The Experimental Study Of Subthalamic Orexin-B Modulating Motor Behaviors Via Orexin-2 Receptors In MPTP Parkinsonian Mice

The basal ganglia are situated in the white matter at the base of the cerebral hemisphere which play important roles in movement regulation.The subthalamic nucleus,the unique excitatory nucleus in the basal ganglia,is an important information-integrating nucleus.The abnormality in any part of the functional pathway of the basal ganglia leads to movement disorders such as Parkinson's disease.Orexin produced predominantly in the hypothalamus is a bioactive peptide involved in various biological effects such as motor regulation.It is reported that orexin increases the spontaneous firing rate of the subthalamic neurons and regulates body swing behaviors in normal rats.The subthalamic nucleus expresses abundant orexin-2 receptor(OX-2R).Objective: To investigate the modulation of orexin-B in the subthalamic nucleus on motor behaviors in MPTP parkinsonian mice,and to elucidate the possible mechanisms of subthalamic orexin-B at the single cell level.Methods: Open field test and pole test were used to observe the effects of intrasubthalamic application of orexin-B on motor behaviors in parkinsonian mice.Further in vivo extracellular single unit recordings were performed to observe the effects of orexin-B on the spontaneous firing rate of the subthalamic neurons.Finally,immunohistochemistry was used to observe the expression of OX-2R in the subthalamic nucleus of both normal and MPTP parkinsonian mice.Results: 1.In the behavioral tests of normal mice,bilateral microinjection of orexin-B into the subthalamic nucleus significantly reduced the total travelled distance(t=3.14,P=0.006),and significantly increased the T-turn time(t=2.40,P=0.050)and the T-LA time(t=4.59,P=0.002)compared with normal saline group.2.In the behavioral tests of MPTP parkinsonian mice,the total travelled distance of MPTP parkinsonian mice was significantly reduced(t=4.24,P=0.000),and the T-turn time(t=3.16,P=0.001)and the T-LA time(t=4.10,P=0.004)were significantly increased compared with that of normal mice.In MPTP parkinsonian mice,bilateral microinjection of orexin-B into the subthalamic nucleus significantly reduced the total travelled distance(t=4.73,P=0.000),and significantly increased the T-turn time(t=5.41,P=0.002)and the T-LA time(t=3.14,P=0.006)compared with that of normal saline.However,bilateral microinjection of TCS-OX2-29,the selective OX-2R antagonist,into the subthalamic nucleus significantly increased the total travelled distance(t=3.97,P=0.002),and significantly reduced the T-turn time(t=1.57,P=0.050)and the T-LA time(t=3.77,P=0.000)compared with that of normal saline.Bilateral co-injection of orexin-B and TCS-OX2-29 into the subthalamic nucleus significantly increased the total travelled distance(t=2.79,P=0.017),and significantly reduced the T-turn time(t=3.23,P=0.039)and the T-LA time(t=2.75,P=0.023)compared with that of orexin-B alone group.3.Furthermore,in vivo extracellular recordings were performed to study the cellular mechanisms of orexin-B-induced behavioral modulation at single cell level.Twenty seven subthalamic neurons were recorded in normal mice.The basal spontaneous firing rate ranged from0.77 Hz to 3.12 Hz,with the average spontaneous firing rate at 1.58±0.16 Hz.Sixteen subthalamic neurons were recorded in MPTP parkinsonian mice.The basal spontaneous firing rate ranged from 2.56 Hz to 12.91 Hz.The average spontaneous firing rate was4.79±1.33 Hz which was significantly faster than that of normal mice(t=9.56,P=0.000).4.In normal mice,microinjection of 0.1?M orexin-B significantly increased the firing rate from 1.92±0.17 Hz to 3.64±0.29 Hz in 13 out of the 17 subthalamic neurons(t=4.59,P=0.001).The average increase was 89.71±24.36%.In MPTP parkinsonian mice,microinjection of 0.1?M orexin-B increased the firing rate from 4.51±0.28 Hz to6.60±0.34Hz(t=4.75,P=0.003)in 6 out of the 7 subthalamic neurons.The average increase was 46.81±13.40%.There was no significant difference in orexin-B-induced excitation of subthalamic neurons between normal and parkinsonian mice(z=-1.84,P=0.072).5.In normal mice,microinjection of 0.05 mM TCS-OX2-29 significantly decreased the firing rate from 1.89±0.12 Hz to 1.50±0.11Hz(t=2.32,P=0.050)in 8 out of the 10 subthalamic neurons.The average decrease was 20.76±2.02%.In MPTP parkinsonian mice,microinjection of 0.05 mM TCS-OX2-29 significantly decreased the firing rate from 5.47±0.75 Hz to 2.52±0.37Hz(t=3.54,P=0.005)in 9 subthalamic neurons.The average decrease was 53.42±3.87%.The significant difference was observed between normal and parkinsonian mice(z=-3.46,P=0.001).6.In co-application set of experiment,co-administration of 0.1?M orexin-B and 0.05 mM TCS-OX2-29 increased the firing rate by 14.23±6.66%(basal: 2.46±0.70 Hz,TCS-OX2-29+orexin-B: 2.81±0.83Hz)in 7orexin-B responsive neurons in normal mice,which was significantly different(z=0.00,P=0.034)compared with that of orexin-B alone treatment(basal: 2.17±0.41 Hz,orexin-B:3.98±0.76 Hz,average increase of 78.41±13.36%).In 4 orexin-B responsive subthalamic neurons in MPTP parkinsonian mice,co-administration of 0.1?M orexin-B and 0.05 mM TCS-OX2-29 changed the firing rate by 8.12±5.71%(basal: 3.32±0.42 Hz,TCS-OX2-29+orexin-B: 3.59±0.56Hz),which was significantly different(z=0.00,P=0.021)compared with that of orexin-B alone treatment(basal: 3.28±0.51 Hz,orexin-B:5.30±1.07 Hz,average increase of 45.73±4.99%).7.The expression of OX-2R in the subthalamic nucleus was observed in both normal and MPTP parkinsonian mice.There was no significant difference in the expression of OX-2R between normal and MPTP parkinsonian mice(z=-1.02,P=0.309).The positive expression of orexin-A neurons was observed in the lateral hypothalamus via immunofluorescence staining.Conclusion: Orexin-B in the subthalamic nucleus is involved in the regulation of motor behaviors in both normal and MPTP parkinsonian mice via OX-2R.Intrasubthalamic application of TCS-OX2-29 alleviates motor deficits in MPTP parkinsonian mice.Exogenous orexin-B increases the excitability of the subthalamic neurons in both normal and MPTP parkinsonian mice,and endogenous orexin modulates the spontaneous discharge of the subthalamic neurons through OX-2R.OX-2R is expressed in the subthalamic nucleus of both normal and MPTP parkinsonian mice.