| Parkinson’s disease(PD)is a neurodegenerative disease caused by abnormal motor circuits in the basal ganglia.Orexin consists of orexin A(3561Da)and orexin B(2899Da),which exerts functions through both orexin type 1 receptor(OX1R)and orexin type 2receptor(OX2R).Orexin plays an important role in motor control,and most of the central motor control areas are dominated by orexinergic fibers.Studies have shown that the orexin system is closely related to PD.The number of orexinergic neurons and the orexin levels in cerebrospinal fluid is decreased in PD.In addition,studies indicated that orexin A exerts protective effects on both cellular model and animal model of PD.In addition,orexin A could relieve the degeneration of dopaminergic(DAergic)neurons in PD model mice and improve the locomotion disorder of PD model mice.It was suggested that orexin B can protect normal rat derived midbrain DAergic neurons from neurodegeneration,and increase the number of tyrosine hydroxylase immunoreactivity(TH-ir)neurons,which suggests that orexin B may exert similar protective effects on DAergic neurons.However,up to now,the direct electrophysiological effects of orexin B on nigral neurons,as well as the modulation on parkinsonian motor disordersare less known.Objective: To observe the effects of orexin B on the motro disorder of MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)PD model mice,the direct electrophysiological effects of orexin B on substantia nigra neurons in PD model mice,the receptor mechanisms of both the electrophysiological and behavioral effects of orexin B.Methods: Open field test and pole test were used to observe the effects of orexin B on motor behaviors in PD model mice.In vivo extracellular single unit recordings were performed to observe the effects of orexin B on the spontaneous firing activity of nigral neurons.Immunohistochemical stainings were used to expression of TH in the substantia nigra.Results:1.Intracerebroventricular administration of orexin B reversed motor disorders in MPTP mice model of PD1.1 Orexin B increased the spontaneous locomotor activity in MPTP PD model mice.PD model mice treated with MPTP were tested in open field test on day 1 and day 7respectively.The results of day 1 showed that there were significant differences in the total distance traveled between the different groups(P<0.001).Compared with the control group,the total distance traveled in NS+MPTP group was significantly reduced(P<0.001).However,the total distance traveled was significantly increased by intracerebroventricular administration of orexin B in MPTP PD model mice(P<0.001 compared with NS+MPTP group).Co-application of the selective OX2 R antagonist,TCS OX2 29,significantly attenuated the increase induced by orexin B in total distance traveled(P<0.001 compared with orexin B+MPTP group).TCS OX2 29 alone had no significant influence on the total distance traveled in MPTP PD model mice(P>0.05 compared to NS+MPTP group).The results of day 7 showed that there were significant differences in the total distance traveled among all the groups(P<0.01).There was a similar trend of change in the total distance traveled with the first day but no statistical difference between different groups.1.2 Orexin B improved motor coordination in MPTP PD model mice.Pole test wasperformed on day 2 and day 8,respectively.The results showed that there were differences in T-turn time(day 2: P<0.001;day 8: P<0.001)and T-LA time(day 2: P<0.001;day 8: P<0.001)between the two experiments.T-turn time(day 2: P<0.001;day 8: P<0.001)and T-LA time(day 2: P<0.001;day 8: P<0.001)of PD model mice were observably prolonged compared with that of normal mice.However,in MPTP PD model mice,intracerebroventricular administration of orexin B reduced T-turn time(day 2: P<0.001 compared with NS+MPTP group;day 8: P<0.001 compared with NS+MPTP group)and T-LA time(day 2: P<0.01 compared with NS+MPTP group;day 8: P<0.001 compared with NS+MPTP group).The protective effects of orexin B in T-turn time(day 2: P<0.01 compared with orexin B+MPTP group;day 8: P<0.001 compared with orexin B+MPTP group)and T-LA time(day 2: P<0.05 compared with orexin B+MPTP group;day 8:P<0.05 compared with orexin B+MPTP group)were attenuated by co-application of TCS OX2 29.TCS OX2 29 alone did not change the T-turn time(day 2: P>0.05 compared with NS+MPTP group;day 8: P>0.05 compared with NS+MPTP group)and T-LA time(day 2:P>0.05 compared with NS+MPTP group;day 8: P>0.05 compared with NS+MPTP group)in MPTP PD model mice.1.3 Orexin B attenuated the degeneration of nigral DAergic neurons in MPTP PD model mice.The number of TH-ir neurons in the substantia nigra of MPTP-induced PD model mice was significantly decreased(n=3,P<0.01),which was about 55.25±2.39% of that of normal mice.Intracerebroventricular administration of orexin B significantly reduced the loss of DAergic neurons in the substantia nigra of MPTP PD model mice.The number of TH-ir neurons in the orexin B+MPTP group increased to 83.50±2.83%(n=3,P<0.05)compared with NS+MPTP group.2.Regulation of orexin B on the firing activity of nigral neurons in MPTP PD model mice2.1 The spontaneous firing activity of nigral neurons between MPTP PD model mice and normal mice was compared firstly.Three firing patterns(regular,irregular and burst)were found in both of the DAergic neurons and gamma-aminobutyric acid-ergic(GABAergic)neurons.The firing rate of nigral DAergic neurons of MPTP PD model mice was higher than that of normal