6-cyano-7-nitroquinoxaline-2-3-dione and alpha-beta-methyleneadenosine-5--triphosphate

Purinergic P2X receptors have been reported to be present in regions of the midbrain periaqueductal gray (PAG). The purpose of this study was to determine the role of presynaptic P2X receptors in modulating excitatory and inhibitory synaptic inputs to the dorsolateral PAG (dl-PAG), which has abundant neuronal connections. First, whole cell voltage-clamp recording was performed to obtain excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) of the dl-PAG neurons. Our data show that alpha, beta-methylene ATP (a P2X receptor agonist), in the concentration of 50 microM, significantly increased the frequency of miniature EPSCs without altering the amplitude of miniature EPSCs in eight tested neurons. The effects were attenuated by PPADS, an antagonist to P2X receptors. Furthermore, alpha, beta-methylene ATP increased the amplitude of evoked EPSCs, and decreased the paired-pulse ratio of eEPSCs in ten neurons. In contrast, activation of P2X had no distinct effect on IPSCs. In addition, immunofluorescent methods demonstrate that P2X labeling was co-localized with a presynaptic marker, synaptophysin, in the dl-PAG. The results of the current study provide the first evidence indicating that P2X receptors facilitate glutamatergic synaptic transmission in the dl-PAG via presynaptic mechanisms.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Analysis of Variance; Animals; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Glutamic Acid; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Periaqueductal Gray; Platelet Aggregation Inhibitors; Presynaptic Terminals; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Synaptic Transmission; Synaptophysin

Ionotropic purine receptors (P2X) have been implicated in nociceptive neurotransmission. In this study, we examine the actions of the P2X receptor agonist alpha,beta methylene adenosine 5'-triphosphate on excitatory neurotransmission in neurons in the deep and superficial laminae of the trigeminal spinal subnucleus caudalis (Vc), which receives nociceptive inputs from the craniofacial region. Alpha, beta methylene adenosine 5'-triphosphate caused an increase in spontaneous excitatory neurotransmission (miniature excitatory postsynaptic currents) in neurons in deep but not superficial laminae of Vc; this effect could be inhibited by the P2X receptor antagonist 2,3-O-2,4,6-trinitrophenyl-ATP. Conversely, the TRPV1 agonist capsaicin caused an increase in miniature excitatory postsynaptic currents in neurons in the superficial but not deep laminae. These data suggest that alpha,beta methylene adenosine 5'-triphosphate acts on presynaptic terminals to increase glutamatergic neurotransmission in deep Vc neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Analgesics, Non-Narcotic; Animals; Animals, Newborn; Capsaicin; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Trigeminal Caudal Nucleus

Hypocretin/orexin (hcrt) neurons play an important role in hypothalamic arousal and energy homeostasis. ATP may be released by neurons or glia or by pathological conditions. Here we studied the effect of extracellular ATP on hypocretin cells using whole cell patch-clamp recording in hypothalamic slices of transgenic mice expressing green fluorescent protein (GFP) exclusively in hcrt-producing cells. Local application of ATP induced a dose-dependent increase in spike frequency. In the presence of TTX, ATP (100 microM) depolarized the cells by 7.8 +/- 1.2 mV. In voltage clamp under blockade of synaptic activity with the GABA(A) receptor antagonist bicuculline, and ionotropic glutamate receptor antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), ATP (100 microM) evoked an 18 pA inward current. The inward current was blocked by extracellular choline substitution for Na+, had a reversal potential of -27 mV, and was not affected by nominally Ca2+-free external buffer, suggesting that ATP activated a nonselective cation current. All excitatory effects of ATP showed rapid attenuation. ATP-induced excitatory actions were mimicked by nonhydrolyzable ATP-gamma-S but not by alpha,beta-MeATP and inhibited by the purinoceptor antagonists suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS). The current was potentiated by a decrease in bath pH, suggesting P2X2 subunit involvement. Frequency and amplitude of spontaneous and miniature synaptic events were not altered by ATP. Suramin, but not PPADS, caused a small suppression of evoked excitatory synaptic potentials. Together, these results show a depolarizing response to extracellular ATP that would lead to an increased activity of the hypocretin arousal system.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Analysis of Variance; Animals; Bicuculline; Choline; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; Green Fluorescent Proteins; Hypothalamus; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Neuropeptides; Orexin Receptors; Orexins; Patch-Clamp Techniques; Pyridoxal Phosphate; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Suramin; Tetrodotoxin

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Bicuculline; Excitatory Amino Acid Antagonists; Fluorescent Dyes; GABA Antagonists; Glycine; Membrane Potentials; Neural Inhibition; Neural Pathways; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Presynaptic Terminals; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X4; Substantia Gelatinosa

The pyramidal neurons in the CA1 area of hippocampal slices from 17- to 19-day-old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 microM 2-amino-5-phosphonovaleric acid (D-APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 +/- 11% of the total EPSC when measured at a holding potential of -50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) 10 microM and bath incubation with non-hydrolysable ATP analogues, ATP-gamma-S and alpha, beta-methylene-ATP at 50 and 20 microM, respectively. The rEPSC was dramatically potentiated by external Zn2+ (10 microM). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one-fifth to one-quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Hippocampus; Neurons; Organ Culture Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X4; Stimulation, Chemical

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of alpha,beta-methylene ATP (alpha,beta-meATP) caused reproducible depolarizations which were depressed by suramin (30 microM) and abolished by suramin (100 microM). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10, 30 microM) also concentration-dependently inhibited the alpha,beta-meATP-induced depolarization, although with a much slower time-course than suramin. Almost complete inhibition developed with 30 microM PPADS. Reactive blue 2 (30 microM) did not alter the effect of alpha,beta-meATP, while reactive blue 2 (100 microM) slightly depressed it. 2. Pressure-applied (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 microM) depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) abolished the response to AMPA. Suramin (100 microM) potentiated the AMPA effect. 3. Pressure-applied noradrenaline hyperpolarized LC neurones. Suramin (100 microM) did not alter the effect of noradrenaline. 4. Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(-)-2-amino-5-phosphonopentanoic acid (AP-5; 50 microM), CNQX (50 microM) and picrotoxin (100 microM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 microM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP-5 (50 microM), CNQX (50 microM), picrotoxin (100 microM), strychnine (0.1 microM), tropisetron (0.5 microM) and hexamethonium (100 microM), a high concentration of suramin (300 microM) almost abolished the p.s.p. without changing the i.p.s.p. 5. In the presence of kynurenic acid (500 microM) and picrotoxin (100 microM), PPADS (30 microM) depressed the p.s.p. Moreover, the application of suramin (100 microM) to the PPADS (30 microM)-containing medium failed to cause any further inhibition. Neither PPADS (30 microM) nor suramin (100 microM) altered the i.p.s.p. 6. It was concluded that the cell somata of LC neurones are endowed with excitatory P2-purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co-transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Kynurenic Acid; Locus Coeruleus; Male; Picrotoxin; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Suramin

Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholine; Adenosine Triphosphate; Animals; Bicuculline; Brain; Chlorocebus aethiops; Evoked Potentials, Somatosensory; gamma-Aminobutyric Acid; Ganglionic Blockers; Glutamates; Glutamic Acid; Hexamethonium; Hexamethonium Compounds; In Vitro Techniques; Ion Channels; Kynurenic Acid; Mecamylamine; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Signal Transduction; Suramin; Tetrodotoxin