| [Objective]As a commonly used clinical analgesic medicine,pregabalin(PGB)is used in the treatment of a variety of neuropathic pain.Besides,PGB can effectively influence the function of nervous system.According to the previous studies,PGB regulates the secretion of neurotransmitters and exerts pharmacologic activity through direction interactions on voltage-gated calcium channel(VGCC)throughout the brain.Large amount of calcium channels are present in the cerebellum.Climbing fiber(CF)triggered complex spikes(CS)are closely related to the activation of calcium channels.However,whether PGB affect the CS of PC in the cerebellum and the related mechanism are still unclear.Therefore,this study used in vivo and in vitro electrophysiological recording methods to examine the effect and mechanism of PGB on PC CS,to explore the possible mechanism of PGB affecting the activity of neurons in cerebellum,in order to provide theoretical and experimental data for the clinical use of PGB.[Methods]1.In vivoAdult C57BL/6 mice(6-8 weeks old)were used for the study.After tracheal intubation,the head of the mouse was fixed on a self-made mouse brain stereotaxic apparatus,and a recording window with a diameter of about 1-1.5 mm was opened at the position of the skull corresponding to the Vermis area of the cerebellum.The body temperature of mice was maintained and the cerebellar superficial perfusion artificial cerebrospinal fluid(ACSF)throughout the operation.We used blind extracellular recording technique and the data were recorded in cell-attached recording mode.2.In vitroThe C57BL/6 mice were deeply anesthetized with halothane.300μM sagittal slices of mouse cerebellar vermis were obtained by vibrating microtome in ice-cold sucrose-based cutting solution.Then the slices were moved to a holding chamber containing oxygenated ACSF at a constant temperature for 1 hour to recovery.The whole-cell recording method was used to collect activity electrical signals of neuron,and CS was evoked by electronic stimulation of climbing fibers(CF)with the stimulating electrodes.3.Biocytin histochemistryAfter electrophysiological recording,the brain slices were fixed and subjected to a series of steps including inhibition of peroxidase,incubation with AB mixture and incubation with3,3’-diaminobenzidine tetrahydrochloride,patching,dehydration,mounting,and photography.Finally,determine the neuron location and morphology.[Results](1)PGB administered by cerebellar surface perfusion could inhibit CS,but it had no effect on the frequency of CS.Specifically,PGB could significantly reduce the number of CS spikelets,shorten the time course and reduce the area under the curve(AUC).(2)In vitro experiment,under current clamp(I=0),perfusion administration of PGB significantly reduced the amplitude of CS spikelets 2,3,4 caused by calcium-dependent component of CS,but did not affect sodium-dependent component of CS.Besides,PGB significantly reduced the half-width of spikelet 2,and inhibited CS amplitude by concentration-dependent,and the half effective concentration(IC50)was 0.324 m M.Under voltage clamp(clamped at-70m V),PGB significantly reduced the number of spikelets,did not affect the amplitude of the sodium spikelet,and decreased the total amplitude,AUC,rise time and decay of the calcium current spikelets.(3)With calcium-free ACSF,although the number of CS spikelets,the calcium current spikelets amplitude,the sodium current spikelet amplitude and AUC were significantly reduced,the addition of PGB had no inhibitory effect on CS.When the calcium chelator BAPTA was added to the intracellular solution,PGB still significantly reduced the number of spikelets,but did not affect the amplitude of the spikelets caused by the first sodium current.PGB also decreased the total amplitude,the area under the waveform and increased rise time and decay time of calcium current spikelets.(4)Blocking GABAA receptor did not affect CS activity nor the inhibitory effect of PGB on CS.Under this condition,PGB still significantly reduce the number of spikelets,but did not affect the amplitude of spikelets caused by sodium current.PGB also reduced the total amplitude and AUC of calcium current spikelets,increasing its rise time and decay time.(5)Although blocking NMDA receptors could attenuate CS activity,PGB still significantly reduced the number of spikelets,reduced the total amplitude of calcium current spikelets,the area under the waveform,and increased rise time and decay time of calcium current spikelets.(6)The inhibitory effect of PGB on CS disappeared after Cd Cl2perfusion,a non-selective calcium channel blocker,completely blocked the calcium current of CS.(7)In the presence of Flunarizine(Flu),a T-type voltage-gated calcium channel blocker,PGB still inhibited the activity of CS.However,the degree of inhibition was weaker than PGB alone.[Conclusion]Pregabalin inhibits significantly the CS activity of mouse cerebellar PC by inhibiting the calcium influx of VGCC,rather than by activating NMDA receptor or GABAA receptor.At least one part of this inhibition is related to T-type VGCC. |