Thalamocortical relationships and network synchronization in a new genetic model "in mirror" for absence epilepsy.

International audienceElectroencephalographic generalized spike and wave discharges (SWD), the hallmark of human absence seizures, are generated in thalamocortical networks. However, the potential alterations in these networks in terms of the efficacy of the reciprocal synaptic activities between the cortex and the thalamus are not known in this pathology. Here, the efficacy of these reciprocal connections is assessed in vitro in thalamocortical slices obtained from BS/Orl mice, which is a new genetic model of absence epilepsy. These mice show spontaneous SWD, and their features can be compared to that of BR/Orl mice, which are free of SWD. In addition, since gap junctions may modulate the efficacy of these connections, their implications in pharmacologically-induced epileptiform discharges were studied in the same slices. The thalamus and neocortex were independently stimulated and the electrically-evoked responses in both structures were recorded from the same slice. The synaptic efficacy of thalamocortical and corticothalamic connections were assessed by measuring the dynamic range of synaptic field potential changes in response to increasing stimulation strengths. The connection efficacy was weaker in epileptic mice however, this decrease in efficacy was more pronounced in thalamocortical afferents, thus introducing an imbalance in the reciprocal connections between the cortex and thalamus. However, short-term facilitation of the thalamocortical responses were increased in epileptic mice compared to non-epileptic animals. These features may favor occurrence of rhythmical activities in thalamocortical networks. In addition, carbenoxolone (a gap junction blocker) decreased the cumulative duration of 4-aminopyridine-induced ictal-like activities, with a slower time course in epileptic mice. However, the 4-aminopyridine-induced GABA-dependent negative potentials, which appeared to trigger the ictal-like activities, remained. Our results show that the balance of the reciprocal connections between the thalamus and cortex is altered in favor of the corticothalamic connections in epileptic mice, and suggest that gap junctions mediate a stronger cortical synchronization in this strain

Mass Spectrometric Detection and Characterization of Atypical Membrane-Bound Zinc-Sensitive Phosphatases Modulating GABA_A Receptors

<div><p>Background</p><p>GABA_A receptor (GABA_AR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I₂ of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABA_AR are deficient.</p><p>Methodology/Principal Findings</p><p>The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABA_AR. We have developed a new analytical tool for <i>in vitro</i> detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABA_AR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABA_AR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABA_AR.</p><p>Conclusion/Significance</p><p>Our results provide new insights on the regulation of GABA_AR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed.</p></div

Effects of okadaic acid on the GABA_A current rundown.

<p>GABA_A currents were measured by whole-cell patch clamp on acutely dissociated cortical pyramidal neurons from Sprague Dawley rats. (A) Micrographs of a patched cell at two different magnifications, showing the rapid application device (left). (B) Rundown in cortical neuron: GABA was applied at a concentration of 100 µM during 1 second every 3 min. The maximal amplitude of GABAergic currents gradually decreased with time in control conditions. In presence of okadaic acid (10 µM in the pipette) variable effects were observed. (C) In some cells the rundown was even totally abolished. (D Left) Rundown profiles of normalized currents in presence (n = 17) or in absence (Control, n = 11) of okadaic acid; (D Right) color-coded hierarchical clustering tree for the recorded okadaic acid-treated cells in which a maximum of 4 groups are significantly distinguished: ‘Very Slow’ (n = 3), ‘Mid Slow’ (n = 5), ‘Mid Rapid’ (n = 4) and ‘Very Rapid’ (n = 5). For each group the plot (Left) is the average of normalized currents. Error bars indicate the SEM. One-way ANOVA with Dunnett’s test of mean currents and Student <i>t</i>-test were used (see in Results) indicating very likely that more than one phosphatase are involved.</p

LC-MS/MS detection and kinetics analysis of GABA_AR α1-subunit phosphatase activities.

