9: 199. doi:10.1007/s13311-011-0078-5
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Hypothermia can terminate epileptiform discharges in vitro and in vivo epilepsy models. Hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy, and it is gaining ground as a potential treatment in patients with drug resistant epilepsy. However, the exact mechanism of action of cooling the brain tissue is unclear. We have studied the 4-aminopyridine model of epilepsy in mice using single- and dual-patch clamp and perforated multi-electrode array recordings from the hippocampus and cortex. Cooling consistently terminated 4-aminopyridine induced epileptiform-like discharges in hippocampal neurons and increased input resistance that was not mimicked by transient receptor potential channel antagonists. Dual-patch clamp recordings showed significant synchrony between distant CA1 and CA3 pyramidal neurons, but less so between the pyramidal neurons and interneurons. In CA1 and CA3 neurons, hypothermia blocked rhythmic action potential discharges and disrupted their synchrony; however, in interneurons, hypothermia blocked rhythmic discharges without abolishing action potentials. In parallel, multi-electrode array recordings showed that synchronized discharges were disrupted by hypothermia, whereas multi-unit activity was unaffected. The differential effect of cooling on transmitting or secreting γ-aminobutyric acid interneurons might disrupt normal network synchrony, aborting the epileptiform discharges. Moreover, the persistence of action potential firing in interneurons would have additional antiepileptic effects through tonic γ-aminobutyric acid release.
Direct cortical application of therapeutic modalities to treat epilepsy has gained significance in the last few years. This includes direct cortical stimulation, cortical delivery of antiepileptic drugs, and application of hypothermia to the brain. Physiological body temperature is an important determinant of neural functions. Therapeutic hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy by reducing the risk of apoptosis and early necrosis, cerebral metabolic rate, and the release of nitric oxide and free radicals. Early hypothermia may result in histological and functional improvement in animal models of perinatal brain injury . Changes in temperature have dynamic influences on hippocampal neural activities. It has been established that changes as little as 2° to 3°C in brain temperature affect brain functions and neuronal properties [2, 3]. Therefore, heat-sensitive molecular compounds seem to be essential for brain functions at physiological temperature. Furthermore, hypothermia has been shown to terminate experimentally induced seizure activity of in vitro and in vivo models of epilepsy within seconds, without causing acute or delayed injury to the cooled brain [4–7]. It is suggested by these studies that cooling could represent a new approach to seizure control in intractable focal epilepsies as an alternative to resective surgery, which may result in suboptimal seizure control and possible neurological deficits.
Temperature levels that must be achieved to obtain seizure suppression have also been studied. It has been found that cooling to <24°C is required to reversibly inactivate general neuronal function . Similarly, inhibition of experimental seizure activity requires cooling to 24°C, whereas complete cessation is only obtained at temperatures of 20° to 22°C. Furthermore, by comparing rapid cooling at rates of 2° to 5°C per second to slower cooling at rates of 0.1° to 1°C per second, we have shown that the cooling rate has significant influence on the efficacy of seizure suppression. We have found that very rapid cooling can abort epileptiform discharges with minor drops in temperature irrespective of the final temperature achieved, whereas slow cooling requires a lower range of temperatures (cooling to 21°-22°C caused a 90% reduction in event frequency and cooling to 14° to 15°C caused terminated discharges). Cooling is effective in diverse in vitro epilepsy models and in diverse brain regions (hippocampus, neocortex, dentate granule layer, and entorhinal cortex). Cooling also inhibits synaptic transmission, as reflected by the field excitatory postsynaptic potentials and population spikes, in parallel with its effect on spontaneous epileptiform activity . However, there is a paucity of data on the cellular mechanisms involved in the effects of hypothermia on epileptiform discharges.
Imaging a fluorescent synaptic marker with 2-photon microscopy, Yang et al.  have proposed a presynaptic mechanism through reduced transmitter release during rapid cooling. In addition, presence of temperature sensitive ion channels in the neurons may mediate the effects of cooling. In hippocampal cultures, the presence of transient receptor potential channel V (TRPV)-4 that is activated at temperatures within the physiological range (>27°-34°C) has been reported .
On the other hand, simultaneous dual recordings in hippocampal pyramidal cells and interneurons have shown a role for alternating activity of these neurons during epileptiform discharges in an in vitro model of epilepsy. It has been reported that in the presence of 4-aminopyridine (4-AP) and decreased magnesium, interneuron activity increased during interictal periods and entered into long-lasting depolarization block during ictal discharges, allowing sustained firing of pyramidal cells . These findings prompted the question of whether hypothermia regulates epileptiform activity by differentially affecting distinct cell types.
