Abstract

A missense mutation of the γ2 subunit of the γ-aminobutyric acid A (GABAA) receptor has been linked to an inherited human generalized epilepsy. As synaptic inhibition in the human brain is largely mediated by the GABAA receptor, we tested the hypothesis that the GABRG2(R43Q) mutation alters cortical excitability. Fourteen subjects affected by the GABRG2(R43Q) mutation (5 males, mean age: 44 ± 15 years) and 24 controls (11 males, mean age: 38 ± 11 years) were studied with transcranial magnetic stimulation (TMS). To assess the specificity of the effect of the mutation, 4 additional family members unaffected by the GABRG2(R43Q) mutation (2 males, mean age: 41 ± 16 years) were included. Subjects affected by the GABRG2(R43Q) mutation demonstrated reduced net short-interval intracortical inhibition and increased intracortical facilitation assessed with paired-pulse stimulation. Subjects with the mutation had similar motor thresholds to controls both at rest and with weak voluntary activation. No significant differences were noted between groups in the cortical silent period. Our findings provide in vivo evidence for increased intracortical excitability in subjects affected by the GABRG2(R43Q) mutation. These findings are also likely to represent an important clue to the mechanisms linking this gene defect and the epilepsy phenotype.

Introduction

Fast synaptic inhibition in the human cerebral cortex is predominately mediated by the GABAA receptor, a ligand-gated chloride channel comprising 5 individual subunits (McCormick 1989). Activation of γ-aminobutyric acid A (GABAA) receptors induces a hyperpolarizing response that reduces cellular excitability, whereas pharmacological blockade results in highly synchronized bursts of action potentials (Chagnac-Amitai Connors 1989; Olsen and Avoli 1997).

Impairment of GABAA receptor function has been implicated in the pathogenesis of epilepsy, and recently, gene mutations affecting specific subunits of the GABAA receptor have been identified in familial forms of generalized epilepsy (Baulac et al. 2001; Wallace et al. 2001; Bianchi et al. 2002; Bowser et al. 2002; Cossette et al. 2002; Crunelli and Leresche 2002; Harkin et al. 2002; Kananura et al. 2002; Dibbens et al. 2004; Macdonald et al. 2004). In a large pedigree with childhood absence epilepsy and febrile seizures, we identified a mutation in the γ2 subunit of the GABAA receptor resulting in substitution of glutamine for arginine, GABRG2(R43Q), in the N-terminus (Wallace et al. 2001). The γ2 subunit of the GABAA receptor regulates key kinetic properties of the receptor, is involved in receptor assembly and clustering and forms part of the benzodiazepine-binding site (Smith and Olsen 1995; Essrich et al. 1998; Haas and Macdonald 1999).

In vitro, expression studies of the GABRG2(R43Q) mutation have revealed loss of GABAA receptor function via a number of mechanisms (Bowser et al. 2002; Macdonald et al. 2004; Sancar and Czajkowski 2004; Kang et al. 2006). Hence, the mutation provides a unique opportunity to study GABAergic influences on brain function. To date, however, the functional consequences of the GABRG2(R43Q) mutation in the living human brain have remained largely unexplored.

One tool for studying the physiological effects of GABRG2(R43Q) mutation is transcranial magnetic stimulation (TMS), which is sensitive to a range of physiological and neurochemical processes underlying cortical excitability (Rothwell 1997). The motor threshold (MT) measured with TMS reflects neuronal membrane excitability and is increased by drugs blocking voltage-gated Na+ or Ca++ channels but is unaltered by GABAergic medication (Ziemann et al. 1996a). The cortical silent period (CSP), transient inhibition of voluntary contraction by TMS, is partly mediated at a cortical level. Evidence for the involvement of the GABAergic system in the CSP is conflicting with some studies showing an effect of intrathecal baclofen, a GABAB receptor agonist (Siebner et al. 1998), no effect of oral and intravenous baclofen (Inghilleri et al. 1996; Ziemann et al. 1996a; McDonnell et al. 2006), and CSP lengthening with the GABA reuptake inhibitor tiagabine (Werhahn et al. 1999). Paired-pulse paradigms have been used to study intracortical inhibition and facilitation. In these paradigms, a subthreshold conditioning stimulus reduces the amplitude of the motor evoked potential (MEP) produced by a succeeding suprathreshold test stimulus at short-interstimulus intervals (ISIs) (1–5 ms) reflecting intracortical inhibition. At longer intervals (6–20 ms) the test MEP is augmented, reflecting intracortical facilitation (Kujirai et al. 1993). Benzodiazepines, positive allosteric modulators of the GABAA receptor, increase intracortical inhibition and depress facilitation (Di Lazzaro et al. 2000; Ziemann et al. 1996a).

