Mechanism Of Noradrenaline Modulates Mouse Cerebellar Cortical Climbing Fiber-purkinje Cell Synaptic Transmission

[Purpose]Noradrenaline(NA)is not only a hormone but also an important neurotransmitter,which widely acts on the central nervous system.Endogenous NA of cerebellum is released by NAergic neurons in locus coeruleus(LC)located in the anterior back of pons and near the rostral end of the fourth ventricle.NA released to cerebellar cortex regulates synaptic transmission and information integration of cerebellar cortical neural circuits through its adrenergic receptor(AR),and participates in cerebellar motor regulation and motor learning.There are abundant ARs in cerebellum.AR is a G protein coupled receptor,including α and βsubtypes.There are 6 subtypes of α-AR,including 1a,1b,1d,2a,2b,2c,and β—AR,including two subtypes of β 1 and β2,in cerebellum.The various receptor subtypes have important physiological functions.The synaptic connection between the climbing fiber(CF)from the inferior olivary nucleus and Purkinje cell(PC),the unique efferent neuron in cerebellar cortex,forms a large number of synaptic connections.CF-PC synapse is an important passway for cerebellar cortex to receive external information.Some reports have shown that NA can regulate CF-PC synaptic transmission,but its receptor and mechanism of regulating CF-PC synaptic transmission in cerebellar cortex are still unclear.Therefore,in vivo and in vitro electrophysiology combined with neuropharmacology were used to study the receptor mechanism of NA affecting CF-PC synaptic transmission in the cerebellar cortex of anesthetized mice,and to explore the intracellular pathway and mechanism of NA affecting CF-PC synaptic transmission.[Methods](1)in vivo experimentsAfter the mice were anesthetized with urethane(1.3 g/kg body weight i.p.),the craniotomy was drilled to expose the cerebellar surface corresponding to vermis.The cerebellar surface was constantly superfused with oxygenated artificial cerebrospinal fluid.Rectal temperature was monitored and maintained at 37.0 ± 0.2℃ using body temperature equipment.In current clamp recording mode(I=0),Purkinje cell(PC)cell-attached recording is realized.All drugs were dissolved in ACSF and administered by peristaltic pump.(2)In vitro experimentsAfter anesthesia with isoflurane,mice were immediately decapitated quickly.The brain were taken out and immersed in ice-cold ACSF.The sagittal slices of cerebellar cortex(250 μm thick)were prepared using a Vibratome.The slices were incubated for≥1 h in a chamber filled ACSF at room temperature(24-25℃)before electrophysiological recording.The whole cell recording was used in the physiological recording of PC.For activation of climbing fiber(CF),a glass electrode containing was placed in the granule cell layer of the cerebellar cortex.The complex spike(CS)and excitatory postsynaptic potential(EPSC)were induced by electrical stimulation.The ACSF included picrotoxin(50 μM)during all recordings to prevent GABAA receptor-mediated inhibitory.The electrophysiological data were analyzed by Clampfit 10.4 software.Values are expressed as the mean ± S.E.M.Student’s paired t-test and One-way ANOVA(SPSS software)was used to determine the level of statistical significance among groups of data.P-values below 0.05 were considered statistically significant.[Results]Part Ⅰ(1)Cerebellar surface perfusion of NA inhibited the spontaneous CS activity without significantly affect the spontaneous CS firing rate of cerebellar PCs in vivo in mice.(2)The NA-induced decrease in the AUC of CS was concentration-dependent.The 50%effective concentration(EC50)of NA was 23.7 μM.(3)Perfusion of αl-AR antagonist,prazosin(PRA),failed to block the NA induced inhibition of CS activity.Perfusion of α2-AR antagonist,yohimbine(Yohim),had less effect on the activity of spontaneous CS activity,but completely blocked the NA-induced depression of spontaneous CS activity.Perfusion of any type ofα2a-,α2b-andα2c-AR could not block the effect of NA on CS activity.However,the combination of α2a(BRL44408,BRL)and 2b-AR(Imiloxan,Imi)antagonist,but notα2a-and 2c-AR or α2b-and 2c-AR antagonist,completely blocked the inhibitory effect of NA on spontaneous CS activity.(4)Perfusion of β1 and β2-AR blockers did not affect the inhibitory effect of NA on spontaneous CS activity.Part Ⅱ(1)Application of NA significantly inhibit the decrease of CS activity induced by electrical stimulation of CF,and the number of N1 spikelets decreased significantly,but the number of N2 spikelets did not change significantly in absence GABAA receptors activity.(2)NA could significantly inhibit CF-PC synaptic transmission induced by electrical stimulation of CF,and increase the paired-pulse ratio(PPR).(3)In the presence of a2-AR specific antagonist yohimbine(yohim),NA could not inhibit CF-PC synaptic transmission induced by electrical stimulation of CF.(4)PKA signaling pathway inhibitors H-89 and kt5720 could completely block NA induced inhibition of CF-PC synaptic transmission.However,forskolin(for),an AC activating agonist,could reverse the inhibitory effect of NA on CF-PC synaptic transmission.On the other hand,NA could inhibit the enhancement of CF-PC synaptic transmission induced by forskolin.AC inhibitors could also block the inhibitory effect of NA on CF-PC synaptic transmission.However,blocking PKA in postsynaptic cells failed to affect NA induced inhibition of CF-PC synaptic transmission in cerebellar cortex.(5)Inhibition of PKC signaling pathway had no significant effect on NA induced inhibition of CF-PC synaptic transmission.[Conclusions]These studies showed that NA inhibited AC-cAMP-PKA pathway by activating both α2a-and α2b-AR,then regulating the release of neurotransmitters to regulate CF-PC synaptic transmission in cerebellar cortex.It is suggested that the neurons of the locus coeruleus nucleus(LC)can influence the information transmission and integration of cerebellum by adjusting the information from the olivary nucleus to the cerebellum.This manner of influencing cerebellar function by adjusting presynaptic activity in cerebellum may be the inherent mechanism of fine regulation of cerebellar movement coordination.These results might provide some theoretical data for the interaction between cerebellum and external information and the rapid processing of information,as well as diagnosis and treatment of cerebellar ataxia.