Study In The Mechanisms Underlying Orexin-A Inhibition On GABA Currents Of The Spinal Cord Ventral Horn Neurons Of Neonatal Rats

OBJECTIVE:To investigate the effect of orexin-A on the functions of ionotropicγ-aminobutyric acid（GABA）receptors in neonatal rats’spinal cord ventral horn neurons and its mechanisms.METHODS:Neonatal SD rats aged 7-12 days were selected.The spinal cord containing lumbosacral enlargement was isolated and sliced after anesthesia.The slices were digested with papain（0.18 g/30 ml artificial cerebrospinal fluid）and incubated for 40-60min,then the ventral horns were retained under the microscope.The fire-polished Pasteur pipettes with different calibers were used to separate individual cells acutely and mechanically.After the cells adhered to the bottom of Petri dishes,the neurons in good condition were chosen and recorded with patch-clamp technique combined with pharmacological methods.In current-clamp mode,the spontaneous action potential（AP）of ventral horn neurons in the spinal cord was recorded.The drugs were applied through the rapid perfusion drug delivery system,respectively.In the voltage-clamp recording mode,the GABA currents were firstly recorded byγ-aminobutyric acid,the GABA receptor agonist,in the spinal ventral horn neurons,then the effect of pretreatment of orexin-A for 2 minutes on the GABA currents was tested.Based on this,OX₁R selective antagonist SB334867,OX₂R selective antagonist TCSOX229 and PKC activator as well as inhibitor were respectively applied to analyze the mechanisms underlying the effect of orexin-A on the GABA currents.RESULTS:（1）The neurons isolated from spinal cord ventral horns maintained good morphology with diverse cell bodies,smooth surfaces and long protrusions.（2）The basic electrophysiological parameters of spontaneous action potentials of spinal ventral horn neurons:resting potential,（-58.85±1.94）mV;threshold,（-48.31±1.82）mV;spike potential amplitude,（61.75±2.11）mV;overshoot,（12.96±3.32）mV;discharge frequency,（10.50±2.69）Hz（n=4）.（3）The current amplitude induced by 0.3 mmol/L GABA in the isolated ventral horn neurons（n=49）in the spinal cord was（734.57±423.76）p A,pretreatment with 100nmol/L orexin-A for 2 min could significantly inhibit the current amplitude to（228.02±141.83）p A（P<0.001）,the inhibition rate was（67.48±12.50）%.（4）In 6 neurons of the ventral horn,simultaneous presence of orexin-1 receptor（OX₁R）selective antagonist SB334867（10μmol/L）and orexin-2 receptor（OX₂R）selective antagonist TCSOX229（10μmol/L）showed the suppressive effect of orexin-A on the GABA currents was completely abolished by the two receptor antagonists.（5）Separately applied with SB334867（n=8）or TCSOX229（n=8）,the suppressive effect of orexin-A on the GABA currents was also partially relieved.（6）After intracellular administration of 10μmol/L Bis-IV（PKC inhibitor）in the 5 ventral horn neurons,orexin-A could no longer suppress the GABA currents（P>0.05）;In 6ventral horn neurons pretreatment with 1μmol/L PMA（PKC activator）for 2 min mimicked the effect of orexin-A,which significantly inhibited the GABA currents,the inhibition rate was（60.79±10.94）%,based on this,the GABA currents were not further suppressed after application of orexin-A（P>0.05）.Conversely,intracellular administration of PKA inhibitor Rp-c AMP（50μmol/L）still depressed the GABA currents（n=5,P<0.01）,the inhibition rate was（63.08±12.82）%.（7）The inhibition by orexin-A of the GABA currents was constant in the extracellular Ca²⁺-free solution（n=8）or in the presence of BAPTA（n=7）,an intracellular Ca²⁺chelator.The inhibition rates were（65.97±18.29）%and（67.29±11.35）%,respectively.CONCLUSIONS:（1）The neurons in the ventral horn of the spinal cord obtained in this experiment maintain good morphology with smooth surfaces,transparent cell bodies,which is suitable for applying patch-clamp recording technology to analyze neuronal receptors and intracellular signal transduction mechanisms.（2）The recorded spontaneous action potentials of the neurons in the ventral horn of the spinal cord are continuous and stable.The overshoot indicates that the cells are in good condition.（3）Orexin-A inhibits the GABA currents in the ventral horn neurons of the spinal cord,which is probably caused by activation of OX₁R,OX₂R and non-Ca²⁺dependent PKC.