Regulation of epileptiform activity by two distinct subtypes of extrasynaptic GABAA receptors
© Sun et al.; licensee BioMed Central Ltd. 2013
Received: 19 March 2013
Accepted: 20 April 2013
Published: 1 May 2013
GABAergic deficit is one of the major mechanisms underlying epileptic seizures. Previous studies have mainly focused on alterations of synaptic GABAergic inhibition during epileptogenesis. Recent work suggested that tonic inhibition may also play a role in regulating epileptogenesis, but the underlying mechanism is not well understood.
We employed molecular and pharmacological tools to investigate the role of tonic inhibition during epileptogenesis both in vitro and in vivo. We overexpressed two distinct subtypes of extrasynaptic GABAA receptors, α5β3γ2 and α6β3δ receptors, in cultured hippocampal neurons. We demonstrated that overexpression of both α5β3γ2 and α6β3δ receptors enhanced tonic inhibition and reduced epileptiform activity in vitro. We then showed that injection of THIP (5 μM), a selective agonist for extrasynaptic GABAA receptors at low concentration, into rat brain also suppressed epileptiform burst activity and behavioral seizures in vivo. Mechanistically, we discovered that low concentration of THIP had no effect on GABAergic synaptic transmission and did not affect the basal level of action potentials, but significantly inhibited high frequency neuronal activity induced by epileptogenic agents.
Our studies suggest that extrasynaptic GABAA receptors play an important role in controlling hyperexcitatory activity, such as that during epileptogenesis, but a less prominent role in modulating a low level of basal activity. We propose that tonic inhibition may play a greater role under pathological conditions than in physiological conditions in terms of modulating neural network activity.
KeywordsExtrasynaptic GABAA receptor, α5 subunit δ subunit Tonic inhibition Epileptogenesis Epileptiform activity THIP Seizure behavior
Many antiepileptic drugs are targeting GABAergic synaptic transmission, but may cause certain side effects [1, 2]. GABAA receptors (GABAA-Rs) are abundant not only at synaptic sites, but also at extrasynaptic sites. Synaptic GABAA-Rs have low affinity for GABA, are activated in a transient manner by GABA released form presynaptic vesicles, and primarily mediate phasic inhibitory transmission. In contrast, extrasynaptic GABAA-Rs exhibit high affinity for GABA, are persistently activated by low concentration of ambient GABA, and mediate tonic inhibition [3–5]. There are two distinct subtypes of extrasynaptic GABAA-Rs in the brain, one contains the α5 subunit [6–10] and the other contains the δ subunit [11–13]. The α5-GABAA-Rs are sensitive to a specific inverse agonist L-655,708 [14, 15], while the δ-GABAA-Rs are typically insensitive to benzodiazepine [16, 17] but highly sensitive to THIP (gaboxadol) [18, 19].
Functional deficit of synaptic GABAergic inhibition plays an important role in the etiology of epilepsy [2, 20–22]. Recent studies revealed a possible role of tonic inhibition in modulating epileptic seizures [23, 24]. A significant reduction of GABAA-R α5 and δ subunit level has been reported in the hippocampus of animals with temporal lobe epilepsy (TLE) [8, 25, 26]. Interestingly, the decrease of δ subunit may be compensated by an increase of α4 and γ2 subunits [26–28]. Mutations in the δ subunit of GABAA-Rs have been mapped in human epilepsy patients [29, 30]. Increased δ subunit level during diestrus stage of ovarian cycle has been associated with less seizure activities in kainic acid-induced epilepsy models . However, in the pyramidal neurons of hippocampal CA1 region, the α5 GABAA-R mediated tonic current was reduced but overall tonic inhibition was not changed or even increased in pilocarpine epilepsy model . Furthermore, enhanced tonic inhibition in thalamocortical neurons was reported to induce absence seizure , suggesting that different tonic inhibition may play different roles in different brain regions.
