| Objective:The spinal cord is the primary center of human motion control.There is a complex cholinergic nervous system that can directly or indirectly participate in spinal motor control.Nicotinic acetylcholine receptors are widely distributed in the ventral horn of the spinal cord and have important modulation functions for spinal cord motor control.To explore the effect of etomidate on the electrophysiological properties of spinal ventral horn neurons and nAChR.This will not only help to deepen the understanding of the spinal cord mechanism of etomidate’s anesthetic effect,but also provide a new theoretical basis for guiding clinical medication.Methods:Neonatal Sprague Dawley(SD)rats aged 7-12 days were selected.After anesthesia,the spinal cord containing lumbosacral enlargement was isolated and sliced.The slices were digested with papain(0.18 g/30 ml artificial cerebrospinal fluid)at constant temperature for 20-25 minutes and incubated for 40-50 min.At the ventral horn,acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes,and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons.In current-clamp mode,the spontaneous action potential(AP)of ventral horn neurons in the spinal cord was recorded firstly,then the effects of different concentrations of ET on AP in ventral horn neurons were recorded with pretreatment administration.In voltage-clamp mode,nicotine was applied to induce inward currents in the ventral horn neurons.Then,combined with pretreatment administration,the effects of ET on inward currents induced by nicotine in the ventral horn neurons were recorded under different concentrations of ET,different holding potentials and different use time.Results:(1)The isolated ventral horn neurons were in good condition with large diverse somata and intact processes.(2)The isolated spinal ventral horn neurons(n=21)had spontaneous action potentials,and were continuously perfused for 2 min by ET with concentration of 0.3,3.0 and 30.0?mol/L.Compared with those before administration,AP amplitude,spike potential amplitude and overshoot were concentration-dependently suppressed(ANOVA,P(27)0.01,n=12),and spontaneous discharge frequency was obviously reduced(P(27)0.01).The APs of other 9 neurons were completely abolished by ET at 3.0 or 30.0?mol/L.(3)At the same holding potential(V_H=-70 mV),after pretreatment respectively with 0.3,3.0 and 30.0?mol/L ET for 2 min,the current amplitude induced by 0.4 mmol/L nicotine showed a concentration-dependent suppression(P(27)0.01,n=7).(4)The holding potentials were set to-30,-50,and-70 mV,respectively.After pretreatment with 30.0?mol/L ET for 2 min,the current amplitude induced by 0.4mmol/L nicotine was voltage-dependently suppressed(P(27)0.01,n=6 for each holding potential).(5)During the 6 min of 30.0?mol/L ET pretreatment,the clamped cells were exposed to 0.4 mmol/L nicotine four times at 0,2,4,and 6 min(each exposure time was 2 s),respectively,the nicotinic current amplitude was gradually decreased as the number of exposure increased.However,if the same concentration of nicotine exposed only twice at the beginning(0 min)and the end(6 min)during the 6 min pretreatment,the inhibition rate was significantly reduced than that when nicotine exposed four times(P(27)0.01,n=6).Conclusion:The neurons in the ventral horn of spinal cord after acute mechanical separation are in good condition,and suitable for patch-clamp experiments.ET reduces excitability of spinal ventral horn neurons in a concentration-dependent manner and suppresses nAChR-mediated currents in concentration-dependent,voltage-dependent,and use-dependent manners. |
PDF Full Text Request