P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21–26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4−/− mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to −120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4−/− mice; this potentiation was not affected by nifedipine, but was abolished by 10 mm 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 μm, at −100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mm), ifenprodil (3 μm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). In P2X4−/− mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 μm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.

We have characterized a mechanism by which the amplitudes of miniature EPSCs (mEPSCs) in CA1 pyramidal neurons in rat hippocampal organotypic slice cultures are potentiated by approximately twofold after a series of depolarizing voltage pulses from -80 to ϩ20 mV. The increase in mEPSC amplitudes is triggered by the activation of L-type calcium channels and is independent of NMDA receptor (NMDAR) activation but also requires calcium release from intracellular stores. The potentiation induced by depolarizing pulses does not alter the kinetic parameters of mEPSCs. The induction phase of this potentiation involves phosphatidylinositol 3 kinase (PI3 kinase) activation because it is blocked completely in the presence of the PI3 kinase inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY294002). Furthermore, we show that the maintenance phase of depolarizing pulse potentiation requires continued PI3 kinase activity because the application of either wortmannin or LY294002 results in a reversal to control levels of the amplitudes of mEPSCs. Finally, we demonstrate that the increase in mEPSC amplitudes is mediated by the increased expression of functional AMPA receptors (AMPARs) because the potentiation is blocked by N-ethylmaleimide, botulinum toxin A, and a variety of short-sequence peptides that disrupt the interaction of AMPAR subunits with proteins involved with the trafficking of these to the cell membrane. Our data are consistent with the notion that PI3 kinase and membrane fusion/trafficking events play a pivotal role in coordinating changes in synaptic strength, mediated by AMPA receptors, which are triggered by alterations in postsynaptic calcium concentrations whether these changes are initiated via NMDAR-dependent or NMDAR-independent routes.

γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

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Die Pyryliumperchlorate (I), (III), (V) und (VIII) lagern sich bei der Photolyse gemäß Formelschema in isomere Pyryliumperchlorate um.