Roles Of Mice Cerebellar Parallel Fiber To Purkinje Cell Synaptic Transmission In Morphine-Conditioned Place Preference

[Objective] Our present study used brain stereotactic technology,immunofluorescence staining,animal behavior,whole-cell patch clamp and neuropharmacology to investigate the roles of mouse cerebellar synaptic transmission from parallel fibers(PF)to Purkinje cells(PC)in morphine conditioned place preference(CPP).[Materials and Methods] 1.6-8 weeks old adult male Kunming mice with a weight of 20-24 g were selected as the experimental animals and were randomly divided into Morphine group and Saline group.Mice of the Morphine group were received morphine conditional place preference training for 5 consecutive days,and they were immediately placed on the one side of the preference box(this side was defined as normal saline side)for 30 min after intraperitoneal injection of saline(10 mg/kg)in the morning,and after intraperitoneal injection of morphine hydrochloride(10 mg/kg)in the afternoon,they were immediately placed on the other side of the preference box(this side was defined as morphine side)for 30 min;and the mice of the Saline group underwent the same experimental operation but injected saline in the morning and afternoon.During the testing phase,two groups of mice were separately placed into conditional place preference boxes without separators for 15 min to explore freely and the duration of their stay on each side was recorded.The time difference obtained by the residence time on the morphine side minus the residence time on the saline side is the morphine conditional place preference score.2.Within 48 h after CPP testing,the brains of mice that had formed preferences were rapidly severed after isoflurane inhalation anesthesia,and the 300 μm thickness cerebellar sagittal slices were prepared in Fully Automated Vibratory Microtome,and then the cerebellar slices were rapidly transferred to artificial cerebrospinal fluid(ACSF)under room temperature condition and containing sufficient O2(95%)and CO2(5%)mixture of gases.After the cerebellar slices were incubated for at least 60 min,wholecell patch clamp recording was performed.The mini excitatory postsynaptic current(m EPSC)of cerebellar PCs were recorded by voltage clamping at-70?m V in the presence of picrotoxin(GABAA receptor antagonist)and TTX(sodium channel blocker).3.Mice were randomly divided into control group and h M4D(Gi)group,and p AAV-Ca MKIIa-MCS-m Cherry and p AAV-Ca MKIIa-h M4D-m Cherry viruses were injected at the cerebellar granule cell layer(GCL)of the mice,respectively.On the 17 th day after the virus injection,a nuclear tube was implanted.Behavioral experiments were conducted 3 days after recovery.In the morphine CPP experiment,15 min before each morphine CPP condition,0.3 μl clozapine-N-oxide(CNO)was injected into the GCL of the mice cerebellum by a micro-syringe pump at a rate of 180 nl/min.The openfield experiment was performed before virus injection(day 0),half month after virus expression(day 16),and 3 days after nuclear tube surgery(day 20)to assess the motility of two groups of mice before and after the injection of the virus and before and after the surgery of nuclear tube.4.As above,mice in the control group and h M4 D group formed the preference after perfusion,the brains were perfused and collected.Cryosections were cut at 15 μm.for immunofluorescence staining to detect virus expression.5.Virus-injected mice were anesthetized by isoflurane inhalation within 48 hours after the morphine CPP test and whole-cell patch clamp recording were performed,the spontaneous excitatory postsynaptic currents(s EPSCs)of PCs in control and h M4D(Gi)groups were recorded by perfusion CNO under the condition of the whole existence of picrotoxin.In the presence of picrotoxin and CNO,the changes of s EPSC in the control group and h M4D(Gi)group were observed by perfusion of DAMGO in cerebellar slices.6.All data were expressed in mean ± SEM and were analyzed for statistical significance by one-way ANOVA,two-way ANNOA,Mann-Whitney test and two independent samples T test.P-values less than 0.05 were considered statistical significance.[Results] 1.After morphine CPP training,the Morphine group mice exhibited significantly elevated CPP score compared with the Saline group mice(P < 0.05).2.The amplitude of PCs m EPSC in the mice of Morphine group was greater than that of PCs m EPSC in the mice of Saline group(P < 0.05),but the frequency was lower than that of the Saline group(P < 0.05).3.Immunofluorescence results showed that chemogenetic virus-labeled neurons overlap with cerebellar granular layer glutamatergic neurons.4.The injection of chemogenetic virus and the implantation of intracranial cannula into the cerebellar GCL did not affect the total distance of movement of control and h M4D(Gi)mice in the open field experiment(P > 0.05)and specific inhibition of cerebellar PF-PC synaptic transmission increased morphine CPP scores in h M4D(Gi)mice(P < 0.05).5.In control group mice cerebellar slices,perfusion of CNO in the presence of picrotoxin did not change the frequency and amplitude of PC s EPSC(P > 0.05),whereas the frequency of PC s EPSC was significantly reduced(P < 0.05)but the amplitude was unchanged(P > 0.05)in the h M4D(Gi)group of mice brain slices perfused with CNO.6.After surface perfusion of MOR-selective agonist DAMGO in cerebellar slices in the presence of picrotoxin and CNO,the frequency of PCs m EPSC in h M4D(Gi)group mice decreased(P < 0.05)and the amplitude remained unchanged before and after DAMGO perfusion(P > 0.05).In the presence of picrotoxin and CNO,the cerebellar slices of control group mice perfused with DAMGO did not affect the amplitude of PCs s EPSC(P > 0.05)but the frequency of PCs s EPSC was reduced(P< 0.05).The decrease of PCs EPSC frequency in h M4D(Gi)group was significantly greater than that in control group(P < 0.05).[Conclusion] 1.Cerebellar PF-PC synaptic transmission in mouse may be involved in the formation of morphine CPP.2.Inhibition of mouse cerebellar cortex PF-PC synaptic transmission promotes the formation of the rewarding effect of morphine,which may be associated with presynaptic mu opioid receptor(MOR).