Here we report the results of our investigation of mechanisms underlying the effectiveness of hypothermia in an in vitro model of epilepsy. We have examined a number of potential target mechanisms involving TRPV channels, and selective effects on transmitting or receiving γ-aminobutyric acid (GABAergic) interneurons that alter network synchrony in hippocampal and extrahippocampal brain regions.
Hippocampal Slice Preparation
Coronal brain slices (250–300 μm) containing hippocampus and parts of adjacent cortex were prepared from postnatal day 14 to 27 C57BL/6 J mice. In some experiments glutamic acid decarboxylase-green fluorescent protein (GAD-GFP) mice were used to identify GABAergic interneurons Mice were sacrificed by decapitation in agreement with the guidelines of the American Veterinary Medicine Association (AVMA) Panel on Euthanasia and the Georgetown University Animal Care and Use Committee. These slices included the hippocampus, thalamus, entorhinal cortex, and neocortex. After cutting, the slices recovered for 30 minutes at 37°C and were continuously perfused with carbogen-bubbled (5 ml/min) artificial cerebrospinal fluid (aCSF) (in mM): NaCl (120), KCl (3.1), Na2HPO4 (1.25), NaHCO3 (26), dextrose (5.0), MgCl2 (1.0), and CaCl2 (2.0) 305 mOsm, pH 7.4. During experiments, slices were submerged in aCSF at various temperatures. Cells were visualized with an upright microscope (E600FN Nikon; Tokyo Japan) using infrared-differential interference contrast video microscopy. Patch pipettes were filled with the following (in μM): K-gluconate (145), EGTA (1.1), MgATP (5.0), and HEPES-KOH (10) to pH 7.2, 295 mOsm.
Hippocampal Cell Culture
Primary cultures were donated by Daniel T. Pak (Dept. of Pharmacology & Physiology Georgetown University, Washington DC, USA) and were prepared from rat embryos as previously described .
Patch electrodes (5–7 MΩ) were pulled (PP – 83; Narishige, Tokyo, Japan) from borosilicate glass capillary (Drummond, Broomall, PA). No fire polishing or Sylgard coating were used. Series resistance (15 MΩ), in whole cell configuration, was compensated and monitored for consistency throughout the experiment. Current and voltage signals at the head stage of the patch-clamp amplifier (Axopatch 1D; Molecular Devices Co., Sunnyvale, CA) were filtered at 2 kHz with a low-pass Bessel filter and digitized at 5 to 10 kHz using a personal computer equipped with Digidata 1322A data acquisition board and pCLAMP 10 software (both from Molecular Devices Co.). Off-line data analysis, curve fitting, and figure preparation were performed with Clampfit 10 software (Molecular Devices). Membrane input resistance was monitored with repetitive injection of a 240 ms-25 pA current pulse every 5 seconds. Patch-clamp recordings were made from 20 CA3 and 17 CA1/CA2 cells from 17 mice; statistical comparisons were performed with paired or independent t test, accordingly.
Simultaneous Dual Recording
An additional patch-clamp amplifier (Axopatch 1D) was used in dual recordings for whole cell voltage and current clamp, juxtacellular loose cell attached, and field recordings. Recordings were carried out from visually identified mouse hippocampal pyramidal cells and interneurons. Hippocampal neurons were visually localized in the CA1/2 and CA3 areas and were identified as pyramidal neurons or GABAergic interneurons on the basis of their anatomical location and action potential firing pattern. Spontaneous epileptiform discharges were recorded extracellularly with glass micropipettes (tip resistance, 2–5 MΩ) filled with aCSF. The field responses consist of field excitatory postsynaptic potentials and population spikes. Spontaneous discharges were recorded from the pyramidal layers of the hippocampal CA1-3 areas. Data from patch-clamp recordings from visually identified cells in CA1/2 and CA3 pyramidal cells and interneurons grouped across different subregions were compared during baseline and various stages of cooling.
Multi-Electrode Array Recording
Simultaneous field recordings through a 60-channel perforated multi-electrode array (MEA) (Multichannel Systems, Reutlingen, Germany) were carried out from the hippocampus and cortex as previously described . Off-line data analysis and figure preparation were performed with MCRack (Multichannel Systems, Reutlingen, Germany) and MEA tools, with routines written for MATLAB . Spike sorting with principal component analysis of single- or multi-unit discharges was done on each channel using Spike2 version 7.04 software (Cambridge Electronic Design Ltd, Cambridge, UK). These discharges of different morphology and amplitude are action potentials arising from one or more neurons. On each channel, these discharges were reviewed off-line based on amplitude and guided by visual inspection to quantify the effect of cooling in different cell types.