Here we investigated the functional role of two distinctly different subtypes of extrasynaptic GABAA-Rs in hippocampal epileptogenesis. We demonstrated that enhancing tonic inhibition by overexpressing either the α5β3γ2 or α6β3δ extrasynaptic GABAA receptors significantly inhibited the formation of epileptiform activity in hippocampal cultures. Furthermore, in vivo injection of selective extrasynaptic GABAA-R agonist THIP also inhibited epileptiform bursting activity in anesthetized rats and seizure behaviors in freely moving rats. Interestingly, low concentration of THIP did not affect basal level of neuronal activity, but significantly suppressed higher frequency neuronal firing. Therefore, our data suggest that tonic inhibition mediated by extrasynaptic GABAA-Rs may play a more prominent function in pathological conditions such as during epileptogenesis.
Molecular enhancement of tonic GABA currents after overexpressing α5β3γ2 GABAA receptors
Tonic inhibition mediated by the α5β3γ2 receptors suppresses epileptiform activity
Inhibition of epileptiform activity by the α6β3δ receptors
Tonic inhibition on in vivo epileptic seizures
Besides epileptiform activity, we further studied whether THIP can directly modulate CTZ-induced seizure behavior in freely moving rats . CTZ was injected repeatedly each day (0.25 μmol i.c.v. for 3 consecutive days, total dose of 0.75 μmol) to induce seizure behavior without or with a pre-injection of THIP. The administration of THIP (5 or 10 mg/kg, i.p.) at 10 min before CTZ injection dose-dependently attenuated the convulsant seizures induced by CTZ. The seizure score was 4.5 ± 0.3 (n = 6) after CTZ injection alone, and significantly reduced to 2.6 ± 0.7 (n = 7) in THIP-preinjected (10 mg/kg) animals (p < 0.05; Figure 5B). The lower dose of THIP pretreatment (5 mg/kg) also reduced seizure score but not reaching statistical significance. Therefore, THIP may be used as a potential anticonvulsant drug to suppress seizure behaviors in living animals.
Tonic inhibition and basal GABAergic neurotransmission
Mechanism of tonic inhibition in modulating neuronal activity
In this study, we have demonstrated that two distinct subtypes of extrasynaptic GABAA-Rs both play an important role in regulating the formation of epileptiform activity in hippocampal cultures. Enhancing tonic inhibition in living animals in vivo also modulates epileptiform activity and behavioral seizures in a dose-dependent manner. More importantly, we demonstrated that tonic inhibition potently inhibits high frequency action potentials under stimulated conditions but not at basal low frequency firing condition, suggesting a novel mechanism of tonic inhibition in regulating neural network activity.
Extrasynaptic GABAA-Rs regulate epileptogenesis
Due to its continuous activation by ambient GABA, the charge transfer of tonic currents mediated by extrasynaptic GABAA-Rs have been estimated to be several folds more than phasic currents mediated by synaptic GABAA-Rs [4, 45, 46]. Tonic GABA conductance controls the overall gain of neuronal input–output [5, 45–48]. Therefore, when GABAA receptor α5 and δ subunits were found significantly reduced in the hippocampus of animal TLE models [8, 25, 49, 50], it was realized that downregulation of tonic inhibition might have contributed to epileptogenesis. Consistently, our previous work has also demonstrated that tonic GABA inhibition was downregulated after chronic epileptogenic stimulation in cultured hippocampal neurons . However, later studies found that despite a reduction of α5 and δ subunit expression in epileptic mice, tonic inhibition in the hippocampus was largely maintained or even increased, possibly mediated by increased expression of α4γ2-containing GABAA-Rs [26, 28, 32]. Thus, tonic inhibition might have undergone homeostatic changes during and after epileptogenesis . Our current study demonstrated that enhancing tonic inhibition by increasing the expression level of either α5β3γ2 or α6β3δ receptors can effectively suppress epileptiform activity. We have further demonstrated that seizure behaviors are attenuated by enhancing tonic inhibition in vivo. Based on previous and our own studies, we attribute an important role to tonic inhibition in modulating hippocampal epileptogenesis: enhancing tonic inhibition will inhibit epileptiform activity, while reducing tonic inhibition will increase the susceptibility of epileptic seizures [31, 44].
