Bidirectional Regulatory Effects And Receptor Mechanisms Of Cannabinoids On The Globus Pallidus Neurons Of Rats

Endocannabinoids（eCBs）are bioactive substances involved in central motor control,which play important physiological functions in pain,appetite,addiction,mood,habit formation,learning and memory,reward and motivational behavior.The globus pallidus（GP）is involved in regulating body movement and forms basal ganglion circuit together with other nuclei.Both cannabinoid type 1 receptor（CB₁R）and cannabinoid type 2 receptor（CB₂R）are expressed in the globus pallidus,suggesting that the globus pallidus is the area of action for eCBs.The globus pallidus neurons receive gamma-aminobutyric acid innervation from the medium spiny neurons in the striatum and the axon collaterals of neighboring pallidal neurons.CB₁R is mainly located in the axonal terminals of striatopallidal projection neurons in the globus pallidus.The biphasic pattern of changes in the eCBs system during the Parkinson’s disease stages,suggests that the eCBs system plays an important role in Parkinson’s disease.Previous studies showed that the level of anandamide in cerebrospinal fluid of primary parkinsonian patients without dopamine treatment is more than twice as high as that in the treatment group.Objective:To investigate the electrophysiological effects and receptor mechanisms ofcannabinoid on pallidal neurons in rats,and to explore the effects and receptor mechanisms of cannabinoid in globus pallidus on motor behavior.Methods:In vivo extracellular single unit electrophysiological recording,elevated bodyswing test,pole test,haloperidol-induced postural test and immunohistochemical staining were performed in the present study.Results:1.The CB₁R and CB₂R non-selective agonists,WIN 55,212-2（0.01 mM）,had different effects on the spontaneous firing rate of 101 pallidal neurons.The spontaneous firing rate of 55 neurons was significantly increased from 13.46±0.98 Hz to 17.54±1.13 Hz（t=-12.727,df=54,P<0.001）after the globus pallidus was injected with WIN 55,212-2,the average increase was 37.08±2.97%.The spontaneous firing rate was significantly decreased from 16.41±1.25 Hz to 9.94±0.93 Hz（t=8.004,df=16,P<0.001）in 17neurons,the average decrease was 38.68±3.74%.In 12 pallidal neurons,the spontaneous firing rate was firstly increased from 18.74±3.97 Hz to 23.02±4.81 Hz（t=-3.583,df=11,P<0.01）with the average increase of 33.05±7.01%,and then decreased from 18.74±3.97 Hz to 12.33±3.35 Hz（t=3.576,df=11,P<0.01）with the average decrease of 45.99±8.48%.In the remaining 17 pallidal neurons,the change in firing rate was less than two standard deviations（basal:12.30±2.41 Hz,WIN 55,212-2:12.76±2.47 Hz,average change:+6.58±2.85%）.There was no significant difference in the firing rate before and after injection of artificial cerebrospinal fluid in control group（basal:13.30±1.23 Hz,vehicle:13.76±1.25 Hz,t=-3.409,df=34,P>0.05,average change:+4.53±1.38%）.After injection of WIN 55,212-2 into pallidal neurons,the increase in firing rate was negatively correlated with the basal firing rate（r=-0.580,P<0.001）.After injection of WIN 55,212-2 into pallidal neurons,the decrease in firing rate did not show any correlation with the basal firing rate（r=0.164,P>0.05）.In the neurons with WIN 55,212-2-induced increase and then decrease in firing rate,the increase of firing rate was negatively correlated with the basal firing rate（r=-0.626,P<0.05）,while the decrease of firing rate was positively related with the basal firing rate（r=0.597,P<0.05）.2.After injection of the CB₁R antagonist,AM 251（0.01 mM）,into pallidal neurons,thespontaneous firing rate was significantly decreased from 13.83±2.08 Hz to 9.35±1.84 Hz（t=6.006,df=11,P<0.001）in 12 neurons,with the average decrease of 37.93±6.43%.The spontaneous firing rate was significantly increased from 12.97±5.49 Hz to 16.62±5.24 Hz（t=-6.289,df=2,P<0.05）in 3 neurons,with the average increase of 39.92±16.47%.In the remaining 8 pallidal neurons,the change in firing rate was less than two standard deviations（basal:14.10±1.57 Hz,AM 251:13.76±1.54 Hz）.In the artificial cerebrospinal fluid group,vehicle did not change the firing rate significantly（basal:15.68±1.97 Hz,vehicle:15.90±1.98 Hz,t=-0.699,df=9,P>0.05,average change:+2.02±2.10%）.Administration of the CB₂R antagonist,AM 630（0.01 m M）,did not change the firing rate（basal:15.35±2.30 Hz,AM 630:15.72±2.37 Hz,t=-1.967,df=18,P>0.05）significantly in total 19 neurons.In addition,a high concentration of AM 630（1 m M）remained unchanged of the firing rate of the pallidal neurons（basal:11.28±3.13 Hz,AM630:11.53±3.02 Hz,t=-0.293,df=4,P>0.05）.The above results suggested that pallidal eCBs modulated the spontaneous firing activity of neurons via CB₁R.3.In 8 pallidal neurons,application of WIN 55,212-2 significantly increased thespontaneous firing rate from 14.10±2.47 Hz to 17.27±2.40 Hz（t=-8.376,df=7,P<0.001）,with the average increase of 31.80±10.59%.Co-application of AM 251 blocked WIN 55,212-2-induced increase in firing rate（basal:12.80±2.20 Hz,AM 251+WIN55,212-2:12.52±2.23 Hz）,with the average change of-2.22±3.08%（Z=-3.361,P<0.001 compared with WIN 55,212-2 alone,n=8）.To eliminate the possible desensitization of the second injection of WIN 55,212-2 in pallidal neurons,WIN 55,212-2 was injected twice in the same neurons（from 3 