Roles Of Opioid Receptors On Parallel Fiber-Purkinje Cell Synaptic Transmission In Cerebellum In Mice

[Objective]Opioids exert pharmacological effects by acting on their receptors.There are three types of opioid receptor in the central nervous system,namely mu-opioid receptor(MOR),delta-opioid receptor(DOR)and Kappa-opioid receptor(KOR).The distribution density of opioid receptor in different brain regions is different,activation of opioid receptor can participate in learning and memory,emotional response,addiction and other physiological functions.In recent years,cerebellum has received more and more attention in non-motor coordination functions,such as participating in cognition,learning and memory,and reward.Purkinje cell(PC)is the only efferent neuron in cerebellum,which is synaptically connected with the parallel fiber(PF)formed at the axon teminal of granule cell.PF-PC synaptic transmission plays a key role in storing the input signals of cerebellar cortex and dynamically regulating the release of glutamate transmitters.Studies have shown that opioid receptors are distributed and expressed during the growth and development of cerebellum cells.However,the distribution of opioid receptors and their subtypes in PF-PC synapses in mice cerebellum and their effects on synaptic transmission are unclear.Therefore,we use whole-cell recording technique and pharmacological methods to investigate the effect of activating opioid receptors on the cerebellar PF-PC synaptic transmission,and to clarify the opioid receptor subtypes on PF-PC synapses and the mechanisms of related synaptic transmission.[Methods]1.KM male mice(6-8 weeks old)weighing 28-33 g were selected as experimental animals.The mice were weighed and placed in an anesthesia machine,and inhaled isoflurane for anesthesia,and then quickly severed and the brain was extracted.Then,sagittal slice of cerebellar cortex was prepared after automatic vibration slicing machine,and the slice thickness is 300 microns.After the cerebellar sections were incubated in an artificial cerebrospinal fluid(aCSF)mixed with 95%O2 and 5%CO2 at room temperature,Its pH value is 7.4 and osmotic pressure is about 295-305 mOsm.Last incubation for 60～90 min for electrophysiological recording.2.Whole cell patch clamp recording of cerebellar slices that the stimulation electrode was placed in the molecular layer to stimulate parallel fibers,with the paired stimulus pulse of 0.5 Hz(0.2 ms;10-100 A;duration:50 ms),The recording electrode was used to clamp PC cells in the voltage clamp mode,and the excitatory postsynaptic currents(EPSC)of PC were recorded under stable condition.3.The Clampfit 10.4 software was used to analyze the electrophysiological data.All results are expressed as mean ±SEM.Statistical significance was estimated with Student’s paired t-test or one-way ANOVA using SPSS software.P values below 0.05 were considered to indicate a statistically significant difference.[Results]1.Application of MOR agonist,DAMGO(2μM)on the cerebellar slices induced a decrease in the amplitude,AUC and an increase in PPR of PF-PC excitatory postsynaptic currents(PF-PC EPSCs)in the presence of picrotoxin.(P<0.05)2.The decrease of DAMGO-induced in the amplitude of PF-PC EPSCs was concentration-dependent,and the half-inhibitory concentration(IC50)of DAMGO for inhibiting PF-PC EPSCs was 5.17 μM.3.Perfusion of selective MOR antagonist,CTOP(5 μM)alone obviously increased the amplitude,AUC of PF-PC EPSCs,accompanied with a significant decrease in PPR in the presence of picrotoxin.(P<0.05).4.Perfusion of co-application of CTOP and DAMGO(2 μM),blocked the changes of DAMGO-induced inhibition in amplitude,AUC and PPR of PF-PC EPSCs in the presence of picrotoxin.5.Application of DOR agonist,DPDPE(500 nM)on the cerebellar slices induced a decrease in the amplitude,AUC and an increase in the PPR of PF-PC EPSCs in the presence of picrotoxin.(P<0.05)6.The decrease of DPDPE-induced in the amplitude of PF-PC EPSCs was concentration-dependent.And the half-inhibitory concentration(IC50)of DPDPE for inhibiting PF-PC EPSCs was 0.66 μM.7.Perfusion of selective DOR antagonist,naltrindole(1 μM)alone obviously increased the amplitude,AUC of PF-PC EPSCs,accompanied with a significant decrease in PPR in the presence of picrotoxin.(P<0.05)8.Perfusion of co-application of naltrindole and DPDPE(500 nM),the changes of DPDPE-induced in the amplitude,AUC and the PPR of PF-PC EPSCs were abolished by a selective DOR antagonist,naltrindole.9.Application of KOR agonist,U50488(1 μM)on the cerebellar slices perfusion failed to change the amplitude,AUC and PPR of PF-PC EPSCs in the presence of picrotoxin.(P>0.05)[Conclusion]1.MOR and DOR may be present at the axonal teminal of PF in the mouse cerebellar cortex,but but there is none or a small amount of KOR.2.Activation of MOR and DOR regulate PF-PC synaptic transmission via inhibiting the release of glutamate in cerebellar cortical in mice.