Under normal conditions, the γ2 subunit is expressed in brain regions that modulate the excitability of the motor cortex via thalamocortical (Deschenes et al. 1979), interhemispheric (Asanuma and Okamoto 1959), and corticocortical circuits. We have shown that the GABRG2(R43Q) mutation produces changes in benzodiazepine receptor binding in brain regions which influence cortical excitability, with the frontal regions being particularly affected (Fedi et al. 2006). Consequently, in this study, we investigated the effect of the GABRG2(R43Q) mutation on human cortical excitability using TMS.

Methods

Subjects

We studied 24 normal controls (11 male, mean age: 38 ± 11 years, range: 21–71 years) and 14 subjects with the GABRG2(R43Q) mutation (5 male, mean age: 44 ± 15 years, range: 18–71 years). Four additional family members without the mutation (2 male, mean age: 41 ± 16 years, range: 31–65 years) were examined to exclude a family effect unrelated to the GABRG2(R43Q) mutation. The MT in 3 subjects with the mutation and 5 controls was too high to allow completion of the paired-pulse TMS protocol. All participants were right handed by the Oldfield (1971) handedness questionnaire except for 4 controls and 3 individuals with the GABRG2(R43Q) mutation (Wallace et al. 2001; Marini et al. 2003). All subjects underwent urine testing for benzodiazepines.

Of the 14 subjects with the mutation, none were on medication or had active epilepsy at the time of the study. Seven had a past history of epilepsy: 2 had childhood absence epilepsy, typical 3-Hz spike-wave activity on electroencephalography (EEG) and seizure offset by the age of 15 years; 3 had febrile seizures, a normal scalp EEG (2) or left temporal spikes (1) and seizure offset before the age of 7 years; 1 had generalized tonic-clonic seizures associated with bisynchronous polyspike and slow waves; and 1 had an unclassified form of epilepsy and a normal EEG. All subjects gave written consent, and the protocol was approved by the local Human Research Ethics Committee.

Transcranial Magnetic Stimulation

We used a figure-of-eight–shaped coil (outer diameter of each loop 70 mm; peak magnetic field 2.4 T) and 2 Magstim 200 magnetic stimulators connected by a Bistim module (The Magstim Company, Dyfed, UK). Conditioning and test stimuli were delivered through the same coil that was placed tangentially over the dominant hand motor cortex with the handle pointing back and laterally 45° away from the midline at the optimal site for the activation of the abductor pollicis brevis (APB). This is thought to be the best position for activating the pyramidal cells transsynaptically and preferentially elicits late I-waves (Brasil-Neto et al. 1992; Sakai et al. 1997). The direction of current induced in the brain was anterior to posterior.