Overexpression of extrasynaptic GABAA-Rs regulates epileptiform activity
While THIP has been used previously to modulate epileptiform activity, the target receptors were usually not specifically identified because higher concentrations of THIP might activate different combinations of synaptic and extrasynaptic GABAA-Rs. Our current study provides more direct evidence on extrasynaptic regulation of epileptogenesis by demonstrating that overexpression of both α5β3γ2 and α6β3δ receptors can effectively attenuate epileptiform activity. We showed that neurons transfected with both α5β3γ2 and α6β3δ receptors had enhanced tonic currents compared to control neurons. Mutations in the δ subunit of GABAA receptors have been mapped in human epilepsy patients [29, 30], indicating the clinical relevance of δ-GABAA receptors. Our molecular expression studies suggest that extrasynaptic GABAA-Rs may be a potential therapeutic target for developing antiepileptic drugs to treat TLE.
THIP regulation of neuronal activity
Previous studies have reported that THIP may inhibit neuronal activity [52–56]. However, these studies used concentrations much higher than our current work. It is known that high concentration of THIP may directly activate γ2-containing synaptic GABAA-Rs [42, 57, 58]. In this study, we used a low concentration of THIP (5 μM) that did not affect mIPSCs, indicating that at this concentration THIP did not activate synaptic GABAA-Rs. Correspondingly, we found that 5 μM THIP did not affect basal neuronal firing in the majority of neurons tested. More importantly, we discovered a strong inhibitory effect of THIP on elevated neuronal activity induced by both CTZ and KA. It is possible that elevated neuronal activity may induce substantial release of GABA , which will act together with THIP to enhance tonic inhibition and reduce neuronal activity. Another possibility is that tonic current may be outward rectifying at depolarized membrane potential , making the effect of THIP more potent when neurons are hyperexcitatory. Our discovery of the preferential inhibition of THIP on elevated activity makes it an ideal candidate for anticonvulsant drug, because it may have less side effects comparing to those affecting basal neural activity.
We employed both molecular and pharmacological tools to demonstrate that tonic inhibition modulates epileptiform activity both in vitro and in vivo. The overexpression of both α5β3γ2 and α6β3δ receptors inhibited the formation of epileptiform activity in hippocampal neurons, establishing unambiguously a solid ground for extrasynaptic modulation of epileptogenesis. Furthermore, we discovered a more prominent role of tonic inhibition in inhibiting hyperexcitatory activity rather than low frequency basal activity, suggesting that extrasynaptic GABAA-Rs are ideal drug targets for developing anti-convulsant drugs that may specifically act against epileptiform activity without much side effect on normal brain functions.
Primary neuronal culture
Primary hippocampal neurons were prepared from embryonic day 18 Sprague–Dawley rat embryos of either sex, similar to our previous work with modifications . Briefly, after dissection of the hippocampi, the tissue was rinsed in cold HBS and then digested with 0.05% trypsin-EDTA for 20 min at 37°C, followed by trituration with pipettes in the plating medium (DMEM with 10% FBS and 10% F12). After rinsing for twice, cells were counted and plated onto coverslips precoated with 0.1 mg/ml poly-D-lysine (Sigma). After culturing for 1 day, media were changed into neuronal culture media (neurobasal media containing 2 mM GlutaMAX™-I Supplement and 2% B-27). AraC (1 μM, Sigma) was added 6–8 days after plating, and cells were fed twice weekly thereafter and maintained at 37°C and in 5% CO2 incubators. Trypsin-EDTA, DMEM, FBS, F12, Neurobasal media, GlutaMAX™-I Supplement and B-27 were purchased from Invitrogen Corporation. Some experiments were also performed using mouse hippocampal cultures.
Calcium-phosphate transfection was performed similar to the protocol previously described . Neurons were transfected at 10 days in vitro (DIV). The plasmids of rat α5, α6, β3, γ2, δ subunits of GABAA receptors (gifts from Drs. Robert Macdonald, Matthias Kneussel, and Dr. Bernhard Luscher) were co-transfected with pEGFP or mCherry (Clontech). Transfection with EGFP or mCherry alone served as controls. Most of the experiments were performed around 2 weeks of culture unless otherwise indicated.
