| ObjectivesEpilepsy is a common chronic neurological disease with recurrent seizures.Recurrent seizures induce neuronal damage,resulting in a variety of brain dysfunction.At present,disruption of the balance between excitation and inhibition is the consensus mechanism leading to seizures.A variety of antiepileptic drugs,enhancing the inhibitory effect mediated by GABA receptor,restore the excitation and inhibition balance and reduce brain damage.However,due to the limitation of technology,this mechanism can not be studied in cellular,subcellular and receptor levels in vivo.Using two-photon calcium imaging,two-photon uncaging,two-photon targeted and shadow patching,and receptor tagging in vivo,we observed the effect of rapid GABA intervention to inhibit the epileptic discharge of single neuron and the epileptic calcium signals of dendrites and spines as well as delayed the injury of the AMPA receptor.MethodsIn the first part,we inhibited epileptic discharge of single-neuron by rapid GABA intervention in the living animals.(1)Using two-photon shadow patching,we recorded the high frequency action potentials induced by current injection in vivo.(2)We uncaged the Rubi-GABA to release GABA by two-photon stimulation.(3)We labeled cortical neurons by multicell bolus loading in vivo.(4)We recorded the calcium signals with two-photon imaging.(5)We recorded action prtentials with cell-attached recording in vivo.Part two,we inhibited epileptic calcium signals of dendrites and spines by rapid GABA intervention in vivo.(1)Using two-photon shadow patching,we recorded the membrane potential of neuron in the marmoset cortex.(2)With two-photon calcium imaging,we observed the calcium signals in neuronal dendrites and spines.(3)We labled the cortex neurons with gene-encoded calcium-sensitive protein by virus transfection.In the third part,the AMPA receptor injury were delayed by rapid GABA intervention.(1)Layer 2/3 progenitor cells were transfected by in utero electroporation of E15 embryos from pregnant mice.(2)With two-photon targeted and shadow patching,electrophysiological characteristics and synaptic transmission were recorded in transfected and non-transfected cortex neurons.(3)AMPA receptor injury were observed by two-photon imaging.ResultsIn the first part,with two-photon shadow patching,we recorded action potentials of single-neuron by current injection in vivo.Using two-photon uncaging,we accurately and rapidly inhibited action potentials of single-neuron in vivo.With cellattached recording,we recorded the incresing frequency of neuronal action potentials induced by 4-AP and the inhibition of discharge by GABA intervention.With twophoton calcium imaging,we observed the increasing frequency of calcium signals in 4-AP-induced seizures,and there was no significant change in amplitude of calcium signals.The epileptic calcium signals were inhibited by GABA intervention.Part two,with two-photon shadow patching,we found that there was no significant difference in electrophysiological characteristics in cortical neuron between marmoset and mice.Using two-photon calcium imaging,we recorded the characteristics of dendrites and spines calcium signals in the cortical neurons of marmoset,and it was proved that the local calcium signal was dependent on the NMDA receptor.With gene-encoded calcium-sensitive protein by virus transfection,we recorded the calcium signals induced by 4-AP and inhibited them by rapid GABA intervention.In the third part,combined with two-photon targeted and shadow patching and in utero embryo electroporation,we found that exogenous AMPA receptor did not change the electrophysiological characteristics and synaptic transmission function of adult living cells.Using two-photon imaging,we observed the rapid injury of AMPA receptors which was delayed by rapid GABA intervention.ConclusionUsing two-photon shadow patching and uncaging,we inhibited action potential of single-neuron accurately and rapidly in vivo.The results show that we can accomplish inhibition of single-neuron accurately and rapidly with two-photon uncaging.Using cell-attach recording,multi-cell bolus loading and two-photon calcium imaging,we can inhibited epileptic discharge and epileptic calcium signals by GABA intervention in cortical neurons in vivo.We used gene-encoded calcium-sensitive protein and twophoton calcium imaging to observe the dendritic calcium signal inhibition by rapid GABA intervention in living animals.The results showed that the inhibitory effect by GABA receptor-mediated,is a direct evidence of the mechanism of antiepileptic drugs in cellular and subcellular levels.The visualization of AMPA receptors in vivo enables us to observe AMPA receptor trafficking after drug-induced seizures which were delayed by rapid GABA intervention.This result provides an experimental proof of the mechanisim of excitation and inhibition imbalance which gives an important experimental basis for epilepsy treatment and antiepileptic drug selection.Marmoset is an invaluable primate model to the study of the pathogenesis and treatment of various human diseases and we extended two-photon shadow patch and calcium imaging to the nonhuman primates,providing technical support for the study of nonhuman primate in cellular and subcellular levels,as well as establishing the basis for exploring the mechanism of epileptic seizures in nonhuman primates. |
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