We recorded the surface electromyogram (EMG) from the contralateral APB with disposable disc electrodes in a tendon-belly arrangement. Band-pass filtering (10–5000 Hz) was used. Auditory EMG feedback was given to ensure complete, voluntary relaxation of the target muscles. We measured: 1) resting motor threshold (RMT)—the minimum intensity eliciting a reproducible MEP at rest with peak-to-peak amplitude ≥100 μV on 5/10 consecutive trials at 1% increments and a gain of 200 μV/div; 2) silent period threshold—the lowest intensity at which at least 50% of the trials produced a silent period ≥10 ms duration while the subject performed a maximal contraction; 3) duration of the silent period at a stimulus intensity of 1.2 × silent period threshold—the time from the beginning of the magnetic stimulus to the resumption of uninterrupted EMG activity at a display gain of 500 μV/div; 4) duration (measured from the onset of the signal to the end of the slow terminal phase) and latency of the MEP; 5) the number of phases of the MEP—the number of times the signal crosses the baseline minus one; 6) the mean evoked response amplitude for unconditioned stimulus (μV); 7) the mean area of the rectified EMG activity for unconditioned stimulus (μV); and 8) intracortical inhibition and facilitation measured using a paired-pulse paradigm at randomly presented ISIs of 2, 3, 4, 5, 6, 7, 10, and 15 ms (Kujirai et al. 1993). We used a conditioning stimulus of 0.8 × RMT and a test stimulus of 1.2 × RMT for 10 trials at each ISI and 10 trials without a preconditioning stimulus. The level of background EMG activity was constantly monitored on the oscilloscope with auditory and visual feedback given to the subjects to ensure that the resting state was achieved. The amplitude ratio between the mean evoked response for conditioned and unconditioned stimuli was calculated at each ISI using both peak-to-peak amplitudes and MEP areas on single-trial rectified data.

Statistical Analysis

MTs and silent period duration were compared using 1-way analysis of variance (ANOVA). Statistical analysis of the intracortical inhibition and facilitation data was performed with analysis of variance for repeated measurements. Repeated-measures (within subject) ANOVA was used to assess the effect of group (controls and GABRG2(R43Q) mutation positive) and ISI on the size of the conditioned MEP. Group by ISI interactions were examined. To study the relationship between cortical excitability and the degree of genetic similarity between subjects, intracortical inhibition (ISI 3 ms) and facilitation (ISI 15 ms) were correlated with the coefficient of relationship for members of the pedigree (e.g., ½ for siblings) using Spearman's rank order correlation coefficient (rs). Differences were regarded as significant when P < 0.05. The software used for statistical analysis was SPSS 11.5 for Windows (SPSS, Chicago, IL).

Results

MT measured at rest, silent period threshold measured during isometric contraction, and the duration of the CSP did not differ between controls, subjects with the mutation, and family members without the mutation (Table 1). Mean size of unconditioned test MEPs did not differ between groups (F2,30 0.1; P > 0.9). The number of phases of the unconditioned test MEP was higher in subjects affected by the mutation (controls 2.2 ± 0.9; mutation: 3.6 ± 1.6; P = 0.001) (Fig. 1). No differences were seen in latency (controls: 22.5 ± 0.9 ms; mutation: 22.1 ± 1.3 ms; P = 0.66) and duration (controls: 15.9 ± 3 ms; mutation: 17.9 ± 4.5 ms; P = 0.41) of the MEPs. Using a paired-pulse paradigm (Fig. 2), intracortical inhibition (ISI 2–5 ms) was found to be reduced in subjects with the mutation compared with controls (F2,30 = 26; P < 0.001) with no interaction between group membership and ISI (F2,30 = 1.2; P = 0.30). Intracortical facilitation (ISI >5 ms) was found to be increased in subjects with the mutation compared with controls (F2,30 = 5; P = 0.003) with no interaction between group membership and ISI (F2,30 = 1.1; P = 0.3).

Figure 1.

Effect of the GABRG2(R43Q) mutation on the MEP morphology. MEPs evoked by a single suprathreshold (+20% of the MT) pulse in 3 representative controls, subjects with the GABRG2(R43Q) mutation, and intrafamilal controls (GABRG2(R43Q) −) are shown. Note the increased number of phases in the patient with the GABRG2(R43Q) mutation compared with the control subjects.

Figure 1.

