Effect And Mechanism Of Glutamate Ion Channel Receptor Subtype AMPARS On Hippocampal Gamma Oscillations In Rats

BackgroundGamma oscillation is a kind of brain neural network oscillation,with a frequency of30～80Hz,which participates in the regulation of brain cognition,emotion,reward and other functions.Glutamate is the most abundant excitatory neurotransmitter in the brain.It acts on glutamate ion-type AMPA receptors and mediates rapid synaptic signal transmission between neurons.According to whether there is GluA2,subunit AMPAR is divided into calcium permeability（CP-AMPAR）and calcium impermeability（CI-AMPAR）.Glutamatergic neuron AMPAR mostly contains GluA2,which belongs to CI-AMPAR.Studies have shown that CP-AMPAR mediates Ca²⁺influx,triggers the release of ER Ca²⁺regulated by RYR,and participates in the regulation of synaptic function of interneurons（Fast-Spiking,FS）that play a key role inγoscillations.The role and mechanism of AMPAR onγoscillations are still unclear,especially CP-AMPAR and its related intracellular Ca²⁺signaling molecules,calmodulin-dependent protein kinase kinase（CAMKK）and calmodulin-dependent protein II（CAMKII）The effect onγoscillation has not been reported.ObjectivesTo explore the effects and mechanisms of AMPAR,CAMKK and CAMKII and other related signal kinases on rat hippocampalγoscillations.Methods1.Record theγoscillations in the hippocampal CA3 area of isolated brain slices of 4-week-old SD rats by field potential recording technology:Take four-week-old SD male rats,weigh,and administer intraperitoneal injection anesthesia with chloral hydrate（0.4ml/100g）Open the thoracic cavity quickly,use pre-cooled cerebrospinal fluid to perfuse the heart,quickly decapitate,and completely peel off the brain tissue,put it into the frozen section fluid filled with 95%O₂+5%CO₂mixed gas,and cut the brain tissue horizontally with a CAMPDEN INSTRUMENTS 5100mz vibrating microtome,The thickness is 400μm.Transfer the sliced brain slices to the incubation tank,the incubation tank has circulated the cerebrospinal fluid in advance,and the mixed gas of 95%O₂+5%CO₂is continuously injected.After incubating for one hour,transfer the brain slice to the recording tank,record the field potential of the hippocampus CA3 area of the brain slice with a glass electrode,add carbachol（CCH）to the circulating cerebrospinal fluid to induceγoscillation,and wait for theγoscillation to stabilize in the circulating cerebrospinal fluid Add glutamate,AMPAR agonist AMPA,CI-AMPAR antagonist NBQX,CP-AMPAR blocker IEM1460,CAMKK inhibitor STO-609 and CAMKII inhibitor KN93 respectively,and observe the changes ofγoscillation after different drugs act on brain slices.2.Data analysis:Analyze theγoscillation data with Sigma State software.Theγoscillation data of different drug treatment groups induced by CCH are subjected to paired t test between groups and One way RM ANOVE test.The statistical results are expressed as mean±standard error.If P<0.05,the result is considered to b e statistically significant.Results1.Glutamate dose-dependently regulates rat hippocampalγoscillations,with low doses increasing,and high doses inhibitingγoscillations.2.The glutamate ionotropic receptor AMPAR agonist AMPA（50μM）quickly and reversibly inhibited theγoscillation in the CA3 region of the rat hippocampus.3.The low-concentration competitive CI-AMPAR antagonist NBQX has no effect on rat hippocampal gamma oscillation,but partially blocked the decrease of rat hippocampal gamma oscillation caused by AMPA.4.The selective CP-AMPAR blocker IEM1460 and the CAMKK inhibitor STO-609downstream of CP-AMPAR significantly increased rat hippocampalγoscillations.The CAMKII inhibitor KN93 had no significant effect on rat hippocampalγoscillations,indicating CP-AMPAR And CAMKK is involved in the negative regulation of gamma oscillation.5.STO-609 pretreatment of brain slices partially blocked the effect of IEM1460 onγoscillation;KN93 pretreatment of brain slices did not affect the regulation ofγoscillation of IEM1460.6.The use of IEM1460,STO-609 or KN93 alone cannot block the decrease in rat hippocampalγoscillation caused by AMPA,indicating that CP-AMPAR and its downstream signaling pathways do not involve the decrease inγoscillation caused by AMPA;combined application of CP-AMPAR and CI-AMPAR blockers IEM1460 and NBQX partially blocked the inhibition ofγoscillation by AMPA,indicating that the decrease ofγoscillation caused by AMPA is mainly related to the activation of CI-AMPAR rather than CP-AMPAR.Conclusion1.The activation of the glutamate ionotropic AMPAR receptor subtypes CP-AMPAR and CI-AMPAR negatively regulates theγoscillation,the activation of the former involves the influx of Ca²⁺and the activation of the downstream intracellular signal kinase CAMKK;the latter’s Activation involves Na⁺internal flow.2.The rapid decline ofγoscillation caused by AMPA has nothing to do with CP-AMPAR,but is related to CI-AMPAR.