| The subthalamic nucleus plays an important role in the indirect pathway of the basal ganglia,whose functional activity transformation directly impacts on Parkinson's disease.In clinical,deep brain stimulus is used as an effective way to treat Parkinson's disease.The exact target of deep brain stimulus is the subthalamic nucleus,which can greatly improve the movement symptoms of Parkinson's disease.The orexins are neuropeptides produced by a specific group of neurons.Orexinergic neurons are located in lateral hypothalamic area which widely innervate central motor control system,such as the basal ganglia.Morphologic studies have already confirmed that the subthalamic nucleus receives orexinergic innervation and expresses both orexin-1 and orexin-2 receptors.Objective: In our study,we aimed to investigate the modulation of exogenous and endogenous orexins on the spontaneous firing rate and pattern of the subthalamic neurons in the normal wistar rats.Furthermore,we observed the regulation of motor activity through orexins effects in the subthalamic nucleus.Methods: In vivo extracellular single unit recordings were used to observe the effects of orexins on the spontaneous firing rate,and firing patterns including the co-efficient of variance(CV)and the fano factor(FF)of the inter-spike interval(ISI).The elevated body swing tests(EBSTs)were performed to observe the effects of microinjection of orexins and receptor antagonists on motor activity.Results: 1.Total 109 subthalamic neurons were recorded in present electrophysiological experiment.The basal spontaneous firing rate ranged from 0.18 Hz to 16.22 Hz with the average firing rate of 6.88±0.30 Hz.Based on the ISI histogram and autocorrelogram,three firing types,regular firing(32.11%),irregular firing(44.95%)and bursty firing(22.94%),were identified in the 109 neurons.2.In total 25 subthalamic neurons recorded,micro-pressure ejection of orexin-A increased the basal spontaneous firing rate in 16 out of the 25 subthalamic neurons.The average increase was79.71±12.86%,which was statistically different compared to vehicle injection(normal saline,Z=-4.342,P=0.000).In the remaining 9 subthalamic neurons,orexin-A did not change the firing rate significantly(t=-1.876,P=0.097).3.Micro-pressure ejection ofSB-334867 significantly decreased the firing rate by 45.27±3.18% in 7 out of the total 16 subthalamic neurons recorded(t=5.603,P=0.001),which was statistically different compared to vehicle injection(Z=-3.000,P=0.003).SB-334867 did not change the firing rate significantly in other 9 neurons(t=2.187,P=0.060).4.Co-application of orexin-A and SB-334867 changed the firing rate by 29.62±3.93% in only 5 out of the total 24neurons(t=-5.636,P=0.005),while did not change the firing rate(5.59±2.92%)significantly in the remaining 19 out of the 24 subthalamic neurons(t=-1.784,P=0.091).Chi-square test revealed that the percentage of orexin-A responsive neurons(64%)was significantly higher(?2=9.317,d.f.=1,P=0.002)than that of SB-334867 with orexin-A responsive neurons(20.83%),suggesting that SB-334867 could block orexin-A-induced effects.Further analysis revealed that the average increase of co-application of SB-334867 and orexin-A was significantly different from the excitation induced by orexin-A alone(Z=-3.64,P=0.000).In the remaining 5 neurons,application of SB-334867 and orexin-A did not block orexin-A-induced excitation.The result suggested that orexin-A increased the firing rate of the subthalamic neurons mainly through orexin-1receptor.5.Micro-pressure ejection of orexin-B increased the firing rate by71.80±15.22% in 17 out of the total 21 neurons,which was significantly different from that of vehicle injection(Z=-4.777,P=0.000).Furthermore,a medium negative correlation between orexin-B-induced excitation and the basal firing rate was observed in the subthalamic neurons(r=-0.679,P=0.007).The firing rate of the other 4 neurons showed no significant difference before and after drug application(t=-2.496,P=0.088).6.Micro-pressure ejection of TCS-OX2-29 decreased the firing rate by 47.42±7.32% in 10 out of total 16 neurons,which was significantly different from that of vehicle injection(Z=-3.78,P=0.000).The firing rate of the other 6 neurons tended to decrease but the difference was not significant(t=1.387,P=0.224).7.Co-application of orexin-B and TCS-OX2-29 changed the firing rate by 6.79±4.86% in the total 7 neurons,which was significantly different from that of orexin-B alone treatment(Z=-3.263,P=0.001).8.In the EBSTs,microinjection of different drugs showed significantly different motor activities(F=16.919,P=0.000).Micro-injection of orexin-A and orexin-B caused contralateral-biased swing(q=10.54,P=0.000;q=7.029,P=0.000),while micro-injection of SB-334867 and TCS-OX2-29 caused ipsilateral-biased swing(q=8.614,P=0.000;q=8.565,P=0.000).Conclusion: The present in vivo electrophysiological studies revealed thatexogenous application of orexin-A and orexin-B increased the spontaneous firing rate of the subthalamic neurons through orexin-1 and orexin-2 receptors,respectively.Endogenous orexins increased the excitability of the subthalamic neurons through both orexin-1 and orexin-2 receptors.Subthalamic orexinergic system modulated motor activity in awake rats.Our present studies may provide a rationale and evidence for further investigations in the involvement of subthalamic orexinergic systems in Parkinson's disease. |
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