Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

Abstract

The properties of ␥-aminobutyric acid (GABA) type A receptors (GABA A receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABA A receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat ␣1␤2␥2-GABA receptors exhibited a much less pronounced GABA-current rundown. Moreover, the GABA A receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABA A receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABA Areceptor ␤1, ␤2, ␤3, and ␥2 subunit mRNAs were significantly overexpressed (8-to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABA A receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy.temporal lobe epilepsy ͉ microtransplantation into Xenopus oocyte ͉ okadaic acid ͉ ␥-aminobutyric acid-current run-down ͉ human tissue slices

Cannabis in epilepsy: From clinical practice to basic research focusing on the possible role of cannabidivarin

“…This impairment was recorded as a strong use‐dependent desensitization (i.e., rundown) of the GABA A receptor mediated currents that was recovered by blocking the protein kinase C (PKC) pathway 12. In agreement with previous results,10 application of GABA (500 μ m ) to transplanted oocytes elicited bicuculline‐sensitive inward currents (I GABA amplitude range: −10 nA to −100 nA). When oocytes were injected with hippocampal membranes of TLE patients and RE tissues, we found a consistent I GABA rundown following repetitive application of GABA (I% TLE = 45% ± 12; 49 oocytes; 4 patients; Fig.…”

“…Considering some recent data emphasizing the possible role of GABA‐mediated action of cannabis, we decided to test CBDV effect on GABAergic transmission in human tissue 10, 11. However, because of the unavailability of the patient's own surgical specimens (temporal lobe tissue was already fixed and embedded in paraffin) and the rarity of his pathology, we performed our experiments on the hippocampal specimens resected from four drug‐resistant temporal lobe epilepsy (TLE) patients (for details, see Fig.…”

“…Membrane and oocyte preparation/injection procedures were performed as previously detailed 8, 10. GABA current rundown (I%) was defined as the decrease, calculated in percentage, of the current peak amplitude after six 10‐s applications of 500 μ m GABA at 40‐s intervals;9 the percentage of the original current amplitude remains.…”

“…Oocytes were incubated with CBDV (50 n m ) for 120–150 min after the control rundown protocol. For an internal control, other cells were incubated with oocytes Ringer's solution8, 10 in the same conditions. All results are given as mean ± standard error (SEM).…”

“…Considering the proven effect of cannabinoids on GABA‐evoked currents11 (I GABA ), we focused our attention on the modulation exerted by CBDV on GABA A ‐Rs, previously reported to be impaired in these patients 10. This impairment was recorded as a strong use‐dependent desensitization (i.e., rundown) of the GABA A receptor mediated currents that was recovered by blocking the protein kinase C (PKC) pathway 12.…”