Address correspondence to Thomas J. Feuerstein, Section of Clinical Neuropharmacology, Department of Neurosurgery, Albert-Ludwigs University, Breisacher Strasse 64, D-79106 Freiburg, Germany. E-mail: firstname.lastname@example.org
To learn whether epileptic seizures in Rasmussen encephalitis (RE) may be promoted by insufficient γ-aminobutyric acid (GABA) release. 3H-GABA was released from neocortical synaptosomes through transporter reversal following intrasynaptosomal Na+ accumulation by veratridine that prevents inactivation of Na+ channels. Tissues of three RE patients were compared with those of nine non-RE. In RE, the release was markedly reduced. In non-RE, the extracellular Ca2+ concentration ([Ca2+]e) was inversely related to the amount of release. In RE, the percental decline of additional release upon withdrawal was linked with the presurgical duration of epilepsy. Permanent opening of Na+ channels by veratridine resembles maximal frequency of action potentials corresponding to epileptic seizures. These are preceded by a fall in [Ca2+]e. Zero [Ca2+]e increased release through the Na+/Ca2+ exchanger additionally elevating intrasynaptosomal Na+. This enhanced GABA release probably reflects an antiseizure mechanism. In RE, the additional release gets lost over epilepsy duration.
Rasmussen encephalitis (RE) is associated with progressive inflammation and cell loss in mostly one cerebral hemisphere. Apart from seizures, RE leads to an irreversible loss of cerebral functions. The immune system is presumably affected (Bien et al., 2005). Mutated voltage-gated Na+ (Nav) channels were also reported (Ohmori et al., 2008).
Stimulation with the Nav channel opener veratridine was compared in neocortical synaptosomes in three RE and nine non-RE patients. Veratridine was used because permanently active Nav channels may reflect a maximum frequency of action potentials, as typical for epileptic seizures. Synaptosomes were incubated with 3H-γ-aminobutyric acid (GABA) and superfused to determine 3H-GABA release. RE showed a strongly reduced 3H-GABA release upon stimulation and, in contrast to non-RE, the percental decline of additional 3H-GABA release upon withdrawal corresponded exactly to both the actual amount of release and the presurgical epilepsy duration (PSED). Our results support the hypothesis of altered GABA release as cause for seizures in RE.
We investigated neocortical tissue of three RE patients. PSED varied between 9, 2, and 0.375 years. Nine non-RE patients served as controls (epilepsy in six, brain tumor in three). PSED in non-RE epileptics varied from months to 50 years. In tumor patients, PSED was defined as zero. In two additional non-RE patients (one epilepsy, one tumor) the experimental setting was different. Written consents were obtained according to the Declaration of Helsinki, as requested by the local Ethics Committee (No. 187/04).
The gray matter was separated from the white and homogenized in 10 volumes (w/v) of sucrose (0.32 m)/HEPES (2.5 mm) buffer (pH 7.4), centrifugation of the homogenate and of the resulting supernatant (10,000 g, 10 min, 4°C, each) followed. The supernatant was discarded and the pellet was resuspended in buffer. The buffer contained (mm) NaCl 121, KCl 1.8, CaCl2 1.3 (or 2.6), KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 10, saturated with 95% O2/5% CO2; pH 7.4. Synaptosomes were incubated (15 min, 37°C, 95% O2/5% CO2) in 2 ml buffer containing 0.04 μm3H-GABA. Eighty microliter of the synaptosomal suspension were transferred to GF/B filters (Whatman, Dassel, Germany) in 24 superfusion chambers in a water bath (37°C) and covered with filters. After 35 min preperfusion, collection of fractional samples followed (5-min intervals, 0.4 ml/min flow rate). Synaptosomes were stimulated (2 min) by 10 μm veratridine. Ca2+-free buffer contained 100 μm ethylene glycol tetraacetic acid (EGTA). The amount of radioactivity was determined by scintillation spectrometry.
The fractional rate, determined as 3H-content of a superfusion fraction divided by the synaptosomal 3H-content at its beginning, was used to calculate the veratridine-evoked 3H-overflow (difference between total 3H-outflow from start of stimulation to end of the following 15 min and basal 3H-outflow, both expressed as fractional rate). The evoked 3H-overflow was assumed to represent the amount of veratridine-evoked 3H-GABA plus endogenous GABA and was called GABA release. The basal ratio “b2/b1”—the quotient of the fractional rate in the last 5-min period of superfusion (b2) and that of the period before stimulation (b1)—indicated how fast 3H-substances leaked spontaneously.
The difference of veratridine-evoked 3H-GABA release between RE and non-RE was evaluated by comparison of the mean of the individual mean stimulation (S) values in RE (N = 3 patients) and the mean of the individual mean S values in non-RE (N = 9 patients).