Induction of Epileptiform Activity
Spontaneous epileptiform discharges were elicited in CA3 or CA1/2 hippocampal regions by switching to a perfusion medium containing 50 μM 4-AP, according to methods previously described for rat brain slices . In some experiments, the perfusion solution contained reduced amount (0.5 mM) of Mg2 [11, 16].
Stock solutions of all drugs (all from Sigma) were diluted in the extracellular medium to the concentrations desired. Drugs were locally applied through a Y tube  modified for optimal solution exchange in brain slices . Bicuculline (25 μM), sodium channel blocker tetrodotoxin (TTX, 0.5-1 μM), 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor antagonist NBQX (5 μM), glycine receptor antagonist strychnine (5 μM), γ-aminobutyric acid (GABA) analogue, and selective GABAA receptor agonist Gaboxadol (10 μM), transient receptor potential (TRP) channel agonists menthol and capsaicin (12.5 μM), and antagonists capsazepine (10 μM) and ruthenium red (RR, 0.1, 1, and 10 μM) were applied in some experiments; TRPV4 agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 20 μM, Sigma) was dissolved in ethanol and diluted 1:1000 in aCSF.
Rapid Cooling and Rewarming
Rapid cooling was induced by switching from the 32o to 34°C perfusion solution to cold (8o to 14°C) aCSF of similar composition, which allowed the brain slice temperature to be reduced to as low as 11°C. However, the majority of experiments were performed at 18o to 22°C. Slice temperature was continuously monitored with a submerged miniature thermistor probe situated within the recording chamber adjacent to the slice (Warner Instruments, Hamden, CT). The temperature response time constant determined by rapidly switching the thermistor probe from 25° to 0°C was 0.5 seconds. Solution from the Y-tube applicator was kept at the same temperature as that in the perfusion bath. During the perforated multi-electrode array (pMEA) experiments, cooling was induced at a rate of 0.1° to 0.5°C per second by switching to cold aCSF to lower the temperature to <20°C, and warming was achieved at a slower rate through an inline heater. Although cooling was performed several times on each slice, only the first cooling experiment was counted and included in this study.
These results were then compared to the results from spike sorting of single- or multi-unit activity recorded through pMEA electrodes located in CA1 and CA3 areas. Units with basal frequencies higher than 5 Hz pooled from both areas were identified as putative interneurons. The putative interneurons showed higher baseline frequency rates of multi-unit discharges recorded on the pMEA (29 ± 24 Hz), compared to the direct recordings from visually identified interneurons (9.1 ± 2.5 Hz). The multi-unit discharge frequency increased toward the peak of the cooling effect, although this change was not statistically significant at different time zones (Fig. 8B, bottom).
In this work, we have attempted to further characterize the effects of hypothermia on neuronal activity and its cellular and network mechanisms. Intracellular current clamp recordings in CA1 and CA3 pyramidal neurons showed that hypothermia induced depolarization or hyperpolarization, or a combination of both, that were accompanied by a blockade of action potential firing. The depolarization was eliminated by TTX in CA1/CA2 neurons. These effects of hypothermia observed with whole cell recordings in mouse hippocampal slices expand what was previously reported in rats with field recordings .
It has been hypothesized that a loss of function caused by alterations in sodium channel gating properties during hypothermia could interrupt neuronal depolarization and abort the epileptiform discharges [5, 22]. Indeed, we observed blockade of action potentials in hippocampal pyramidal neurons that was associated with either depolarization or hyperpolarization. However, in the presence of TTX, cooling only caused hyperpolarization associated with an outward current, raising the possibility of closure of a tonically active inward current triggered by warmer temperatures in the recorded neuron. This was further confirmed by remarkable increases in membrane input resistance in response to hyperpolarizing and depolarizing current injections.
As TRPV4 has been shown to be expressed in hippocampal neurons in primary culture  and is activated by warm temperatures (>30°C), this cationic channel was an ideal candidate to mediate the effect of cooling. However, its selective agonist 4α-PDD failed to induce currents during cooling in slices, which suggests that the expression of TRPV4 channel is restricted to the culture model. Our results also excluded activation of TRPV1, as agonist capsaicin or antagonist capsazepine failed to activate any currents in CA3 neurons at 32°C.
Similarly, the possible involvement of TRPM8 channels, which are activated by cooling with a threshold of ~25°C , was ruled out. The results with RR, which blocks several TRPV-mediated responses , but failed to alter cooling responses in hippocampal neurons, provides further evidence against the possible involvement of temperature-sensitive TRP channels in aborting epileptiform discharges.
