Effects Of Noradrenaline On Long Term Plasticity Of Climbing Fiber-Purkinje Cell Synaptic Transmission In Mouse Cerebellum

[Purpose]Noradrenaline（NA）is a monoamine neurotransmitter,which plays an important role in the regulation of functional activities of the central nervous system by modulating the release of GABA,glutamate and other neurotransmitters.The NAergic projections of the central nervous system mainly come from the locus coeruleus（LC）.The activity of NAergic neurons induces NA secretion and release to a wide range of the central nervous system,and regulates the activity of the central nervous network through adrenergic receptor（AR）.The climbing fiber（CF）originating from the inferior olivary nucleus can form excitatory synaptic connection with Purkinje cell（PC）.It transmits external information to PC and plays a key role in motor regulation and motor learning.Previous studies found that NA significantly down regulated CF-PC synaptic transmission in mouse cerebellum,suggesting that NA may be involved in regulating the induction and maintenance of long-term plasticity of CF-PC synaptic transmission,but its mechanisms are still unclear.Therefore,we here studied the mechanism of long-term plasticity of CF-PC induced by electrical stimulation of CF and investigated the effects of NA on CF-PC transmission and long-term plasticity by whole-cell patch clamp recording technology combined with pharmacological methods.[Methods]For the experiment,adult ICR mice（4-6 weeks old）were decapitated after isoflurane inhalation anesthesia,the cerebellum was prepared into 300μm thick acute cerebellar sagittal slices.At room temperature,cerebellar slices were incubated in artificial cerebrospinal fluid（ACSF）filled with 95%O2 and 5%CO2 for more than 60minutes prior to electrophysiological recording.Whole cell patch clamp recordings from PC were visualized by Nikon microscope through its infrared visual system.The electrophysiological data were collected and obtained by Axopatch 700 B amplifier,and acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.4 software.Paired stimulation of CF induced PC to produce excitatory postsynaptic currents（EPSCs）,N1 and N2.The test frequency was 0.05 Hz,for the induction of CF-PC LTD,we applied 5 Hz（150 pulses）CF electrical stimulation.The drugs used in the experiment were dissolved in ACSF and perfused to the surface of slices.Clapfit 10.4 software was used to analyze electrophysiological data,and the collected data were expressed as mean±S.E.M.Using student’s paired t-test and one-way ANOVA,P values below 0.05 were considered statistically significant.[Results]Part I:Mechanism of long-term plasticity of CF-PC synaptic transmission（1）CF stimulation with tetanic stimulation at 5 Hz（150 pulses）induced CF-PC long-term depression（LTD）.The amplitude of EPSCs of N1 was significantly decreased,which could express for more than 40 minutes,but there was no significant change in PPR before and after the tetanic stimulation.（2）In the presence of JNJ16259685,a metabolic glutamate type I receptor（m Glu R1）blocker,tetanic stimulation could not induce CF-PC LTD.The amplitude of N1amplitude and PPR did not change significantly.（3）Under the condition that BAPTA was added to the recording electrode to block postsynaptic Ca²⁺,the tetanic stimulation failed to induce CF-PC LTD.The amplitude of N1 and PPR after stimulation was similar to that of baseline（before tetanic stimulation）.The above results show that 5 Hz,150-pulses tetanic stimulation can induce a CF-PC LTD,which depends on m Glu R1 and intracellular Ca²⁺,suggesting that CF-PC LTD occurs postsynaptically.Part II:Effects of NA on synaptic transmission and long-term plasticity of CF-PC（1）Perfusion of NA could significantly inhibit CF-PC EPSCs,resulting in the decrease of N1 amplitude,but PPR increased significantly.Also,the inhibitory effect of NA on N1 amplitude was concentration-dependent,and its half effective inhibitory concentration(IC₅₀)was 5.04μM.（2）In the presence of NA（500 n M）,tetanic stimulation（5 Hz,150 pulses）could induce a stronger CF-PC LTD,the expression of CF-PC LTD was enhanced and the amplitude of N1 was even lower,but PPR increased significantly.These results suggest that NA can promote the CF-PC LTD.（3）Blocking m Glu R1-dependent CF-PC LTD,the presence of NA could trigger the tetanic stimulation to induce a CF-PC LTD.The amplitude of N1 is significantly reduced,accompanied by a significant increase in PPR,indicating that after blocking m Glu R1-dependent LTD,tonic stimulation can induce CF-PC LTD mediated by NA.（4）Under the condition of blocking m Glu R1-dependent CF-PC LTD,we further studied the mechanism of CF-PC LTD mediated by NA.Blockingα-AR with phentolamine（Phen）,NA could not trigger the induction of CF-PC LTD.The amplitude of N1 and PPR after stimulation was similar to that in pre-stimulation conditions.However,perfusionβ-AR antagonist propranolol（Pro）,NA could still trigger the induction of CF-PC LTD.The amplitude of N1 were significantly decreased accompanied with PPR increased.Blockingα1-AR with prazosin（Pra）,NA could still trigger the induction of CF-PC LTD.The amplitude of N1 were significantly decreased accompanied with PPR increased.However,perfusionα2-AR antagonist yohimbine（Yoh）,NA could not trigger the induction of CF-PC LTD.The amplitude of N1 and PPR after stimulation was similar to that in pre-stimulation conditions.In addition,perfusionα2-AR agonist,UK14304,could also trigger the induction of CF-PC LTD,and the amplitude of N1 was significantly decreased accompanied with PPR increased.The LTD induced by UK14304 prevented the effect of NA on CF-PC LTD.The above results show that NA triggers tetanic stimulation to induce presynaptic CF-PC LTD viaα2-AR,and then enhances the expression of CF-PC LTD.[Conclusions]（1）The tetanic stimulation of CF can induce CF-PC LTD,which not only depends on the activation of m Glu R1,but also requires the increase of intracellular Ca²⁺concentration,indicating that the tetanic stimulation of CF can induce postsynaptic CF-PC LTD.（2）NA triggers tetanic stimulation to induce presynaptic CF-PC LTD viaα2-AR,and then enhances the expression of CF-PC LTD.The results suggest that NA regulates the synaptic transmission long-term plasticity of CF-PC viaα2-AR,which may play an important role in motor regulation and motor learning.