Comparative analysis of the effects of orexin-A and RJR-2403 on spontaneous action potentials of spinal ventral horn neurons
Objective:To investigate different effects of orexin-A and α4β2-N-type acetylcholine receptor(α4β2-nAChR)selective agonist RJR-2403 on the spontaneous action potentials of isolated spinal ventral horn neurons in neonatal rats.Methods:The spinal cord from cervical to sacral segments was initially dissociated from neonatal Sprague-Dawley rats(8-12 days old)after anesthesia,and the lumbosacral enlargement segment was preserved to prepare slices.The slices were 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 excised along the central canal,and the neurons were acutely mechanically dissociated using Pasteur pipettes of large to small calibers and left for 20 min for cell adhesion.Good neurons were recorded using patch clamp under inverted microscope,and functional studies were conducted by patch-clamp recordings combined with pharmacological methods.Under the current-clamp recording mode,the effects of orexin-A and RJR-2403 on the spontaneous action potential of spinal ventral horn neurons in vitro were observed,and the differences of various parameters were compared.Results:①In this experiment,the acutely isolated spinal ventral horn neurons were recorded by patch-clamp recordings.The recorded neurons had intact somata,diverse morphologically,and could emit many protrusions with branches;②The spontaneous action potential frequency of ventral horn neurons was increased significantly from the initial(3.55±0.66)Hz to(8.26±3.31)Hz after application of orexin-A(P<0.01),and action potential amplitude was decreased from the initial(95.28±7.21)mV to(85.15±5.80)mV(P<0.05),yet no significant changes were observed in the half-amplitude duration of resting potential and action potential;③After application of RJR-2403,the resting potential of ventral horn neurons depolarized from the initial(-66.67±43.38)mV to(-60.48±3.38)mV(P<0.01).The firing frequency was significantly accelerated from the initial(2.74±1.60)Hz to(8.94±3.14)Hz(P<0.001),whereas the action potential amplitude was notably reduced from the initial(84.96±4.85)mV to(73.77±5.42)mV(P<0.01),and the half-width was increased from(4.34±1.52)ms to(5.39±1.80)ms(P<0.01).Conclusion:Both orexin-A and RJR-2403 have positive modulatory effects on spontaneous action potentials in spinal ventral horn neurons,yet produce different effects on the resting potentials and half-width,which is probably due to the differences in the effects mediated by metabotropic receptors and ionotropic receptors.