| OBJECTIVE: To investigate the effects of orexin-A on basic electrophysiological parameters and nicotinic acetylcholine receptors(n ACh Rs)in spinal ventral horn neurons.METHODS: Neonatal SD rats of 7-12 days were used.After anesthesia with ether-assisted ice bath,the spinal cord containing the lumbosacral enlargement was freed,and 4-5 pieces of spinal cord slices with thickness of 350-500 ?m were prepared.After incubation for about 0.5 h,digest the sections for 20-25 minutes with digestive enzyme(Papain,0.18 g/30 ml artificial cerebrospinal fluid ACSF).Stop digestion and incubate for50 minutes.The ventral horn was selected for acutely mechanical dissociation of neurons with fire-polished micro-Pasteur pipette.Single cells were dissociated and perforated patch-clamp recordings were performed.RESULTS:(1)The isolated ventral horn neurons were in good condition with large diverse somata and intact processes.(2)Nine acutely isolated spinal ventral horn neurons have spontaneous action potentials,of which five neurons have a discharge frequency of(5.88 ± 1.87)Hz and the other four neurons have a discharge frequency of(16.75 ± 5.40)Hz.There was a significant difference(P < 0.01).The resting potential of the low-frequency discharge neurons was (-66.54 ± 6.48)m V,the action potential amplitude was(76.08 ± 10.07)m V,and the resting potential of the high-frequency discharge neurons was(-52.74 ± 5.27)m V,and the action potential amplitude was(55.54 ± 7.46)m V,both were significantly different(P <0.05).There were no significant differences in the remaining electrophysiological parameters of the cells.(3)After 2 minutes of continuous perfusion of orexin-A(OXA),the spontaneous action potential discharge frequency of the four spinal ventral horn neurons was increased(63.64 ± 5.25)%,with no significant change in other parameters,such as amplitude.(4)In 12 of the 15 neurons,0.3 mmol/L nicotine induced inward current with amplitude of(142.97 ± 75.02)p A,and bath application with 100 nmol/L orexin-A for 2 minutes significantly inhibited the current amplitude to(69.07 ± 61.07)p A(P < 0.001),that is,inhibition ratio was(54.75 ± 22.62)%.(5)In 5 of the 7 neurons,the nicotine-induced current amplitude was(127.94 ± 68.78)p A,100 nmol/L orexin-A was co-administered to the neurons,and the nicotine-induced current amplitude decreased to(64.58 ± 34.03)p A.On this basis,the inhibitory effect of orexin-A on nicotine-induced current was blocked by administration of the orexin-1receptor(OX1R)antagonist SB334867(10 ?mol/L)for 2 minutes.The current amplitude was(111.06 ± 58.25)p A.Therefore,SB334867 completely nullified the inhibition of nicotine current by orexin-A.But for the other two neurons,SB334867 did not prevent the inhibition of orexin-A.CONCLUSION: The ventral horn neurons of the spinal cord isolated from the experiment are large and three-dimensional,with many protrusions and multiple branches,and the cell state is good.The recorded spontaneous action potentials of neurons can be divided into low frequency and high frequency according to the discharge frequency.The resting potential and action potential amplitude of these two types of cells are significantly different,suggesting that there may be two or more cells clamped in this experiment;orexin-A can increase the discharge frequency of neurons in the ventral horn of the spinal cord,but other electrophysiological parameters have no significant changes.Orexin-A,probably via OX1 Rs,has an inhibitory action on n ACh Rs in most of ventral horn neurons in spinal cord,but the possibility of OX2 R involvement in the orexin-A effects cannot be excluded either. |
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