A common effect of cooling in all neurons tested is an increase in membrane input resistance. Previous studies have reported temperature-dependent increases in input resistance [24, 25]. The underlying mechanisms of thermal-induced changes in resistance are not very clear, but one intriguing suggestion that still awaits demonstration points at the alteration of membrane structure by temperature .
The results of dual-patch clamp recordings during cooling at baseline in the presence of 4-AP showed a significant degree of synchrony between distant CA1 and CA3 pyramidal neurons, and to a lesser extent between principal cells and interneurons. The synchrony and rhythmic action potential discharges in CA1 and CA3 pyramidal neurons were disrupted by hypothermia. Cooling also blocked rhythmic discharges in interneurons, but did not abolish action potential firing. This desynchronizing effect would last for the duration of cooling, but was reversible in all cell types on rewarming. The differential effects of cooling on action potential firing between pyramidal neurons and interneurons was not due to the presence of 4-AP, as it was observed on depolarizing current injections even in the absence of 4-AP.
The pMEA recordings revealed independent interictal and ictal-like discharges in the cortex and hippocampus, possibly due to the coronal slicing orientation. Hypothermia reversibly abolishes 4-AP-induced epileptiform discharges in all hippocampal and cortical sites while preserving spontaneous multi-unit firing. Spike sorting of the multi-unit discharges recorded by pMEA showed a consistent number of cells with higher firing rates being differentially affected by cooling. In contrast to the cessation of epileptiform discharges in response to cooling, as seen with local field potentials, putative fast spiking interneurons did not show significant reductions in frequency. This suggests a different threshold between interneurons and pyramidal neurons in response to cooling, as confirmed with patch-clamp recordings.
The underlying mechanisms for the preferential effects of cooling on pyramidal cells versus interneurons might be related to differences in intrinsic action potential firing mechanisms in these neurons, varying thresholds in voltage-gated channels, or differences in synaptic afferents to these cells. However, the differential effects of cooling, seen even in the absence of 4-AP when spontaneous synaptic activity is considerably less pronounced, suggests that the unique spiking properties of interneurons are probably the key. Indeed, several studies  have shown that hippocampal interneurons are rich in expression of the NaV1.1 subunit variant of voltage-gated Na2+ channels, whereas pyramidal neurons express NaV1.6 subunit. Although detailed biophysical studies of temperature sensitivity of these distinct channels are not available, point mutations of NaV1.1 channels have been clearly implicated in the pathogenesis of clinical conditions, such as generalized epilepsy with febrile seizures plus .
The role of brief high-frequency discharges in synchronizing neuronal activity has been recognized. It has been shown that GABAergic neurons can play both antiepileptic and ictogenic roles in different networks in the brain by synchronizing neural networks . Therefore, this finding raises the possibility that the observed decrease in fast interneuron synchronous discharges on cooling might contribute to the antiepileptic activity of cooling by disturbing the normal synchrony between different cell types and networks in the hippocampus, hence aborting epileptiform discharges within in vitro models of ictogenesis. Furthermore, the persistence of action potential firing in interneurons would have an additional antiepileptic effect through tonic GABA release, and cooling will induce prolongation of decay of phasic inhibitory synaptic current, similar to the effect of benzodiazepines and barbiturates .
The effect of high temperature in triggering seizures has been known in the clinical setting. The decay, and to a lesser extent, the amplitude of GABAA-mediated inhibitory synaptic currents are very sensitive to temperature changes. Indeed, hyperthermia by decreasing charge transfer at the synapse acts as a convulsant agent . Although the exact ictogenic mechanism in childhood febrile seizures is unclear, it has been associated with temperature-dependent changes in synaptic efficacy consequent to mutations involving the GABAA γ2 subunit [29, 30]. Our findings of distinct effects of cooling on interneurons suggest that the anti-seizure effect of hypothermia is likely to be modulated through GABA receptors. Further studies will help define the subtypes of interneurons that may potentially play a pivotal role in the therapeutic effects of cooling.
Despite accumulating in vitro and in vivo data, the clinical use of implanted cooling devices as a treatment for epilepsy and other neurological disorders is currently hampered by technical problems. In particular, a safe and efficient method for heat transfer and dissipation, as well as an effective method of power consumption seem to be among the major obstacles. With further elucidation of cellular mechanisms involved, it might be plausible to design clinical studies safe enough to test the potential therapeutic effects of cooling in humans.
This work was supported by funding from the CONACyT of the Mexican Government and the National Science Foundation Partnerships for International Research and Education (A.G.S.), the National Science Foundation Partnerships for International Research and Education (PIRE) grant (S.V., and A. G.S.), and in part by the Luce Foundation (R.D.). The authors do not have any real or perceived conflict of interest. Full conflict of interest disclosure is available in the electronic supplementary material for this article.