Effect of the GABRG2(R43Q) mutation on the MEP morphology. MEPs evoked by a single suprathreshold (+20% of the MT) pulse in 3 representative controls, subjects with the GABRG2(R43Q) mutation, and intrafamilal controls (GABRG2(R43Q) −) are shown. Note the increased number of phases in the patient with the GABRG2(R43Q) mutation compared with the control subjects.

Figure 2.

Effect of the GABRG2(R43Q) mutation on intracortical inhibition and facilitation. (A) Paired-pulse TMS comparing normal controls (blue), individuals carrying the GABAA mutation (red), and family members unaffected by the mutation (green). The data represent the mean MEP amplitude ratio normalized to the control stimulus at different ISI; error bars are for the standard error of the mean. Ratios less than 100% indicate inhibition and ratios greater than 100% indicate facilitation. We observed higher ratios in the subjects affected by the mutation (repeated-measures ANOVA, between subjects: F2,30 = 26; P = 0.001). No significant interaction between group membership and ISI was observed (F2,30 = 2.4; P = 0.1). (B) Mean evoked response amplitude ratio normalized to the control stimulus for ISIs of 2–5 ms representing inhibition (left) and 6–15 ms representing facilitation (right) in controls, subjects affected by the GABRG2(R43Q) mutation, and family members unaffected by the mutation. Individual subject values are shown. The box represents the mean (indicated by heavy line) ± 2.5 standard deviations.

Figure 2.

Effect of the GABRG2(R43Q) mutation on intracortical inhibition and facilitation. (A) Paired-pulse TMS comparing normal controls (blue), individuals carrying the GABAA mutation (red), and family members unaffected by the mutation (green). The data represent the mean MEP amplitude ratio normalized to the control stimulus at different ISI; error bars are for the standard error of the mean. Ratios less than 100% indicate inhibition and ratios greater than 100% indicate facilitation. We observed higher ratios in the subjects affected by the mutation (repeated-measures ANOVA, between subjects: F2,30 = 26; P = 0.001). No significant interaction between group membership and ISI was observed (F2,30 = 2.4; P = 0.1). (B) Mean evoked response amplitude ratio normalized to the control stimulus for ISIs of 2–5 ms representing inhibition (left) and 6–15 ms representing facilitation (right) in controls, subjects affected by the GABRG2(R43Q) mutation, and family members unaffected by the mutation. Individual subject values are shown. The box represents the mean (indicated by heavy line) ± 2.5 standard deviations.

Table 1

Effect of the GABRG2(R43Q) mutation on cortical excitability

 RMT (%) CSP threshold (%) CSP duration (ms) MEP amplitude for unconditioned stimulus (μV) 
Controls (n = 11) 70 ± 10 57 ± 8 92 ± 23 742 ± 549 
GABRG2(R43Q) mutation (n = 11) 71 ± 8 56 ± 8 91 ± 20 662 ± 510 
Family members (n = 4) 62 ± 8 53 ± 10 89 ± 28 782 ± 454 
P Value 0.7 0.9 0.2 0.9 
 RMT (%) CSP threshold (%) CSP duration (ms) MEP amplitude for unconditioned stimulus (μV) 
Controls (n = 11) 70 ± 10 57 ± 8 92 ± 23 742 ± 549 
GABRG2(R43Q) mutation (n = 11) 71 ± 8 56 ± 8 91 ± 20 662 ± 510 
Family members (n = 4) 62 ± 8 53 ± 10 89 ± 28 782 ± 454 
P Value 0.7 0.9 0.2 0.9 

Note: RMT, CSP threshold and duration, and MEP amplitude for unconditioned stimulus in controls and individuals with and without the GABRG2(R43Q) mutation (mean ± standard deviation). Thresholds are expressed as a percentage of maximum coil output. P values are for the main effect of group membership in 1-way ANOVA.