<p>(A) Dose-dependency at various protein concentrations; (B) time course of phosphatase activities using 50 µg/ml of total proteins of washed cortical membranes from bovine brain and (C) from human epileptic tissue. The N-terminal phosphopeptide (pI₂α1N-P) used as substrate at 10 µM was incubated during 10 min at 30°C in 10 mM Hepes (pH 7.4) in the presence of 1 mM Mg²⁺. The enzymatic reactions were stopped with 10% acetic acid and the samples were analyzed by LC-MS/MS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100612#pone-0100612-g001" target="_blank">figure 1E, F</a>. The dephosphorylation rate was quantified by measuring simultaneously the quantity of the produced native peptide (pI₂α1N) and of the remaining phosphorylated substrate (pI₂α1N-P). Error bars represent SEM of two experiments. Kinetic analysis of GABA_AR α1-subunit phosphatase activities was investigated using N-terminal (pI₂α1N-P) and C-terminal (pI₂α1C-P) phosphopeptides. All experiments were carried out with 50 µg/ml membrane proteins concentration, incubated at 30°C during 10 min, in presence of different concentrations of substrates. (D) Saturation plot of the initial velocity V_i versus [pI₂α1N-P] and Lineweaver-Burk plots of 1/V versus 1/[pI₂α1N-P] (inset) showing two significantly different slopes. (E) Saturation plot of V_i versus [pI₂α1C-P] and Lineweaver-Burk plot of 1/V versus 1/[pI₂α1C-P] (inset). The data points are means ± SEM of 3 experiments.</p

Effects of various inhibitors on the membrane-bound phosphatase activities.

<p>The phosphatase activity was performed in presence of reaction mixture contained 50 µg/ml of washed cortical membrane with 10 mM Hepes (pH 7.4) and 1 mM Mg²⁺, in addition to the inhibitor being tested. Incubations were performed at 30°C during 10 min. The activities are expressed as a percentage of that of control samples without inhibitors. Inhibition profiles of PPase activities by okadaic acid were performed using N-terminal phosphopeptide (A) or C-terminal phosphopeptide (B) as substrate. The data points are means ± SEM of 3 experiments. The red arrows indicate the different IC₅₀. Using N-terminal phosphopeptide pI₂α1N-P as substrate, membrane phosphatase activities were inhibited by both Br-t (+) and (−) enantiomers with close IC₅₀ values of 0.2 and 0.1 mM respectively (C), thus excluding alkaline phosphatase. Data points are the average ± SEM of duplicate assays. (D) Effect of Ca²⁺ ions, fluoride (F⁻) and spermine (SPM⁴⁺) on PPase activity using N-terminal phosphopeptide as substrate.</p

Kinetics parameters of the phosphatase activities dephosphorylating the N- and C-terminal phosphopeptides.

<p>Experimental conditions are detailed in Materials and Methods. Values are means ± SEM of two or three experiments.</p

Mass spectrometry (MS) and liquid chromatography (LC)-MS/MS analysis of N- and C-terminal peptides.

<p>The insert (A) shows the representation of the α1 subunit TM3-TM4 intracellular loop (I₂α1) of type A gamma-amino butyric acid receptor (GABA_AR). The sequences indicate the synthetic N- and C-terminal peptides (AA334–346 and AA407–420 respectively) used in this study, with the two identified phosphorylation sites (PO₄) on threonine (337Thr) and serine (416Ser) residues involved in the endogenous phosphorylation. EXT, extracellular; INT, intracellular. These peptides are detected in positive mode electrospray ionization (ESI) from m/z 200 to 2000 (mass in Da to charge ratio). The most abundant peptides (in blue) are selected for the subsequent ESI-MS/MS studies (parent peptides) and the other labeled ions have two, three or four charges. MS of native (A), and phosphorylated (B) N-terminal peptides. MS of native (C) and phosphorylated (D) C-terminal peptides. The major fragment ions produced by collision induced dissociation of parent peptide ions are labeled and the nature of fragmentation is indicated by “b” (N-flanking) or “y” (C-flanking) when the ion is broken at peptide bounds (break position in the amino acid sequence) or by losses (−) or gains (+) of small neutral molecules (water, ammonia). The highest intensity peaks used for identification and quantification of the different peptides are indicated in blue. LC-MS/MS analysis was performed simultaneously for the native and phosphorylated peptides. (E) Spectrum of native (F) phosphorylated N-terminal peptides. (G) Spectrum of native (H) phosphorylated C-terminal peptides. The inserts show the chromatograms of the chosen fragmentation products.</p