| Object:The subthalamic nucleus（STN）is one of the major nuclei of the basal ganglia and the only excitatory nucleus in which the changes in neuronal electrical activity play an important role in regulating the excitability of motor cortex neurons.Apelin was originally extracted from bovine gastric secretion,its precursor peptide containing 77amino acids was cleaved to produce active peptides such as apelin-13,apelin-17 and apelin-36.Apelin-13 has the strongest activity.The apelin receptor APJ belongs to G protein-coupled receptor family.Apelin,as a bioactive peptide,plays extensive and important roles in both the central nervous systems and peripheral organ systems.It has been reported that apelin regulates the spontaneous discharge activity of neurons in the hypothalamus and other nuclei,and then participates in the regulation of related physiological functions.Morphological studies have revealed the expression of APJ in the subthalamic nucleus,but the regulation of apelin on spontaneous discharge of subthalamic neurons and its effects on motor behaviors are not clear.Therefore,using the immunohistochemistry,in vivo single unit electrophysiological recordings and behavioral tests,we investigated the electrophysiological effects of apelin in the STN and its involvement in the regulation of motor behaviors in the present study.Method:1.Immunofluorescence labeling technique was used to observe the expression of both apelin and APJ in the STN.2.In vivo extracellular single unit recordings were used to study the effects of both exogenous and endogenous apelin on the spontaneous discharge of rat subthalamic neurons.3.The behavioral tests,including elevated body swing test（EBST）,open field test and haloperidol induced rigidity test,were used to reveal the effects of microinjection of apelin into STN on motor functions in rats.Results:1.Immunofluorescence staining showed that both apelin and APJ were expressed in the STN,suggesting that apelin may regulate neuronal electrical activity in an autocrine or paracrine manner.2.Effects of different concentrations of exogenous apelin-13 on the spontaneous discharge of STN neuronsThe spontaneous discharges of total 129 rat subthalamic nucleus neurons were recorded in the present extracellular electrophysiological experiments using three-barrel microelectrodes.The discharge frequency ranged from 0.73 Hz to 33.44 Hz,and the average discharge frequency was 5.52±0.53 Hz.（1）Effects of 10μM apelin-13 on the spontaneous firing rate of the subthalamic neurons.In 26 out of the 45 STN neurons recorded,the firing rate was significantly increased from 4.38±0.81 Hz to 8.07±1.24 Hz（t=-5.511,P=0.000）after micro-pressure administration of 10μM apelin-13,the average increase was 120.43±17.23%,which was significantly increased（z=-3.718,P=0.000）compared to that of vehicle（normal saline）group（basal:10.18±2.78 Hz,vehicle:10.29±3.01 Hz,average change:-0.19±5.41%,n=6）.However,in another 13 neurons,the firing rate was decreased from 10.12±2.68 Hz to 2.07±0.57 Hz（t=2.835,P=0.016）,the average decrease was71.99±6.61%,which was significantly decreased（z=3.378,P=0.000）compared to that of vehicle（normal saline）group.Apelin-13 had no significant effect on the spontaneous firing rate in the remaining 6 STN neurons.（2）Effects of 1μM apelin-13 on the spontaneous firing rate of the subthalamic neurons.Application of 1μM apelin-13 increased the frequency of spontaneous firing from 3.46±1.01 Hz to 6.82±1.32 Hz in 8 out of the 17 subthalamic neurons（t=-3.406,P=0.011）.The average increase was 164.04±45.75%.Apelin-13 at 1μM decreased the firing rate from 12.52±3.54 Hz to 7.00±0.24 Hz in other 2 out of the17 subthalamic neurons（t=1.461,P=0.382）,with the average decrease of 38.63±19.25%.In the remaining 7 STN neurons,1μM apelin-13 did not alter the firing rate significantly.（3）Effect of 0.1 m M apelin-13 on the spontaneous firing rate of subthalamic neurons.After microinjection of 0.1 m M apelin-13,a total of 13 STN neurons were recorded,of which the spontaneous firing rate of 6 neurons increased from 7.70±3.13Hz to 10.94±3.65 Hz（t=-3.906,P=0.011）with an average increase of 105.06±44.46%,and the spontaneous firing rate of another 3 neurons decreased with an average change 67.35±18.51%.（4）Effects of 1 m M apelin-13 on the spontaneous firing rate of neurons.Among the 12 STN neurons recorded,1 m M apelin-13 could increase the spontaneous firing rate in 10 neurons,with an average increase of 114.67±17.80%,but no inhibitory effect was recorded at this concentration.The apelin-13-induced percentage of reaction were58.82%（10/17）,86.67%（39/45）,69.23%（9/13）,66.67%（8/12）over a concentration range of 1μM to 1 m M,respectively.According to the percentage of cells responding to different concentrations of apelin-13,the proportion of positive cells of 10μM apelin-13 was the highest.The above experimental results suggested that exogenous apelin-13 in the STN exerted a dual effect of excitation and inhibition on the spontaneous discharge of neurons,mainly the excitation effect.3.Effects of endogenous apelin on the spontaneous discharge of STN neuronsMorphological study showed that the neurons in the STN expressed both apelin and APJ.To further observe whether the endogenous apelin in the STN participate in the regulation of neuronal spontaneous discharge,ML221,the apelin receptor blocker,was directly given to block the effects of endogenous apelin and then observe the effects on neuronal spontaneous discharge.In a total of 36 STN neurons recorded,micro-pressure administration of ML221（10μM）decreased the spontaneous firing rate of 13neurons from 7.66±1.27 Hz to 2.97±0.92 Hz（t=5.311,P=0.000）.The average decrease was 