Similar results were obtained when MEP area was measured on single-trial rectified EMG data and substituted for peak-to-peak amplitudes. Representative indices of intracortical inhibition (ISI 3 ms) and intracortical facilitation (ISI 15 ms) were found to be unaffected by age (F2,30 3.5; P = 0.09) and clinical phenotype (F2,30 0.4; P = 0.55). No correlation between the coefficient of relationship between members of the pedigree and the mean intracortical inhibition at an ISI of 3 ms (rs = −0.13; P = 0.6) or facilitation at an ISI of 15 ms (rs = 0.2; P = 0.3) was seen.

Discussion

Using TMS, we observed reduced net short-interval intracortical inhibition and increased facilitation in individuals with a GABRG2(R43Q) receptor mutation associated with generalized epilepsy. The mutation was associated with an increase in polyphasia of MEP waveforms but MTs and CSP were unaffected.

The GABRG2(R43Q) mutation affects several aspects of GABAA receptor function that are likely to affect cortical excitability. In vitro, the mutation manifests functionally as a reduction in inhibition, although different mechanisms have been reported including decreased GABA-induced inhibitory postsynaptic currents, slower GABAA receptor deactivation, and increased fast desensitization (Bianchi et al. 2002; Bowser et al. 2002; Macdonald et al. 2004). The mutation also leads to impaired trafficking and reduced expression on the cell surface of functional receptors containing R43Q γ2 subunits in single cell in vitro preparations (Sancar and Czajkowski 2004) and in knock-in mice with the GABRG2(R43Q) mutation (Mulligan et al. 2004). Such reduced expression may be represented in humans by the widespread reduction of [11C]-flumazenil binding on positron emission tomography studies in individuals affected by the GABRG2(R43Q) mutation, with peak differences occurring in the frontal regions (Fedi et al. 2006).

Subjects with the GABRG2(R43Q) mutation had reduced net short-interval intracortical inhibition and enhanced facilitation. At the ISIs used in this study, there is extensive evidence that intracortical inhibition and facilitation are modulated by the GABAA receptor. In vitro, the GABAA receptor has been shown to mediate intracortical inhibition (McCormick 1989) and GABAA receptor blockade increases synchronization and horizontal propagation of neural activity within the neocortex (Chagnac-Amitai and Connors 1989). It appears likely that the changes in intracortical excitability observed in individuals with the GABRG2(R43Q) mutation reflects a reduction in the gain of inhibitory intracortical circuits mediated by GABAergic interneurons.

The mutation had a greater effect on intracortical facilitation than on net short-interval intracortical inhibition. A similar effect, with a greater influence on facilitation, is seen when the paired-pulse paradigm is used to study the effects of GABAergic drugs such as benzodiazepines, vigabatrin, and ethanol on intracortical excitability (Ziemann et al. 1996b). An explanation for this difference in effect size may, in part, be related to the observation that inhibition and facilitation are mediated by distinct pools of interneurons with different anatomical orientations, thresholds, and drug sensitivities (Kujirai et al. 1993; Ilic et al. 2002).

Studies employing conditioning pulse intensity curves have demonstrated that the inhibition observed at short ISIs (<5 ms) is the net effect of 2 components. One component is inhibitory and is most evident at lower conditioning stimulus intensities. The other is facilitatory and becomes evident for conditioning stimulus intensities close to MT. Diazepam affects both these components (Ilic et al. 2002). Hence, it is possible that the observed reduction in net inhibition in the subjects with the GABRG2(R43Q) mutation is caused by a reduction of short-interval intracortical inhibition, an enhancement of short-interval intracortical facilitation, or both. Future studies with stimulus response curves at a range of intensities will be required to disentangle effects on the different components of net short-interval intracortical inhibition (Butefisch et al. 2003). Differently directed induced currents may also be of value in dissecting out effects on different I-wave components.

