Effects Of Propofol On Climbing Fiber-purkinje Cell Synaptic Transmission And Long Term Plasticity In Mice Cerebellum

[Purpose]Propofol is generally used for the induction and maintenance of anesthesia in clinical procedures.It is commonly accepted that the molecular mechanism of propofol is related to the activation of y-amino butyric acid A(GABAA)receptors and N-methyl-D-aspartic acid(NMDA)receptors and other ion channels.When administered at the clinical dose,the side effects of administration of propofol are associated with movement disorders,including dystonia and ataxia suggesting that propofol administration impacts the function of cerebellar neuronal circuitry.Cerebellar Purkinje cells(PCs)are considered as the sole output of cerebellum that received excitatory inputs through climbing fiber(CF)pathway.Note that CF pathway convey error signals during motor learning and predict the aversive events to cerebellar PCs and trigger parallel fiber(PF)-PC long-term synaptic plasticity.Therefore,the CF-PC signal plays a critical role in movement regulation and motor learning.Although the pharmacological effects of propofol have been widely studied in clinic researches,the effects of propofol on synaptic transmission and plasticity of neurons are still unclear.In this study,we investigated the effects of propofol on CF-PC synaptic transmission and plasticity by whole-cell recording technique and pharmacological methods to elucidate the mechanism of propofol on CF-PC synaptic transmission and long-term depression in vitro in mouse cerebellum slices.[Methods]For all experiments,adult(4-6 weeks old)ICR mice was decapitated immediately after deeply anaesthetization with isoflurane.The cerebellum was sectioned and placed in ice-cold artificial cerebrospinal fluid(ACSF)and bubbled with 95%O2 and 5%CO2.The sagittal slices of cerebellar cortex(300 ?m thick)were prepared using a Vibratome.The slices were incubated at least 1h in a submerged chamber filled with 95%O2 and 5%CO2 equilibrated ACSF at room temperature(24-25?)prior to recording.Whole-cell patch-clamp recordings from PC somas in cerebellar slices were visualized using a 60 X water-immersion lens through a Nikon microscopy.Membrane potentials and/or currents were monitored with an Axopatch 700B amplifier,filtered at 5 kHz,and acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.4 software.Cells were held in voltage clamp mode at-70 mV.Series resistance was monitored by applying voltage pulses(10 ms,5 mV),and only cells with stable series resistance were included in the analysis.For CF electrical stimulation,a stimulating electrode containing ACSF was placed in the granular layer of the cerebellar slice in vicinity of the recorded PC until the CF all-or-nothing response was obtained.Paired-current pulses at 0.05 Hz were delivered through a glass pipette mounted on remote-controlled manipulators.For the induction of CF-PC LTD,we applied 5 Hz stimulation for 30 s after paired-current pulses.The ACSF included picrotoxin(50?M)and saclofen(10 ?M)during all recordings to prevent GABAA,and GABAB receptor-mediated inhibitory responses.All other chemicals were dissolved in solution and kept in frozen in aliquots,and they were applied to the cerebellar slices at 0.5 ml/min in ACSF.After the experiments,the slice was fixed in 4%paraformaldehyde in 0.1 PBS for 24h.The slices were incubated overnight with the avidin-biotin complex at room temperature.Finally,biocytin was detected using 3,3'-diaminobenzidinetetrahydrochloridehistochemistry.Electrophysiological data were analyzed using Clampfit 10.3 software.All data are expressed as the mean ± S.E.M.Student's paired t-test and one-way ANOVA(SPSS software)were used to determine the level of statistical significance between groups of data.P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]Part?(1)In the absence of GABAergic receptors activation,propofol induced an increase in the amplitude of CF-PC EPSCs(P1)and area under curve(AUC),a decrease in paired-pulse ratio(PPR)under voltage-clamp.Also,the propofol-induced increase in the P1 amplitude was concentration-dependent.The half effective concentration(EC50)was 20.9 ?M.(2)NMDA receptor blockade decreased the amplitude of P1 and AUC,and increased PPR significantly.Also,blocking NMDA receptors abolished propofol-induced enhancement in the amplitude of P1 amplitude and AUC and decrease in PPR.(3)Pharmacological activating NMDA receptors significantly increased the amplitude of P1 and AUC with PPR decreased.Additional application of propofol failed to enhance CF-PF synaptic transmission,the P1 amplitude and AUC and PPR were similar to the control conditions(with administration NMDA alone).(4)Blocking of postsynaptic NMDA receptors significantly decreased(not entirely)the propofol-induced the P1 amplitude and AUC enhancement of CF-PC EPSCs,suggesting that CF-PC postsynaptic NMDA receptors also involved in the propofol-induced enhancement in CF-PC synaptic transmission.Part?(1)CF stimulation with 5 Hz for 30 s induced CF-PC long term depression(LTD).The amplitude of P1 and AUC were significantly decreased with no PPR change.Blockade of group 1 metabotropic glutamate receptors(mGluRl)abolished CF-PC LTD.The amplitude of P1,AUC and PPR after stimulation was similar to that in pre-stimulation conditions.(2)Administration of BAPTA in recording electrode to prevent postsynaptic Ca2+failed to induce CF-PC LTD.The amplitude of P1,AUC and PPR after stimulation was similar to that in pre-stimulation conditions.(3)Application of propofol enhanced the CF-PC LTD with a decrease in the amplitude of P1 and AUC and no changes in PPR compared to the control conditions(only convey induction).(4)Blocking mGluR1 did not affect the propofol-induced increase in CF-PC LTD,the amplitude of P1 and AUC and PPR were similar to the control conditions(using propofol alone).(5)Propofol failed to induce CF-PC LTD in the absence of mGluR1 and NMDA receptors activation,the amplitude of P1 and AUC and PPR were similar to the pre-stimulation conditions.(6)Pharmacological activating NMDA receptors induced CF-PC LTD in the absence of mGluR1 activation.The amplitude of P1 and AUC were significantly decreased compared to the blockade of mGluR1 alone.Additional application of propofol didn't change the amplitude of P1 and AUC,so did the PPR.(7)In the absence of mGluR1 activation,pharmacological activating NMDA receptors failed to induce the CF-PC LTD with MK-801 in recording electrode which prevented the postsynaptic NMDA receptors.The amplitude of P1 and AUC and PPR were similar to the pre-stimulation conditions.[Conclusions](1)Propofol enhances CF-PC synaptic transmission via activating NMDA receptors in the absence of GAB Aergic receptors in mice cerebellar cortex.(2)Low frequency stimulation of cerebellar CF induces the mGluRl-dependent long term depression and propofol can increasae CF-PC LTD through activating postsynaptic NMDA receptors.