Electrophysiological recordings in cultured hippocampal neurons
Whole-cell recordings were performed in current- or voltage-clamp mode using a MultiClamp 700B amplifier (Axon Instruments). Patch pipettes were pulled from borosilicate glass (Sutter Instrument, BF150-86-10) and fire polished (4–6 MΩ). The recording chamber was continuously perfused with a bath solution consisting of (mM): 128 NaCl, 30 Glucose, 25 Hepes, 5 KCl, 2 CaCl2, 1 MgCl2, pH 7.3 adjusted with NaOH. The pipette solution for recording action potentials and mEPSCs contained (mM): 125 K-gluconate, 10 KCl, 5 EGTA, 10 Hepes, 10 Tris-phosphocreatine, 4 MgATP, 0.5 NaGTP, pH 7.3 adjusted with KOH. For tonic GABA currents and GABA-induced whole-cell currents, patch pipettes were filled with (mM): 135 KCl, 10 Tris-phosphocreatine, 2 EGTA, 10 Hepes, 4 MgATP, 0.5 NaGTP, pH 7.3 adjusted with KOH. Liquid junction potentials were always corrected before forming giga-ohm seal. The series resistance was typically 10–20 MΩ and partially compensated by 30-50%. Data were acquired using pClamp 10.2 software (Axon Instruments), sampled at 2–10 kHz, and filtered at 1 kHz. Off-line analysis was done with Clampfit 10.2 software (Axon Instruments). Miniature events were analyzed using Mini Analysis software (Synaptosoft). Large depolarization shift resembling paroxysmal depolarization shift is defined here as ≥ 10 mV depolarization and ≥ 300 ms in duration. An epileptiform burst is defined by at least five consecutive action potentials overlaying on top of the large depolarization shift. When quantifying the percentage of neurons showing epileptiform activity, the criterion is at least two epileptiform bursts occurring during 10 min of recording. All of the drugs used were freshly diluted in bath solution to their final concentrations before experiments.
Electrophysiological recordings in anaesthetized rats
Adult male Sprague–Dawley rats weighing between 250–350 g were maintained on an ad libitum feeding schedule and kept on a 12 hr on/off light cycle. During electrophysiological study, rats were anesthetized with urethane (1.2 g/kg, i.p.) and the level of anesthesia was assessed by the absence of a withdrawal reflex, and additional anesthetic (urethane, 0.2-0.6 mg/kg, i.p.) was administered as necessary. Body temperature was maintained at 37 ± 0.5°C with a Harvard Homoeothermic Blanket (Harvard Apparatus Limited, Kent, UK). At the end of experiments, animals were killed with an overdose of urethane. All animal experiments were approved by the local committee of Laboratory Animals, Fudan University and carried out in accordance with Chinese National Science Foundation animal research regulation. Animal preparation was similar to previously reported [36, 37, 39]. Briefly, all the animals had their lateral tail vein cannulated for drug administration and then mounted in a stereotaxic frame. An incision was made in the midline of the head to expose the top part of the skull for the implantation of i.c.v. guide cannula (22GA, Plastics One, USA) into the lateral ventricle (0.3 mm posterior to bregma, 1.3 mm lateral to the midline, and 4 mm below the skull surface), and then secured by the dental cement. For recording and stimulating, a large burr hole was made in the left side of the incised skull above the hippocampal area, and the dura was pierced and removed. A concentric bipolar stimulating electrode (Harvard Apparatus) was placed close to the CA3 region (3.8-4.5 mm posterior to bregma, 3.5-4.0 mm lateral to the midline, and 3.0-3.8 mm below the brain surface) in order to stimulate the Shaffer collateral pathway. For recording in the CA1 pyramidal cell layer, a tungsten electrode (0.5 MΩ, WPI, USA) was placed 3.5-4.2 mm posterior to bregma, 2.0-3.0 mm lateral to the midline. The depth of the recording electrode was approximately 2.0-2.5 mm below the brain surface as determined by the sudden change of electrical noise and the shape of the evoked field excitatory postsynaptic potentials (fEPSPs) and population spike (PS). For stimulation, a constant