| Orexin,including orexin A and orexin B,is a member of neuropeptides produced by the orexinergic neurons located in the lateral hypothalamic and the perifornical area.Targeting to orexin 1 receptor(OX1R)and orexin 2 receptor(OX2R),orexin is involved in the regulation of multiple physiological functions,such as feeding and energy homeostasis,sleep and wakefulness,learning and memory,and motor control.The substantia nigra pars compacta(SNc)is a part of the basal ganglia circuitry involved in central motor control.Selective death of dopaminergic neurons in SNc is responsible for the motor disorders of Parkinson’s disease(PD).Morphological studies suggest that the orexinergic fibers project to the SNc,and the dopaminergic neurons in SNc express both OX1 R and OX2 R.The number of orexinergic neurons and the orexin levels in cerebrospinal fluid are decreased in both parkinsonian patients and animal models of PD.Besides,orexin A and orexin B exert protective effects in cellular and animal models of PD.Objective: To observe the electrophysiological effects and the mechanisms of orexin on nigral dopaminergic neurons and the modulation of nigral orexin on motor behavior in normal rats.Methods: In vivo extracellular single unit electrophysiological recording,elevated body swing test(EBST),haloperidol-induced postural test,high performance liquid chromatography and immunohistochemical staining were performed in the present study.Results: 1.Thirty-six dopaminergic neurons were recorded in orexin A group.Orexin A increased the firing rate from 2.11±0.25 Hz to 3.20±0.32Hz(t=8.380,P=0.000)in 25 out of the 36 dopaminergic neurons,and the average increase was 65.87±7.73%.While the firing rate was increased to 3.68±0.26 Hz from 2.17±0.23Hz(t=6.443,P=0.000)in 25 out of the 33 nigral dopaminergic neurons after the injection of orexin B,and the average increase was 90.49±17.83%.2.Selective OX1 R antagonist,SB 334867,was injected in 28 nigral dopaminergic neurons.The spontaneous firing rate was decreased from 2.99±0.27 Hz to 1.86±0.28Hz(t=5.787,P=0.000)in 21 out of the 28 neurons.The decrease of the spontaneous firing rate was averagely 36.77±6.26%.TCS OX2 29 decreased the spontaneous firing rate to 2.24±0.41 Hz from 3.19±0.43Hz(t=4.424,P=0.003)in 9 out of the 15 nigral dopaminergic neurons,and the average decrease was 32.04±6.12%.3.SB 334867 and orexin A were co-applicated in 6 nigral dopaminergic neurons excited by orexin A.The average increase of firing rate induced by co-application of SB 334867 and orexin A was 14.06±2.18%,which was significantly weaker(z=-2.882,P=0.004)than that of orexin A alone(51.51±4.78%)in the same neurons.TCS OX2 29 was co-injected to investigate the role of OX2 R in orexin B-induced excitatory effects.The average increase induced by orexin B alone was 69.60±24.00%,while co-application of orexin B and TCS OX2 29 hardly increased the firing rate in nigral dopaminergic neurons(z=-2.562,P=0.010,n=6).4.Inhibitor of L type calcium channels,nifedipine,and orexin A were co-applicated in 4 nigral dopaminergic neurons.Orexin A alone increased the firing rate by 113.17±36.48%,while co-application of nifedipine and orexin A could not increase the firing rate with an average change of-26.65±13.58%(z=-2.309,P=0.029).Furthermore,co-application of nifedipine and orexin B decreased the firing rate by 17.70±23.56%,while orexin B alone increased the firing rate by 119.07±39.17% in the same six neurons.Coapplication of nifedipine and orexin B significantly blocked orexin B-induced increase in firing rate(z=-2.402,P=0.015).5.Microinjection of orexin A and orexin B in SNc induced contralateral-biased swing at a rate of 88.0±7.3%(n=5,z=-2.455,P=0.014)and 95.0±2.2%(n=6,z=-2.802,P=0.008)respectively in elevated body swing test(EBST).While SB 334867 and TCS OX2 29 could inhibit the behavioral effects induced by orexin A and orexin B in the EBST.The percentage of ipsilateral-biased swing induced by SB 334867 and TCS OX2 29 was 92.5±4.8%(n=4,z=-2.481,P=0.013)and 88.0±5.8%(n=5,z=-2.635,P=0.008)respectively.6.In haloperidol-induced postural test,orexin A and orexin B induced contralateral deflection posture during 60 minutes and the scores were maintained at 1.0~2.0,which was abolished by SB 334867 and TCS OX2 29 respectively.SB 334867 and TCS OX2 29 alone induced ipsilateral deflection posture,and the scores stayed between-1.5 and-2.0.7.The content of dopamine in the striatum treated with orexin A in the ipsilateral SNc was 9.34±1.37ng/mg,which was higher than that of the other side treated with normal saline(7.48±0.98ng/mg,n=7,t=3.004,P=0.024).Orexin B also promoted dopamine release in the striatum compared with normal saline(orexin B: 10.05±1.16ng/mg,normal saline: 8.0±0.78ng/mg,n=6,t=2.825,P=0.037).8.Immunohistochemical staining showed that both OX1 R and OX2 R were expressed in the SNc.Conclusion: Our present studies indicate positive expression of both OX1 R and OX2 R in the SNc.Exogenous orexin A increases the firing rate of nigral dopaminergic neurons mainly via OX1 R,while exogenous orexin B exerts the excitatory effects through OX2 R.Endogenous orexin participates in the modulation of the spontaneous firing activity of nigral dopaminergic neurons through both OX1 R and OX2 R.L type calcium channel is involved in orexin-induced excitatory effects on nigral dopaminergic neurons.Exogenous orexin A and orexin B promote the release of dopamine in nigrostriatal pathway.Finally,nigral orexin is involved in the central motor control. |
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