Subjects affected by the GABRG2(R43Q) mutation displayed large and abnormally polyphasic MEPs. TMS of the motor cortex activates pyramidal cells transsynaptically resulting in multiple descending volleys in the corticospinal tract (Ziemann and Rothwell 2000). An initial excitatory volley, the D-wave, is followed by subsequent volleys or I-waves. The motor cortical circuits responsible for production of the latter are under the control of GABA-mediated inhibition and GABAA receptor agonists consistently lead to marked I-wave depression (Ziemann and Rothwell 2000). Epidural recordings using a similar double-pulse paradigm indicate that at ISIs of 2–5 ms, this stimulus inhibits markedly the later I-waves evoked by the test stimulus (Nakamura et al. 1997). The ISIs over which this occurs are consistent with the duration of inhibitory postsynaptic potentials in the cat mediated through GABAA. A reduction in GABA-mediated inhibition because of the GABRG2(R43Q) mutation could result in an increased number of I-waves following TMS and may explain the increased MEP polyphasia. An alternative explanation is that polyphasia reflects more diffuse activation as a result of reduced inhibition.

The absence of differences in MTs between patients and controls suggests that the GABRG2(R43Q) mutation did not affect the recruitment pattern of neuronal elements activated by a single stimulus both in the relaxed and active states. It is known that MT relates mainly to neuronal membrane excitability, which largely depends on sodium channel conductivity (Mavroudakis et al. 1997). In keeping with this, GABAA receptor agonists and antagonists do not modify the MT (Jung et al. 2004; Ziemann et al. 1996a, 1996b). The mutation also had no effect on the CSP, the later portion of which reflects activation of intracortical inhibitory neurons probably mediated by the GABAB receptor (Roick et al. 1993). Effects on the CSP produced by benzodiazepines have been inconsistent. Ziemann et al. (1996a) reported that the CSP was prolonged following the administration of lorazepam. In contrast, Inghilleri et al. (1996) observed that diazepam reduced CSP duration, an effect reversed by the benzodiazepine antagonist flumazenil.

The evidence supporting a role for the mutation in epileptogenesis is based on the genetic analysis of the pedigree and on previous observations in experimental models. In this pedigree, febrile seizures segregate as an autosomal dominant trait and the clinical genetic data suggests that the mutation is the major determinant of the febrile seizure phenotype. The second major phenotype, childhood absence epilepsy, is inherited in a complex fashion, and there is evidence that 2 or more genes are operative (Marini et al. 2003). Previous TMS studies in sporadic idiopathic generalized epilepsies have revealed a variety of changes. As in our patients, some studies in idiopathic generalized epilepsy showed reduced intracortical inhibition or increased intracortical facilitation, although the latter was only studied after sleep deprivation or at long ISIs (Brodtmann et al. 1999; Manganotti et al. 2000; Badawy et al. 2006). Other studies showing decreased MT (Reutens et al. 1993) and prolonged CSP (Ertas et al. 2000; Macdonell et al. 2001) in nonfamilial idiopathic generalized epilepsy suggest pathophysiological diversity in phenotypically similar forms of epilepsy.

We have shown that the mutation causes neurophysiological changes although the subjects were all in seizure remission, suggesting a key role for additional factors to produce seizures in the vulnerable age range in childhood. These factors might include temperature-dependent effects on cell surface GABAA receptor expression (Kang et al. 2006) and increases in intracortical inhibition from childhood to adulthood (Mall et al. 2004). During normal development, the number of GABAergic synapses increases and the open times of the GABAA receptor shorten (Heinen et al. 2003; Bosman et al. 2005). Developmental changes in other systems may also counteract the effects of the mutation rendering the brain less vulnerable to seizures after childhood (Huntsman et al. 1999; Bowser et al. 2002).

In summary, we have demonstrated that the GABRG2(R43Q) mutation which reduces GABAA receptor function increases intracortical excitability in the living human brain. The neurophysiological changes observed may be an important clue to the mechanisms linking the mutation and the epilepsy phenotype.

Funding

Postgraduate Research Scholarship awarded by The University of Melbourne to MF; National Health and Medical Research Council of Australia (Program Grant i44105).

We wish to thank the subjects who volunteered their time for the studies. Conflict of Interest: the authors declare that they have no competing financial interests.

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