The number of chambers (n) represents the number of observations in each patient. The effects of [Ca2+]e alterations were evaluated by comparison of normalized S values. Normalization was achieved by dividing all individual S values (n) through the mean S value after veratridine-stimulation (control groups, [Ca2+]e = 1.3 or 0 mm). Results are given as arithmetic means with 95% confidence intervals (CI95). Because comparison of effects as % of corresponding controls requires consideration of the controls' variances, Fieller's Theorem was used for determination of the CI95 of % values.
Significant differences between two means were tested using Student's t-test after one-way analysis of variance (ANOVA). An individual patient assumingly had no influence (Kammerer et al., 2011a,b; see methodical details). was withdrawn in some chambers in all experiments, except in the 10th and 11th non-RE patient, where [Ca2+]e was doubled.
In RE, the veratridine-evoked 3H-GABA release (mean of the individual mean S values) at [Ca2+]e = 1.3 mm was only 31.6% of that in non-RE (Fig. 1A).
In non-RE, , withdrawal increased the release in all patients (Fig. 1B). Herein, epilepsy and brain tumor tissue was similar (+61.2% [42.5, 83.4], n = 22, and +51.2% [30.6, 76.3], n = 24). Heightened [Ca2+]e (2.6 mm) diminished the release (Fig. 1B).
In the RE patient with the shortest PSED , withdrawal increased release by 135.8% [111.9, 159.7], p < 0.001. There were no significant percental changes of the releases in the other RE patients (longer PSEDs, see Fig. 2 for details).
The mean basal ratio (b2/b1) in RE, expressed as % of that in non-RE, was 83.1% [79.5, 86.8], p < 0.001, at [Ca2+]e = 1.3 mm and 89.7% [85.1, 94.4], p < 0.001, at [Ca2+]e = 0 mm. There was no change in the mean basal ratio due to withdrawal, neither in RE nor in non-RE.
The mean synaptosomal 3H-content after superfusion in RE was 72.9%, p < 0.05, of that in non-RE (16052.9 [13893.8, 18212.1] versus 22032.3 [18950.7, 25113.8] decays per minute).
Firing during seizures is characterized by massive Na+ influx. By preventing the inactivation of Nav channels, veratridine stimulation simulates high frequency firing, causing intraneuronal Na+ accumulation. Veratridine-evoked 3H-GABA release from human neocortical synaptosomes is caused mainly by transporter reversal due to increased cytosolic Na+ concentration ([Na+]c; Kammerer et al., 2011b). This type of release may also prevail in vivo during seizures when increased [Na+]c enhances transporter reversal (Wu et al., 2007).
In epilepsy an imbalance between GABA and glutamate may be due to disturbed GABAergic signaling (Conti et al., 2004; Pearl & Gibson, 2004; Galanopoulou, 2010). In addition, in RE, GABA release may be diminished, possibly due to altered transporters or Nav channels.
In RE, both the synaptosomal 3H-content after superfusion and the basal ratio were decreased. Possibly, disturbed transporters diminished 3H-GABA accumulation or endogenous GABA synthesis from glutamine. Alternatively, GABA could continuously leak from hyperexcitable synaptosomes, thereby depleting extravesicular GABA. This would reduce transporter reversal and explain a lower basal ratio.
An increased veratridine-evoked 3H-GABA release upon removal was described (Kammerer et al., 2011b). Correspondingly, an increased [Ca2+]e reduced the release. Zero may activate the Na+/Ca2+ exchanger and thus elevate [Na+]c, enhancing transporter reversal (Kammerer et al., 2011b). Increased GABA release may counteract paroxysmal activity, which is preceded by decreased [Ca2+]e that can induce epileptiform activity (Heinemann et al., 1977, 1986). Indeed, [Ca2+]e decreases just before the onset of epileptic firing in vivo (Pumain et al., 1983). Therefore, selectively increased GABA release subsequent to decreased [Ca2+]e could represent a defense mechanism against hyperexcitability. Notably, withdrawal decreased 3H-glutamate release (Kammerer et al., 2011a). Increased 3H-GABA release upon withdrawal was found in all non-RE patients, which seems pathophysiologically relevant. In RE, increased release was found only in one patient. Its percental magnitude was even higher than in non-RE patients, suggesting that the defense mechanism is active early during the disease. Possibly, RE tissue tried to counteract the RE-typical decrease in GABA release. A lost counteraction might then boost seizures. Our assumption that the decline of additional release upon withdrawal may be linked to both the actual amount of release and PSED was confirmed by nonlinear regression analysis (Fig. 2). It was performed with the exponential function as solution of the differential equation that reflected our assumption. In non-RE, the fitting procedure did not yield any meaningful results.
In RE, veratridine-evoked 3H-GABA release was lower than in non-RE. Veratridine stimulation may simulate a maximum frequency of Nav channel openings like in seizures. Because the release was augmented only by withdrawal in the RE patient with short PSED, the defense mechanism against hyperexcitability may get lost over time. Although a decreased GABA release plus a lost increase upon withdrawal may not completely explain the pathophysiology of seizures in RE, it might promote understanding epileptogenesis.
None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.