rats）.The results showed that the second injection of WIN 55,212-2 exerted similar excitatory effects with the first injection in 5 pallidal neurons（basal:18.74±4.18 Hz,first administration:25.14±4.93 Hz,average increase:40.34±9.54%;basal:18.78±4.26 Hz,second administration:24.37±4.88 Hz,average increase:35.31±8.74%,P>0.05 compared with first administration of WIN 55,212-2）.In addition,in 2 neurons with WIN 55,212-2 induced inhibitory response（-56.22±7.02%）in firing rate,co-application of AM 251 with WIN 55,212-2 significantly blocked WIN 55,212-2-induced inhibition（average change:+10.76±10.58%）.Moreover,in another 8 neurons,application of WIN 55,212-2 significantly increased the spontaneous firing rate from 21.22±3.44 Hz to 26.15±3.97 Hz,with the average increase of 26.84±4.74%,the second time co-application of AM 630 with WIN 55,212-2 could not block WIN 55,212-2-induced increase in firing rate（basal:21.12±3.64 Hz,AM 630+WIN 55,212-2:25.15±4.04 Hz）,with the average increase of 23.41±4.78%,（Z=-0.630,P>0.05 compared with WIN55,212-2 alone,n=8）.The results indicated that exogenous cannabinoids modulated the spontaneous firing activity through CB₁R in the globus pallidus.4.Behavioral tests were applied to further observe whether pallidal cannabinoids areinvolved in motor control in rats.The results of the elevated body swing test showed that unilateral microinjection of artificial cerebrospinal fluid into the globus pallidus did not induce any remarkable biased swing（contralateral-biased swing 50.00±2.36%,n=9）.Compared to the control group,unilateral microinjection of WIN 55,212-2 into the globus pallidus induced a strong contralateral-biased swing in 9 rats（contralateral-biased swing:81.11±2.60%,n=9,Z=3.641,P<0.001）.However,in another 4 rats,unilateral microinjection of WIN 55,212-2 into the globus pallidus induced a strong ipsilateral-biased swing（82.50±4.79%,n=4,Z=-2.874,P<0.01）.Furthermore,co-application of AM251 with WIN 55,212-2 did not induce any clear contralateral or ipsilateral-biased swing（contralateral-biased swing:50.00±2.18%,n=7;Z=3.416,P<0.001 and Z=-2.446,P<0.05 compared with WIN 55,212-2-induced contralateral and ipsilateral-biased swing group,respectively）.While,co-application of AM 630 with WIN 55,212-2 still induced a contralateral-biased swing（contralateral-biased swing:83.33±2.11%,n=6,Z=-0.764,P>0.05 compared with WIN 55,212-2-induced contralateral-biased swing group）.In addition,unilateral microinjection of AM 251 alone induced an ipsilateral-biased swing（ipsilateral-biased swing:76.67±3.33%,n=6,Z=-3.262,P<0.001 compared to that of vehicle）,while AM 630 alone did not induce any remarkable biased swing（contralateral-biased swing:53.33±3.33%,n=6,Z=0.891,P>0.05 compared to that of vehicle）.5.Rats with intraperitoneal haloperidol injection showed stiffness,unilateralmicroinjection of artificial cerebrospinal fluid into the globus pallidus did not induce any specific deflection posture in rats（n=10）.Furthermore,unilateral administration of WIN55,212-2 induced a significantly contralateral deflection posture during 60 minutes in 14rats,and the scores were stayed between 1 and 3.While WIN 55,212-2 induced a significantly ipsilateral deflection posture and the scores were stayed between-1 and-2 in another 3 rats.Microinjection of AM 251 alone induced an ipsilateral deflection and the scores were stayed between-1 and-3（n=6）.But AM 630 did not induce any clear specific deflection posture the scores were about 0（n=6）.Co-application of AM 251 with WIN55,212-2 blocked the deflection posture induced by WIN 55,212-2,the scores were about0（n=7）.While co-application of AM 630 with WIN 55,212-2 did not block the deflection posture induced by WIN 55,212-2,the scores were stayed between 1 and 3（n=6）.6.Bilateral microinjection of WIN 55,212-2 into the globus pallidus significantly reduced the time to climb down the pole compared with the control group（n=6,t=4.941,P<0.001）in normal rats.Microinjection of AM 251 did not change the time for normal rats to climb down the pole compared with the control group（n=6,t=1.222,P>0.05）,and AM 630 also did not change the time of climbing down the pole（n=6,t=2.322,P>0.05）.Furthermore,compared with WIN 55,212-2 group,co-application of AM 251+WIN55,212-2 significantly blocked WIN 55,212-2-induced reduction of time to climb down the pole（n=9,t=3.565,P<0.05）,but co-application of AM 630+WIN 55,212-2 did not change WIN 55,212-2-induced effects on the time to climb down the pole（n=6,t=0.514,P>0.05）.The above behavioral studies（4,5,6）demonstrated that cannabinoids modulated motor behaviors through its regulation of the firing activity of pallidal neurons.7.Immunohistochemical staining showed that the CB₁R was expressed in the globus pallidus.Conclusion:The present in vivo single unit electrophysiological recordings demonstrated that exogenous application of WIN 55,212-2 exerts complex effects,including increase,decrease and increase then decrease,on the spontaneous firing activity of pallidal neurons.Furthermore,behavioral tests showed that application of WIN 55,212-2 into the globus pallidus mainly enhanced motor behavior in normal rats,and eCBs also participated in the regulation of motor behavior in normal rats.Finally,the CB₁R was involved in WIN 55,212-2-induced both electrophysiological and behavioral effects.