Functioanal Alterations In Potassium Channels Involve In Neuronal Hyperexcitability Caused By Exaggerated Group 1 Metabotropic Glutamate Receptor Signaling In A Fragile Mouse Model

Objective Fragile x syndrome(FXS)is the most common form of inherited intellectual disease.One of its prominent characteristic is neuronal hyperexcitability,which results in a variety of clinical symptoms such as epilepsy,hyperactibity and increased sensitivity to sensory stimuli.Fragile x syndrome is caused by lack of fragile x mental retardation protein(FMRP),which is an RNA-binding protein.FMRP can bind to a variety of RNAs and negatively regulate the expression of multiple RNAs as a translational inhibitor.Metabolic glutamate receptor(mGluR)is closely related to the pathophysiological mechanism of FXS.FMRP is an important inhibitory protein of group 1 mGluR signaling pathway.FMRP acts downstream of group 1 mGluR signal to inhibit protein synthesis.Lack of FMRP enhances the function of Group 1 mGluR,which may closely relate to neuronal hyperexcitability in Fmr-1 KO mice which is a excellent animal model for human FXS.Potassium currents is the most important current in the action potential repolarization process and is closely related to the neuronal excitability.Therefore,we speculate that potassium currents are the downstream target of group 1 mGluR and this pathway may modulate neuronal hyperexcitability by altering t potassium current in KO mice.In this study,wild type(WT)and Fmr1 knockout(KO)mice(at the age of 10 days,p10)were used to study the effect of Group 1 mGluR on firing properties,synaptic transmission and potassium current in neurons,for the purpose of clarifying the involvement of Group 1 mGluR and potassium currents on the neuronal hyperexcitability in FXS.Methods 1.Acute brain slices were prepared from KO and WT mice.2.The whole-cell patch clamp technique was used to record the action potential and EPSC in the hippocampus CA1 pyramidal neurons of KO and WT mice.The group 1 mGluR agonists DHPG and antagonist MPEP were used to evaluate the regulation of mGluR on characteristic of neuronal input and output.3.The voltage clamp technique was used to study whether group 1 mGluR antagonist MPEP can regulate potassium currents of hippocampus CA1 pyramidal neuron of KO and WT mice.Evaluate the regulation of mGluR on potassium currents by analyzing kinetics of activation,inactivation and recovery.Result 1.To evaluate discharge ability,slices were perfused with low-Mg2+ ACSF.Intracelluar recording were performed for up to 40 mins with the treatments of the mGluR agonist DHPG and antagonist MPEP.We found DHPG significantly increased the frequency of spontaneous discharges and burst firing in WT slices(P<0.05,paired t test.n=8),but showed no significant effects on the KO slices.On the contrary,MPEP significantly decreased frequency of spontaneous discharges and burst firing in KO group(p<0.01,paired t test.n=9)and decreascd frequency of burst firing in WT group(P<0.01,paired t test.n=8),but there were no statistically significant effect on the frequency of spontaneous discharges in WT group.Further study on action potentials(AP)evoked by current injection found that KO neurons had a higher proportion of IB neuron either in P10 or P30 groups(p=0.04,for P10;p=0.036,for P30.Chi-square test).MPEP can significantly decreased the frequency of APs in both KO and WT group(p=0.0090,for P10;p=0.0308,for P30,one-way ANOVA and post-hoc LSD test.n=7,for WT for P10;n=8 for KO for P10 and KO for P30;n=9 for WT P30).MPEP treatment in the KO neurons resulted in an transition from burst spiking to regular spiking,and there were significant differences in the probability of the transition between KO and WT neurons in P10(p=0.0419,Chi-square test).Our results suggest that exaggerated group 1 metabotropic glutamate receptor signaling plays an inportent role in the abnormal discharges in the neurons of KO mice and the antagonism of MPEP can significantly inhibit discharges and burst firing properties.2.Voltage-clamp technique was used to record s EPSC of the hippocampal CA1 neurons of KO and WT mice.Compared with WT group,KO group had higher level of average amplitudes,numbers and average frequency of s EPSC(p<0.05,for average amplitude;p<0.01,for average frequency and numbers;t test.n=6,for WT;n=5,for KO).MPEP decreased the numbers and average frequency of s EPSC in KO group(p<0.05,paired t test.n=6,for WT;n=5,for KO)and decreased the numbers and average amplitudes of s EPSC in WT group(p<0.01,for numbers;p<0.05,for average amplitude,paired t test.n=6,for WT;n=5,for KO).The percentage reduction in the average amplitude of the KO group was significantly different from that of the WT group(p<0.05,t test.n=6,for WT;n=5,for KO).Our results suggest that exaggerated group 1 metabotropic glutamate receptor signaling is one of the reasons for the increased excitatory synaptic transmission of KO mice.3.To evaluate evoked EPSC(e EPSC),the stimulus was placed in the Schaffer collateral and t hippocampal CA1 neurons of WT and KO mice were recorded.The results show that the amplitude and decay time of e EPSC in KO group was significantly increased(p<0.05,t test.n=4,for WT;n=5,for KO),as compared to that of WT group.A potassium current antagonist 4-AP can significantly increased the amplitude,decay time and paired-pulse ratios(PPR)of e EPSC in WT group(p<0.05,for amplitude;p<0.01,for delay time and PPR;paired t test.n=4,for WT;n=5,for KO),but there were no statistically significant effect on the KO group.Our results suggest that KO and WT neurons have a significant difference in synaptic transmission.Using 4-AP to reduce potassium current of WT neurons can produce corresponding changes in synaptic transmission and facilitation properties of KO neurons,suggesting that decreases in potassium currents are involved in the abnormal synaptic transmission properties of KO neurons.4.We further measured changes in current density and kinetics of potassium channels in response to MPEP treatment.It showed that either peak or steady-state amplitudes of potassium current were decreased in KO group(p<0.01,one-way ANOVA and post-hoc LSD test.n=5,for WT;n=6,for KO).MPEP significantly increased peak conductance of potassium current(p<0.01,one-way ANOVA and post-hoc LSD test.n=5,for WT;n=6,for KO)and decreased time constant of recovery(p<0.01,one-way ANOVA and post-hoc LSD test.n=5,for WT;n=6,for KO)in KO group,but the effects in WT group was not significant.MPEP did not affect the kinetic parameters of activation and inactivation.Our results suggest that antagonism of mGluR can partially rescue the reduction of potassium current of KO neurons,suggesting that the modulation of potassium current may be an important mechanism of MPEP in the treatment of fragile X syndrome.Conclusion 1.Neuronal hyperexcitability of hippocampal CA1 pyramidal neurons of KO mice are resulting from the aberrant signaling via group 1 mGluR.2.Antagonizing the mGluR signaling pathway can effectively reduce the synaptic transmission efficiency of hippocampal CA1 neurons in Fmr1 knockout mice,leading toinhibition of burst discharges in KO mice.3.Neurons of Fmr1 knockout mice show weaken potassium currents.Impairments in potassium currents are associated with neuronal hyperactivity and facilitated synaptic trasnsmission caused by exaggerated group 1 mGluR signaling pathway.Modulation of potassium current may be an important mechanism of MPEP in the treatment of fragile X syndrome.