6-cyano-7-nitroquinoxaline-2-3-dione and 8-cyclopentyl-1-3-dimethylxanthine

Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine-regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin neurons by activation of presynaptic adenosine A1 receptors, which may finely regulate the excitability of these neurons as well as eventually modulate the sleep-wakefulness.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Animals; Animals, Newborn; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Agents; GABAergic Neurons; Green Fluorescent Proteins; Hypothalamus; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Neural Inhibition; Neuropeptides; Orexins; Patch-Clamp Techniques; Presynaptic Terminals; Purinergic Antagonists; Synaptic Transmission; Theobromine; Theophylline; Valine

1. ATP and glutamatergic synaptic currents were compared in slices of rat medial habenula nucleus using whole-cell patch-clamp techniques. 2. In most cells low voltage stimulation resulted in glutamatergic responses and not purinergic responses. In five cells where ATP currents could be stimulated with low voltages, wash out of glutamate antagonists did not reveal evoked glutamate currents. Spontaneous glutamate currents confirmed washout of antagonist. 3. Modulation of release probability of glutamate and ATP, assessed by changes in failure rate of synaptic currents, was compared under conditions of different stimulation frequencies and in the presence of adenosine agonists and antagonists. 4. ATP release, but not glutamate release, was shown to be modulated by increased stimulation frequency which resulted in inhibition of ATP release via A2-like adenosine receptors. A1 receptors caused inhibition of both ATP and glutamate release. 5. Endogenous adenosine inhibited glutamate release via A1 receptors but only inhibited ATP release via A2-like receptors. 6. Attempts to inhibit the degradation of ATP to adenosine did not alter the frequency dependence of the failure rate. 7. We conclude, from the direct demonstration and from the differences in pharmacology and frequency dependence of the modulation of release, that ATP and glutamate responses are due to release from separate neurones.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Adenosine Triphosphate; Aniline Compounds; Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Glutamic Acid; Habenula; Male; Neurons; Nucleotidases; Presynaptic Terminals; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Receptors, Glutamate; Receptors, Purinergic P1; Suramin; Synaptic Transmission; Synaptic Vesicles; Theophylline

The effects of endogenously released ATP on the maturation of developing neuromuscular synapses were investigated in Xenopus nerve-muscle co-cultures. The potentiating action of ATP (1 mM) on spontaneous acetylcholine release was inhibited by P2-purinoceptor antagonists suramin (0.3 mM) and reactive blue 2 (RB-2, 3 microM) in day 1 cultures. Bath application of suramin (10 microM) or RB-2 in day 1 cultures and prolonged treatment for 2 days dramatically decreased the amplitude of both spontaneous synaptic currents (SSCs) and evoked synaptic currents (ESCs) in the same cultures on day 3. Chronic treatment with 8-cyclopentyltheophylline (4 microM) or 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX, 10 microM), P1-purinoceptor and glutamate receptor antagonists respectively, did not exert such an inhibitory effect. Chronic treatment with suramin or RB-2 for 2 days had no significant effect on the amplitude of either iontophoretic acetylcholine-induced whole-cell currents or single acetylcholine channel measurements in 3-day-old cultured myocytes. In addition, prolonged treatment for 2 days with various kinase inhibitors such as H-8 (10 microM), KN-62 (5 microM) and H-7 (10 microM) also decreased the amplitudes of both spontaneous and evoked synaptic currents in natural synapses, but not those of iontophoretic acetylcholine-induced currents. Furthermore, suramin and these protein kinase inhibitors also decreased the amplitude of spontaneous synaptic currents in manipulated synapses of 'vacated' nerve terminals. The results suggest that endogenously released ATP, acting in concert with various protein kinases, is involved in the maintenance and/or development of the quantum size of synaptic vesicles at embryonic neuromuscular synapses.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholine; Adenosine Triphosphate; Animals; Cells, Cultured; Coculture Techniques; Embryo, Nonmammalian; Enzyme Inhibitors; Evoked Potentials; Kinetics; Muscle, Skeletal; Neuromuscular Junction; Neurons; Protein Kinases; Quantum Theory; Suramin; Synapses; Synaptic Transmission; Theophylline; Triazines; Xenopus laevis

A1 adenosine receptors efficiently modulate the excitatory synaptic transmission in hippocampus. Here we report that in addition to previously known modulatory action on the synaptic efficacy, A1 adenosine receptors are also capable of regulating the relative contribution of N-methyl-D-aspartate receptor-mediated component of the excitatory postsynaptic current in CA3-CA1 excitatory synapses, in the rat. When applied externally, a selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine, increases not only the amplitude of excitatory postsynaptic current but also the relative contribution of the N-methyl-D-aspartate receptor-mediated component of postsynaptic current recorded by in situ voltage clamp. This effect develops only at increased external Ca2+ concentration and also depends on the external Ca2+/Mg2+ ratio. The increased ratio of N-methyl-D-aspartate/non-N-methyl-D-aspartate components of excitatory postsynaptic current remains at a new level after the removal of 8-cyclopentyl-1,3-dimethylxanthine, even though the amplitude of excitatory postsynaptic current returns close to control value. These results indicate the existence of a mechanism that preferentially enhances the N-methyl-D-aspartate component of excitatory postsynaptic current when the A1 adenosine receptors are blocked and imprints the newly acquired ratio of corresponding excitatory postsynaptic current components.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Animals; Calcium; Hippocampus; In Vitro Techniques; Ion Channels; Magnesium; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P1; Synapses; Theophylline

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Dizocilpine Maleate; Evoked Potentials; Hippocampus; In Vitro Techniques; Magnesium; Models, Neurological; N-Methylaspartate; Picrotoxin; Potassium Chloride; Purinergic P1 Receptor Antagonists; Pyramidal Cells; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P1; Synapses; Synaptic Transmission; Theophylline; Time Factors