Search

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by [GABA.sub.A] receptors. In particular, the use-dependent decrease (run-down) of the currents ([I.sub.GABA]) evoked by the repetitive activation of [GABA.sub.A] receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of [I.sub.GABA] run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in [I.sub.GABA] run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in [alpha]4-, relative to [alpha]1-containing [GABA.sub.A] receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic. [GABA.sub.A] receptor | pilocarpine rat | Xenopus oocytes www.pnas.org/cgi/doi/10.1073/pnas.0914710107

A study was made of the 'rundown' of [GABA.sub.A] receptors, micro-transplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional [GABA.sub.A] receptors. Upon repetitive activation with GABA (1 mM), 'epileptic' [GABA.sub.A] receptors exhibited a [GABA.sub.A]-current ([I.sub.GABA]) rundown that was significantly enhanced by [Zn.sup.2+] ([less than or equal to]250 [micro]M), and practically abolished by the high-affinity [GABA.sub.A] receptor inverse agonist SR95531 (gabazine; 2.5-25 [micro]M). Conversely, [IGAB.sub.A] generated by 'control' [GABA.sub.A] receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with [Zn.sup.2+]. We conclude that rundown of mTLE epileptic receptors depends on the presence of 'phasic [GABA.sub.A] receptors' that have low sensitivity to antagonism by [Zn.sup.2+]. Additionally, we found that [GABA.sub.A] receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by [Zn.sup.2+] (40 [micro]M) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of [GABA.sub.A] receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE [GABA.sub.A] receptor activity may represent a useful therapeutic approach to the disease. Xenopus oocytes | tonic | epileptic rat

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABAA receptors. In particular, the use-dependent decrease (run-down) of the currents (IGABA) evoked by the repetitive activation of GABAA receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of IGABA run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in IGABA run-down occurs in the natural history of epilepsy. We found: (I) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in a4-, relative to al-containing GABAA receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic. [ABSTRACT FROM AUTHOR]

A study was made of the "rundown" of GABAA receptors, micro- transplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), "epileptic" GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5-25 μM). Conversely, lGABA generated by "control" GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of "phasic GABAA receptors" that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease. [ABSTRACT FROM AUTHOR]

N-terminal labelled fluorescent BODIPY®-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors.BODIPY®TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1–6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1–4 nM) receptor subtypes.BODIPY®FL-PYY(3–36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays.BODIPY®FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays.BODIPY®TMR-[cPP(1–7), NPY(19–23), Ala31, Aib32, Gln34]hPP and BODIPY®TMR-[hPP(1–17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1–0.6 nM).As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties.When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively.These results demonstrate that BODIPY®-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.British Journal of Pharmacology (2005) 146, 1069–1081. doi:10.1038/sj.bjp.0706425; published online 17 October 2005 [ABSTRACT FROM AUTHOR]