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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

We previously found that the endogenous anticonvulsant adenosine, acting through [A.sub.2A] and [A.sub.3] adenosine receptors (ARs), alters the stability of currents ([I.sub.GABA]) generated by GABAA receptors expressed in the epileptic human mesial temporal lobe (MTLE). Here we examined whether ARs alter the stability (desensitization) of [I.sub.GABA] expressed in focal cortical dysplasia (FCD) and in periglioma epileptic tissues. The experiments were performed with tissues from 23 patients, using voltage-clamp recordings in Xenopus oocytes microinjected with membranes isolated from human MTLE and FCD tissues or using patch-clamp recordings of pyramidal neurons in epileptic tissue slices. On repetitive activation, the epileptic GABAA receptors revealed instability, manifested by a large [I.sub.GABA] rundown, which in most of the oocytes ([approximately equal to] 70%) was obviously impaired by the new [A.sub.2A] antagonists ANR82, ANR94, and ANR152. In most MTLE tissue-microtransplanted oocytes, a new [A.sub.3] receptor antagonist (ANR235) significantly improved [I.sub.GABA] stability. Moreover, patch-clamped pyramidal neurons from human neocortical slices of periglioma epileptic tissues exhibited altered [I.sub.GABA] rundown on ANR94 treatment. Our findings indicate that antagonizing [A.sub.2A] and [A.sub.3] receptors increases the [I.sub.GABA] stability in different epileptic tissues and suggest that adenosine derivatives may offer therapeutic opportunities in various forms of human epilepsy. epilepsy | focal cortical dysplasia

We examined how the endogenous anticonvulsant adenosine might influence [gamma]-aminobutyric acid type A ([GABA.sub.A]) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 [micro]M), [GABA.sub.A] receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious [GABA.sub.A]-current ([I.sub.GABA]) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced [I.sub.GABA] run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated [I.sub.GABA] run-down in [approximately equal to]40% and [approximately equal to]20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 [micro]M) potentiated [I.sub.GABA] run-down but only in [approximately equal to]20% of tested oocytes. CGS15943 administration again decreased [I.sub.GABA] run-down in patch-clamped neurons from either human or rat neocortex slices. [I.sub.GABA] run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, [GABA.sub.A]-receptor stability is tonically influenced by A2A but not by A1 receptors. [I.sub.GABA] run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2-A3 receptors alter the stability of [GABA.sub.A] receptors, which could offer therapeutic opportunities. A2A receptor | A3 receptor | microtransplantation into Xenopus oocyte | temporal lobe epilepsy

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human [CD34.sub.+] cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes, hUCB [CD34.sub.+] cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP+ cells were tested for their electrophysiological features by patch clamp and for cytosolic [[Ca.sup.2+]] ([[Ca.sup.2+]][sub.i]) homeostasis by [[Ca.sup.2+]][sub.i] imaging of X-rhodl-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured ceil populations by real-time RT-PCR. EGFP+ cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [[Ca.sup.2+]][sub.i] transients. These cells also exhibited spontaneous sarcoplasmic reticulum [[Ca.sup.2+]][sub.i] oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB [CD34.sub.+] cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR. functional differentiation

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

We studied the properties of GAB[A.sub.A] receptors microtransplanted from the human temporal lobe epilepsy (TLE)-associated brain regions to Xenopus oocytes. Cell membranes, isolated from surgically resected brain specimens of drug-resistant TLE patients, were injected into frog oocytes, which rapidly incorporated human GAB[A.sub.A] receptors, and any associated proteins, into their surface membrane. The receptors originating from different epileptic brain regions had a similar run-down but an affinity for GABA that was [approximately equal to] 60% lower for the subiculum receptors than for receptors issuing from the hippocampus proper or the temporal lobe neocortex. Moreover, GABA currents recorded in oocytes injected with membranes from the subiculum had a more depolarized reversal potential compared with the hippocampus proper or neocortex of the same patients. Quantitative RT-PCR analysis was performed of the GAB[A.sub.A] receptor [alpha]1- to [alpha]5-, [beta]1- to [beta]3-, [gamma]2- to [gamma]3-, and [delta]-subunit mRNAs. The levels of expression of the [alpha]3-, [alpha]5-, and [beta]1- to [beta]3-subunit mRNAs are significantly higher, with the exception of [gamma]2- subunit whose expression is lower, in subiculum compared with neocortex specimens. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE subiculum leads to the expression of GAB[A.sub.A] receptors with a relatively low affinity. This abnormal behavior of the subiculum GAB[A.sub.A] receptors may contribute to epileptogenesis. temporal lobe epilepsy

The functional properties and cellular localization of the human neuronal [alpha]7 nicotinic acetylcholine (AcCho) receptor ([alpha]7 AcChoR) and its L248T mutated (rout) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mut[alpha]7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wt[alpha]7 receptors decay much faster than those elicited by the mut[alpha]7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated [alpha]7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable 'run-down' of the AcCho currents generated by mut[alpha]7-GFP receptors, whereas those of the wtc[alpha]7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mut[alpha]7-GFP oocytes was accompanied by a marked decrease of [alpha]-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wt[alpha]7 and mut[alpha]7 receptors provides powerful tools to study the distribution and function of [alpha]7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

Neuronal nicotinic acetylcholine (AcCho) receptors composed of [alpha]7-subunits ([alpha]7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) [alpha]7-AcChoRs, we identified two classes of channel conductance: a low conductance ([[gamma].sub.L]) of 72 pS and a high one ([[gamma].sub.H]) of 87 pS, with mean open-times ([[tau].sub.op]) of 0.6 ms. The same classes of conductances, but longer [[tau].sub.op] (3 ms), were seen in experiments with chimeric [alpha]7 receptors in which the wt[alpha]7 extracellular C terminus was fused to the green fluorescent protein (wt[alpha]7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing [alpha]7 receptors carrying a Leu-to-Thr 248 mutation (mut[alpha]7) or oocytes expressing chimeric mut[alpha]7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wt[alpha]7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter [[tau].sub.op], than those activated by AcCho. Accordingly, human homomeric wt[alpha]7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mut[alpha]7 receptors open channels of lower conductance and longer lifetime than those gated by wt[alpha]7-AcChoRs, and these parameters are not greatly altered by fusing the mut[alpha]7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function.