Direct excitation of hypocretin/orexin cells by extracellular ATP at P2X receptors.
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.
Wollmann
Guido
G
Department of Neurosurgery, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut 06520, USA.
Acuna-Goycolea
Claudio
C
van den Pol
Anthony N
AN
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002794:Choline / Q000494:pharmacology; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007031:Hypothalamus / Q000166:cytology*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D064446:Orexin Receptors; D000068797:Orexins; D018408:Patch-Clamp Techniques; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D013498:Suramin / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; C546402:P2rx2 protein, mouse; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D049452:Green Fluorescent Proteins; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D013779:Tetrodotoxin; D011732:Pyridoxal Phosphate; D013498:Suramin; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D015763:2-Amino-5-phosphonovalerate; D000255:Adenosine Triphosphate; D002794:Choline; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D001640:Bicuculline