64.31±5.50%.In other 12 neurons,10μM ML221 increased the spontaneous firing rate from 3.08±1.03 Hz to 4.98±1.46 Hz（t=-3.249,P=0.008）,with the average increase of 71.54±10.49%.In the remaining 11 neurons,ML221 had no significant effect on the spontaneous discharge frequency of neurons.These results suggested that the endogenous apelin in the STN also exerted bidirectional effects on the neuronal spontaneous discharge.4.Effects of exogenous and endogenous apelin on the spontaneous discharges of the same STN neuronsThe above electrophysiological experiments showed that both exogenous and endogenous apelin exerted bidirectional effects on the spontaneous discharge of the subthalamic neurons.In further experiments,apelin-13 and ML221 were applied to the same recorded neuron via two glass microelectrodes respectively to observe whether the effects of exogenous and endogenous apelin on the spontaneous discharge of STN neurons were consistent.The results showed that,among the 13 recorded STN neurons,micro-pressure administration of apelin-13 increased the spontaneous firing frequency in 8 neurons.Within 2 out of the 8 neurons,the spontaneous firing rate was also increased after micro-pressure administration of ML221（apelin-13:increased from1.25±0.50 Hz to 2.29±0.29 Hz,with an average increase of 129.15±114.86%;ML221:increased from 1.71±0.05 Hz to 3.64±1.50 Hz,with an average increase of114.69±93.10%）;ML221 inhibited the firing rate in another 2 neurons（apelin-13:increased from 7.27±3.19 Hz to 9.30±4.22 Hz,with an average increase of 26.90±2.49%;ML221:decreased from 7.29±3.51 Hz to 5.01±3.39 Hz,with an average decrease of 39.66±17.44%）.The spontaneous discharge frequency of the other 4neurons did not change significantly after micro-pressure injection of ML221（apelin-13:increased from 2.49±1.45 Hz to 7.29±5.28 Hz,with an average increase of 134.58±37.97%;ML221:changed from 1.87±0.79 Hz to 2.20±0.75 Hz,with an average change of 31.02±17.31%）.The spontaneous firing rate of 2 neurons decreased after micro-pressure administration of apelin-13,and the firing rate of 1 neuron increased after micro-pressure administration of ML221,while the other did not change significantly.Among the remaining 3 neurons,1 neuron had no change in discharge frequency after micro-pressure administration of apelin-13,while ML221 could increase its discharge frequency;the other 2 neurons did not show any change of discharge frequency after injection of apelin-13 and ML221.These results suggested that the effects of exogenous and endogenous apelin on spontaneous discharge frequency of the same STN neuron were not completely consistent.5.Effects of apelin in the STN on motor behaviors in rats（1）In the EBST,rats receiving unilateral intrasubthalamic normal saline injection did not show a significant swing tendency（48.33±7.03%,n=6）,but unilateral microinjection of 10μM apelin-13 into the STN induced a strong contralateral-biased swing in 6 rats（75.00±3.42%,n=6,z=2.622,P=0.009 compared to that of vehicle）.However,in another 4 rats,unilateral microinjection of apelin-13 into the STN induced a strong ipsilateral-biased swing（92.50±4.79%,n=4,z=-2.343,P=0.024 compared to that of vehicle）.In addition,unilateral microinjection of ML221 alone induced ipsilateral-biased swing of 80.00±11.55%（n=4,z=-2.582,P=0.010 compared to that of vehicle）and contralateral-biased swing of 60.00±10.00%（n=3,z=-0.809,P=0.548 compared to that of vehicle）.Furthermore,co-application of 10μM apelin-13with ML221 did not induce any clear contralateral or ipsilateral-biased swing in 6 rats tested（z=-2.463,P=0.004 and z=-2.574,P=0.000 compared with apelin-13-induced contralateral and ipsilateral-biased swing group,respectively）.（2）The total distance traveled of each group was significantly different(F（3,35）=4.835,P=0.006)in the open field experiment.The total distance traveled did not change significantly in the apelin-13-treatment animals compared to the control animals（control:3984.93±393.77 cm,apelin-13:3206.73±496.86 cm,P=0.311）.After micro-pressure administration of ML221,the movement distance changed significantly（ML221:5765.30±624.89 cm,n=12,P=0.019 compared to that of control）.When apelin-13 and ML221 were given in combination,there was no significant change in movement distance compared with apelin-13 treatment group（apelin-13+ML221:3659.79±546.64 cm,P=1.00）.（3）In haloperidol-induced rigidity experiment,unilateral microinjection of vehicle into the STN did not induce any fixed postural asymmetry in rats（n=4）.Furthermore,in the presence of haloperidol,unilateral administration of apelin-13 induced a significant contralateral deflected posture in 6 rats,while a significant ipsilateral deflected posture was induced in another 4 rats.Next,microinjection of ML221 alone induced an ipsilateral deflection dominated posture（n=4）,as well as a contralateral deflection dominated posture（n=3）.Finally,co-application of apelin-13 with ML221had no significant deflection behavior（n=6）.Conclusion:The present morphological,electrophysiological and functional results showed that neurons in the STN expressed both apelin and APJ.Both exogenous and endogenous apelin could bidirectionally regulate the spontaneous discharge frequency of the subthalamic neurons,with mainly excitatory effects.Further behavioral study revealed that the subthalamic apelin participate in the regulation of motor behaviors in rats probably via modulating the spontaneous firing activity of neurons.The present study may provide a theoretical and experimental basis for further exploring apelin and its receptor system in STN as a potential target of movement disorders. |
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