mice(normal: 2.21±0.11 Hz,n=32;MPTP: 2.71±0.09 Hz,n=53;t=3.44,P<0.01).The firing pattern of nigral DAergic neurons did not change significantly(The coefficient of variation(CV)value of normal mice: 0.48±0.05,n=32;CV value of MPTP PD model mice: 0.50±0.05,n=53,t=-0.35,P>0.05).Furthermore,the firing rate of nigral GABAergic neurons was also increased compared with normal mice(control:8.39±0.29 Hz,n=36;MPTP: 10.16±0.32 Hz,n=48;t=81.60,P<0.001).The CV of GABAergic neurons in PD model mice was higher than that in normal mice(normal:0.31±0.06,n=36;MPTP: 0.51±0.07,n=48,t=-2.18,P<0.05).2.2 Orexin B increased the firing rate of nigral DAergic neurons via OX2 R in MPTP PD model mice.Micro-pressure ejection of 100 n M orexin B significantly increased the firing rate from 2.59±0.21 Hz to 3.92±0.40 Hz(t=-6.31,P<0.001)in 9 out of the 18 nigral DAergic neurons in MPTP PD model mice.The average increase was 49.46±5.13%.However,the firing rate of control group treated with normal saline was not changed significantly,and the average change of firing rate was 4.55±2.13%.The difference between the orexin B group and the normal saline control group was significant(z=-3.18,P<0.001).The CV was increased after the injection of orexin B(basal: 0.66±0.09,orexin B: 0.82±0.07,n=9,t=-2.52,P<0.05).Then,we compared the excitability of orexin B to DAergic neurons of normal mice(basal: 2.26±0.21 Hz,orexin B: 3.14±0.32 Hz,n=8,t=-5.34,P<0.01,average change: 38.04±6.30%)and MPTP PD model mice(49.46±5.13%),and no significant difference was observed between the two groups(z=1.35,P>0.05).We investigated the receptor mechanism of the orexin B-induced excitatory effects on nigral DAergic neurons in PD model mice.In total 12 nigral DAergic neurons,application of orexin B in the presence of 10 μM TCS OX2 29 did not change the firing rate significantly(basal:2.79±0.16 Hz,TCS+orexin B: 2.84±0.22 Hz,n=12,t=-0.56,P>0.05).While orexin B changed the firing rate from 2.57±0.16 Hz to 3.26±0.28 Hz in the total 18 neurons of previous orexin B alone group.The change in firing rate induced by orexin B in the presence of TCS OX2 29(0.90±2.65%)was significantly different from that of orexin B alone(26.37±6.23%,n=18,z=-2.582,P<0.05).In a few neurons with long time stable recordings,the second time application of orexin B alone increased the firing rate from2.97±0.45 Hz to 4.03±0.43 Hz(n=2).These results suggested that orexin B increased the spontaneous firing rate of the nigral DAergic neurons via OX2 R in MPTP PD model mice.2.3 Endogenous orexins were involved in the regulation of spontaneous firing activity of nigral DAergic neurons in PD model mice.Micro-pressure administration of selective OX2 R antagonist,TCS OX2 29,decreased the firing rate from 2.79±0.22 Hz to 1.86±0.29Hz(t=4.25,P<0.01,average change:-34.52±7.43%)in 7 out of the 12 nigral DAergic neurons in MPTP PD model mice.The difference between the TCS OX2 29 group and the normal saline control group was significant(z=-2.842,P<0.01).TCS OX2 29 did not significantly change the CV value of DAergic neurons(basal: 0.46±0.14;TCS OX2 29:0.56±0.14;n=7,t=-1.73,P>0.05).The inhibitory effect of TCS OX2 29 on nigral DAergic neurons in MPTP PD model mice(-34.52±7.43%)was not significantly different from that of normal mice(basal: 2.28±0.33 Hz,TCS OX2 29: 1.61±0.21 Hz,n=5,t=4.84,P<0.01,average variation:-28.75±3.32%,z=-0.57,P>0.05).2.4 Orexin B did not affect the spontaneous firing activity of nigral GABAergic neurons.The firing rate of GABAergic neurons in MPTP PD model mice(basal:10.69±0.55 Hz;orexin B: 11.05±0.75 Hz;n=14,t=-1.44,P>0.05;average change:2.65±1.94%)and normal mice(basal: 7.73±0.62 Hz;orexin B: 7.89±0.72 Hz;n=8,t =-0.98,P>0.05;average change: 1.70±1.63%)was not changed by micro-pressure injection of orexin B.Then,we tried to inject 10 n M orexin B and 10 μM orexin B and found that both high and low concentrations of orexin B did not change the firing rate of GABAergic neurons in MPTP parkinsonian and normal mice.2.5 Endogenous orexins were involved in the regulation of spontaneous firing activity of a small part of nigral GABAergic neurons.In the majority of nigral GABAergic neurons(8 out of 12),application of TCS OX2 29 did not change the firing rate in MPTP PD model mice(basal: 9.76±0.87 Hz;TCS OX2 29: 9.66±0.85 Hz;n=8,t=0.93,P>0.05;average change:-0.94±1.10%).While in only 4 nigral GABAergic neurons,the firing rate was decreased by TCS OX2 29 in PD model mice(basal: 8.28±0.18 Hz;TCS OX2 29:6.13±0.39 Hz;n=4,t=4.36,P<0.05;average change:-25.27±5.51%).In normal mice,8nigral GABAergic neurons were application with TCS OX2 29,and the firing rate of 6neurons did not significantly change,while only 2 GABAergic neurons showed decreased firing rate(basal: 12.16±2.52 Hz;TCS OX2 29: 10.21±2.67 Hz;t=13,P<0.05;average change:-17.02±4.76%).Conclusion: We observed for the first time that intracerebroventricular application of orexin B reversed motor impairments and attenuated the loss of DAergic neurons in MPTP PD model mice.Orexin B increased the firing rate of nigral DAergic neurons via OX2 R in MPTP PD model mice.Orexin B did not change the firing rate of nigral GABAergic neurons.Endogenous orexins were involved in the regulation of the spontaneous firing activity of DAergic neurons and GABAergic neurons in MPTP PD model mice.The present studies would provide some theoretical and experimental basis for the involvement of nigral orexin B in PD. |
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