The Effect And Mechanism Of Orexin-A On α₄β₂-nAChR In Spinal Ventral Horn Neurons

Objective:To study the effect of orexin-A onα₄β₂nicotinic acetylcholine receptors（α₄β₂-nAChRs）in isolated ventral horn neurons of the neonatal rat spinal cord and its mechanism.Methods:In this study,newborn SD rats at age of 8-12 days were anesthetized with ether combined with an ice bath,and the spinal cord was rapidly freed from the cervical end to the sacral region,with the lumbosacral dilated segments preserved and slices prepared.The slices were respectively incubated for 30 min,digested with papain for 19-22 min,and incubated at room temperature for 45-60 min.The ventral horn of the spinal cord was cut along the central canal,and the neurons were acutely mechanically separated using Pasteur pipettes from large to small caliber.Functional studies of neurons were performed.In current-clamp mode,the spontaneous action potential（AP）of spinal ventral horn neurons was recorded to determine the good status of acutely isolated neurons;the voltage-clamp mode was applied,and theα₄β₂-nAChR selective agonist RJR-2403 was firstly applied to record the current evoked in ventral horn neurons through a rapid gravity drug delivery system,and orexin-A was applied for 2 min after current stabilization to observe its modulation of RJR-2403 current.RJR-2403 current’s modulation by orexin-A,was tested by sequential administration of OX₁R selective antagonist SB334867,OX₂R selective antagonist TCSOX229,PKC blocker and PKA blocker to analyze the mechanism of orexin-A effect on RJR-2403current.Results:（1）In this experiment,the neurons were recorded from the ventral horn neurons of the spinal cord.The neurons recorded had intact cytosol,diverse morphology,and could send out more neurites and had branches;（2）The AP of neurons in the ventral horn of the spinal cord is stable and continuous with overshoot,indicating that the electrophysiological characteristics of the cells are good;（3）The mean amplitude of current evoked by application of 20μmol/L RJR-2403on spinal ventral horn neurons was（221.00±24.44）p A,and pretreatment of 100nmol/L orexin-A for 2 min significantly reduced the current amplitude to（97.26±18.53）p A（P<0.001）,and the inhibition ratio was（59.97±3.07）%（n=31）;（4）In the eight ventral horn neurons recorded,the co-application of the OX₁R selective antagonist SB334867（10μmol/L）and the OX₂R selective antagonist TCSOX229（10μmol/L）abolished the orexin-A inhibition of RJR-2403 currents（P<0.05）;（5）Application of either SB334867（n=12）or TCSOX229（n=7）alone partially abolished the inhibitory effect of orexin-A on RJR-2403 currents;（6）In 10 neurons,intracellular application of the PKC inhibitor Bis-IV（10μmol/L）,orexin-A still depressed RJR-2403 currents by（35.02±18.90）%,and in other6 neurons,after intracellular application of the PKA inhibitor Rp-c AMP（50μmol/L）,orexin-A suppressed RJR-2403 currents by（44.13±11.40）%（P<0.05）;（7）When the Ca²⁺chelator BAPTA was applied intracellularly,orexin-A inhibited RJR-2403 current with the inhibition ratio of（52.82±10.65）%（n=5,P<0.05）.Conclusions:（1）The spinal cord ventral horn neurons recorded in this experiment are three-dimensional,morphologically rich,with more protrusions and good cellular status,which are suitable for patch-clamp recording and pharmacological mechanism study;（2）Spontaneous action potentials of the neurons recorded with current-clamp recording were continuous and stable,and the overshoot was obvious,suggesting that the electrophysiological properties of acutely isolated neurons were well maintained;（3）RJR-2403 currents mediated byα₄β₂-nAChR in spinal ventral horn neurons can be suppressed by orexin-A.The experimental results suggest that orexin-A may act in combination with both OX₁R and OX₂R,while PKA and PKC are also involved in its modulation.