Electrophysiological Effects And Receptor Mechanisms Of Orexin-b On The Globus Pallidus Neurons In Both Normal And MPTP Parkinsonian Mice

Orexins,the neuropeptides released from hypothalamus,play a significant role in the body.The globus pallidus is an important nucleus in the basal ganglia,which participates in movement regulation.Much evidence indicated that the firing activities of the globus pallidus are closely related to Parkinson's disease.Morphological studies have demonstrated the expression of both orexinergic fibers and orexin receptors?OXR?in the globus pallidus.Objective:To investigate the effects and receptor mechanisms of orexin-B on the spontaneous firing of globus pallidus neurons in both normal and MPTP parkinsonian mice.Methods:In vivo extracellular single unit recordings,immunohistochemical staining and behavioral test were performed in the present study.Results:1.Total 67 globus pallidus neurons were recorded in the electrophysiological test of normal mice.The average spontaneous firing rate of pallidal neurons in normal mice was 6.13±0.58Hz.Three firing types were recorded in the globus pallidus,with 45?67.16%?regular firing,18?26.87%?irregular firing,4?5.97%?bursty firing.2.In normal mice,micropressure ejection of 0.1?M orexin-B significantly increased the firing rate from 6.84±1.11Hz to 8.65±1.11Hz in 29 out of the 45 pallidal neurons?t=-8.85,P=0.000?.The average increase was 53.33±8.88%,which was significantly different from that of normal saline group?z=-4.72,P=0.000?.A negative correlation between orexin-B-induced excitation and the basal firing rate was observed in the globus pallidus neurons?r=-0.67,P=0.000?.Analysis of the firing pattern showed that orexin-B did not change the coefficient of variation?CV??basal:0.75±0.10;orexin-B:0.81±0.14;t=-0.98,P=0.34,n=29?and the fano factor?FF??basal:0.62±0.23;orexin-B:0.89±0.36;t=-0.14,P=0.31,n=29?significantly.3.Micropressure ejection of OX₂R antagonist,TCS-OX₂-29?0.01mM?,slightly decreased the firing rate from 3.75±0.86Hz to 3.32±1.12Hz?more than mean±2 standard deviation of the baseline?in only 3 out of the total 22 pallidal neurons,but there was no significant difference?t=1.48,P=0.28?.In the remaining 19neurons,TCS-OX₂-29 did not change the firing rate significantly?basal:5.53±0.68Hz;TCS-OX₂-29:5.69±0.68Hz;t=-1.15,P=0.27?.The average change was 4.17±2.75%,which was not significantly different from that of vehicle group?z=-1.06,P=0.29?.TCS-OX₂-29 did not change the CV?basal:0.94±0.14;TCS-OX₂-29:1.04±0.20;t=0.97,P=0.35,n=19?and FF?basal:0.58±0.21;TCS-OX₂-29:0.57±0.17;t=0.018,P=0.99,n=19?significantly.4.In another set of experiment,micropressure ejection of orexin-B alone increased the firing rate from 3.28±0.51Hz to 5.22±0.76Hz in 9 orexin-B responsive neurons.The average increase was 62.61±13.36%.After recovery of firing rate completely,co-application of TCS-OX₂-29 and orexin-B only increased the firing rate from 2.91±0.70Hz to 3.36±0.77Hz.The average increase of firing rate was18.11±7.23%,which was decreased significantly?z=-2.34,P=0.019?compared with that of orexin-B alone.5.In MPTP-treated parkinsonian mice,the average firing rate was4.60±0.49Hz in 32 pallidal neurons recorded,which was decreased significantly compared with that of normal mice?t=2.02,P=0.047?.Three firing patterns were analyzed in MPTP parkinsonian mice,16?50.00%?regular firing,13?40.62%?irregular firing,3?9.38%?bursty firing.In MPTP parkinsonian mice,the fraction of irregular firing and burst firing neurons tended to be more than that in normal mice but there was no statistical significance?c²=3.02,d.f.=2,P=0.22?.6.In MPTP parkinsonian mice,micropressure ejection of 0.1?M orexin-B increased the firing rate from 4.78±0.57Hz to6.81±0.72Hz?t=-7.70,P=0.000?in 14 out of the 18 pallidal neurons.The average increase was 48.14±8.21%,which excited the pallidal neurons significantly compared to vehicle injection?z=-3.34,P=0.000?.Firing pattern analysis showed that orexin-B did not change the CV?basal:0.93±0.11;orexin-B:0.86±0.12;t=0.83,P=0.42,n=14?and FF?basal:0.40±0.10;orexin-B:0.33±0.12;t=1.17,P=0.26,n=14?significantly.7.In MPTP parkinsonian mice,micropressure ejection of TCS-OX₂-29 weakly decreased the firing rate from 6.33±2.26Hz to 3.77±1.37Hz?more than mean±2 standard deviation of the baseline?in only 3 out of the total 14 pallidal neurons,but there was no significant difference?t=2.79,P=0.11?.In the remaining 11 neurons,TCS-OX₂-29 did not change the firing rate?basal:4.66±1.02Hz;TCS-OX₂-29:4.46±1.17Hz;t=0.26,P=0.81?significantly.Firing pattern analysis showed that TCS-OX₂-29 did not change the CV?basal:1.12±0.20;TCS-OX₂-29:1.09±0.16;t=0.38,P=0.71,n=11?and FF?basal:0.76±0.33;TCS-OX₂-29:0.66±0.21;t=0.76,P=0.47,n=11?significantly.8.The excitation of orexin-B on globus pallidus neurons showed no significant difference between normal and MPTP parkinsonian mice?z=-0.09,P=0.93?.9.In behavioral pole test,bilateral microinjection of orexin-B into the globus pallidus decreased both the T-turn time?time to turn downward from the top?and the T-LA time?time to descend to the floor?significantly compared to that of normal saline?t=2.38,P=0.03;t=5.12,P=0.000,respectively?.In open field test,bilateral microinjection of orexin-B into the globus pallidus increased total distance travelled in MPTP parkinsonian mice.10.In both normal and MPTP parkinsonian mice,positive expression of OX₂R was observed in the globus pallidus.The expression between normal and MPTP parkinsonian mice was not significantly different?z=-1.17,P=0.24?.Conclusion:Micropressure ejection of orexin-B increased the firing rate of pallidal neurons through OX₂R in both normal and MPTP parkinsonian mice.TCS-OX₂-29 only decreased the firing rate in few pallidal neurons,which suggested that endogenous orexins control the spontaneous firing of globus pallidus neurons rarely through OX₂R.Behavioral tests suggested that pallidal orexin-B could improve motor activity.In addition,the present electrophysiological and morphological studies showed that both orexin-B and OX₂R exerted similar effects in both normal and MPTP parkinsonian mice,which may provide a theoretical basis for further investigations of pallidal orexinergic system in the treatment of Parkinson's disease.