current generator passed a square-wave pulse (0.2 ms in duration) through the stimulating electrode (test pulse) and the stimulation frequency was set at once per minute. The electrophysiological signals were amplified and filtered (0.3-3 kHz) using a NeuroLog System (Digitimer Ltd., Hearts, UK) and visualized and stored in a PC computer through an A-D converter, CED 1401 micro (Cambridge Electronic Design, Cambridge, UK). After both electrodes were in the right place, the fEPSPs and PS were monitored for at least 30 min until a stable recording was achieved. Following a 30 min baseline recording, CTZ (5 μmol, 5 μL) was administered i.c.v. via the pre-implanted guide cannula into the left lateral ventricle. Pharmacologically induced seizure-like activity was monitored after CTZ injection by observing the change of the evoked potentials transforming from single PS into a multi-peaked display, and spontaneous seizure burst activity in CA1 pyramidal neurons [37, 39, 61]. After the epileptiform burst activity was stable for at least 30 min, THIP (4 mg/kg in 1 mL/kg) or vehicles were delivered through the cannula pre-implanted in the lateral tail vein. To confirm correct placement of the electrode and cannula, the brain was taken for histological validation of the injection and recording/stimulating sites. Epileptiform activity within CA1 pyramidal cells was analyzed offline using Spike2 software (an analyzing program for CED 1401, Cambridge Electronics, UK) and specific scripts designed for this study with Spike2. The highly synchronized bursting activity was defined as having high frequency multiple high amplitude spikes (>0.5 mV) with an initial interspike interval of less than 0.1 s, a minimum of 5 spikes, and burst duration over 1 s .
Behavioral test in freely moving rats
CTZ induced seizure behavioral test was carried out similarly as previously reported . Briefly, under general anesthetics with sodium pentobarbital (60 mg/kg, i.p.), a guide cannula was pre-implanted into left lateral ventricle (0.3 mm posterior to bregma, 1.3 mm lateral to the midline, and 4 mm below the skull surface) at least 5 days before the behavioral test. Cannula-implanted animals were randomly divided into following experimental groups: 1) CTZ group: 0.25 μmol (i.c.v.) for one injection per day, three consecutive days; 2) THIP + CTZ group one: 5 mg/kg (i.p.) THIP + 0.25 μmol (i.c.v.) CTZ for one injection per day, three consecutive days; 3) THIP + CTZ group two: 10 mg/kg (i.p.) THIP + 0.25 μmol (i.c.v.) CTZ for one injection per day, three consecutive days. All behavioral tests were carried out between 2:00 pm and 7:00 pm. The animals were first placed in a plastic cage and acclimatized for at least half an hour before experiments. Before and after drug injection, animal behavior was continuously monitored for a period of 1 and 3 hours with video recording, respectively. Behavioral seizures were scored using 5-graded Racine Score system . Briefly, Racine score I, facial clonus; score II, head nodding; score III, unilateral forelimb clonus; score IV, rearing with bilateral forelimb clonus; score V, rearing and falling (loss of postural control).
Group data were expressed as mean ± SEM. Across different groups of data, statistical significance between means was determined using one-way ANOVA with Tukey HSD post hoc analysis. Comparison within a group used a paired or unpaired t test (GraphPad Prism, GraphPad Software Inc.). Pearson Chi-Square test was used for statistical analysis of percentage (SPSS). Significance level was set at p < 0.05.
Drugs and solutions
Cyclothiazide (CTZ) and L655708 were purchased from Tocris (Northpoint, Bristol). THIP (4,5,6,7-tetrahydroisoxazolo[4,5-c] pyridine-3-ol and urethane (25% in distilled water) were purchased from Sigma Aldrich Chemical Co. (Poole, Dorset).
This project was supported by NIH grants NS054858 and MH083911 to G.C., and grants from National Science Foundation of China 31129003 to G.C. and Y.W., and 81171224 to Y.W.
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