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Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. Using both a “microtransplantation technique” and synaptosomes preparations from drug-resistant temporal lobe epilepsies (TLEs), we used the technique of two-electrode voltage clamp to record GABA-evoked currents, focusing selectively on the synaptic “fast inhibition” mediated by low-affinity GABA_A receptors. Here, we report that the use-dependent GABA current desensitization (i.e., GABA rundown, which is evoked by applying to the cells consecutive pulses of GABA, at high concentration), which is a distinguishing mark of TLE, is mainly dependent on a dysfunction that affects synaptic GABA_A receptors. In addition, using the same approaches, we recorded a depolarized GABA reversal potential in synaptosomes samples from the human epileptic subicula of TLE patients. These results, which confirm previous experiments using total membranes, suggest an altered chloride homeostasis in the synaptic area. Finally, the lack of a Zn²⁺ block of GABA-evoked currents using the synaptosomes supports the enrichment of “synaptic fast inhibitory” GABA_A receptors in this preparation. Altogether, our findings suggest a pathophysiological role of low-affinity GABA_A receptors at the synapse, especially during the fast and repetitive GABA release underlying recurrent seizures. Full article

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders. Full article