Effect Of Orexin-A On Glycine Currents In Spinal Cord Ventral Horn Neurons And Its Mechanisms

Objective:To explore the effect and mechanisms of orexin-A on the glycine receptors in the spinal cord ventral horn neurons.Methods:Neonatal SD rats（7-12 d）were used.After anesthesia,the spinal cord containing lumbosacral enlargement was isolated,and several slices with thickness of400～500μm were cut with the aid of a vibrating microtome.The slices were digested with digestive enzyme（Papain,0.18 g/30 mL ACSF）and incubated in ACSF for 40～60min,and then the spinal cord slices were morphologically observed by microscopy.Moreover,the ventral horn was selected for acute mechanical dissociation of neurons by fire-polished micro-Pasteur pipettes with different calibers.Observed with an inverted fluorescent microscope,the neurons adhering to the bottom of petri dish in 10～20 min after dissociation could be selected for electrophysiological recordings.Results:1.Morphology of the spinal cord ventral horn neuronsThe isolated ventral horn neurons were in good condition with large diverse somata and intact processes.2.Electrophysiological parameters of spinal cord ventral horn neuronsThe spontaneous action potentials of 10 acute isolated ventral horn neurons of spinal cord were recorded,and the discharge frequency was continuous and stable,and almost all of them had overshoots.The main electrophysiological parameters were basically consistent with those of the previous studies.3.Effect of orexin-A on the glycine currents in the spinal cord ventral horn neuronsIn the 66 cases of spinal ventral horn neurons recorded,given 100 nmol/L orexin-A pretreatment for 2 min,the amplitude of the glycine currents in the neurons could be significantly increased and stabilized to 171.54±40.46%of control（P<0.01）.4.OX₁R mediates the enhancement of the glycine currents by orexin-AIn 14 cells,100 nmol/L orexin-A was applied to potentiate the glycine currents to176.21±25.91%of control（P<0.01）,and then 10μM SB334867（OX₁R selective antagonist）pretreatment for 2 min could completely nullify the increase of the glycine currents by orexin-A,the current amplitude was 100.90±1.75%of control（P>0.05）.In the other 7 cells,100 nmol/L orexin-A was given to potentiate the glycine currents to166.40±39.87%of control（P<0.001）,and applying 10μmol/L TCSOX229（OX₂R selective antagonist）for pretreatment for 2 min,orexin-A invariably potentiated the glycine current amplitude to 164.05±40.53%of control（P<0.01）.5.The effect of Ca²⁺on the orexin-A-induced glycine current enhancementWhen the isolated neurons were bathed in Ca²⁺-free extracellular solution,orexin-A still increased the current amplitude to 168.95±31.55%of control（n=7,P<0.01）.While,orexin-A effect on the glycine currents was completely blocked by internal infusion of 10 m M BAPTA,a calcium chelator,the glycine currents obtained during the BAPTA infusion were 102.35±6.99%of control（n=11,P>0.05）.The orexin-A effect was totally abolished by Heparin（5 mg/mL）,an IP₃ receptor antagonist,the current amplitude was 98.52±3.77%of control（n=8,P>0.05）.In addition,internal infusion of 20μmol/L Xe-C,another IP₃ receptor antagonist,also acquired a similar result.After pretreatment with orexin-A for 2 min,the glycine currents were still 100.64±1.85%of control（n=5,P>0.05）.While,during internal infusion of 50μM ryanodine,a ryanodine receptor blocker,pretreatment with orexin-A for 2 min still potentiated the glycine current amplitudes to 175.43±32.91%of control（n=9,P<0.01）.6.PKC is involved in the increase of the glycine currents by orexin-AInternal infusion of a PKC inhibitor Bis-IV（10μmol/L）also nullified the orexin-A effect,and the glycine current amplitude was 100.65±2.02%of control（n=17,P>0.05）.In addition,orexin-A did not cause a further enhancement of the currents after bath application of 1μmol/L PMA（n=5,P>0.05）,a PKC agonist.7.PKA is not involved in the orexin-A effect on the glycine currentsDuring internal infusion of 50μmol/L Rp-c AMP,a PKA inhibitor,orexin-A still invariably increased the glycine current amplitude to 177.02±21.31%of control（n=6,P<0.01）.Conclusion:The cells isolated by digestive enzyme are in good condition and fit for patch-clamp recordings.Combined with OX₁R,orexin-A can activate the intracellular IP₃/Ca²⁺/PKC signaling pathway so as to increase the glycine currents of spinal cord ventral horn neurons.