| Objectives:The pathological hallmarks of Alzheimer's disease?AD?are accumulation and deposition of Amyloid ? protein?AP?in brain area such as hippocampus and cerebellum,as well as general loss of cholinergic neurons and nicotinic acetylcholine receptors?nAChRs?in whole brain.Unstill now,it is still unclear to explain the pathological mechanism of AD.Recently,it has been widely investigated the binding and interaction between Ap and a7-nAChR.Studies have shown the interaction between Ap and a7-nAChR caused by pathological increase of A? can lead to the progression of AD.However,very few studies have been reported about the direct effect of A?1-42on pyramidal neurons and interneurons,which was poorly compared between these two kinds of neurons.To elucidate these questions,our project aims to explore the novel effect of A?1-42 on membrane current and excitability of the pyramidal neurons and interneurons,and to explore interaction between A?1-42 and a7-nAChR in pyramidal neurons and interneurons to analyze the similarities and differences of this interacted pattern acting on neural network function.The underlying approaches and mechanisms of central cholinergic system dysfunction or even neuronal network dysfunction triggered by central neuron toxicity caused by A? were also elucidated.Our work allows for new insights on pathological mechanism,prevention and treatment of AD.Methods:1.Hippocampal slices?300?m?from SD rats?14-18d?were prepared to identify the specific pyramidal neurons and interneurons with distinct sites in rat CA1 hippocampal rigion,including cell size,cell shape,morphology of axons and dendrites as well as distribution trend by using slice patch clamp combined with infrared differential interference microscope.With the visual slice patch clamp,the specific pyramidal neurons and interneurons in rat CA1 hippocampal region were under investigation of electrophysiological characteristics and the difference involved based on whole-cell voltage clamp and current clamp technique.2.Whole-cell voltage clamp or current clamp mode were employed in pyramidal neurons and interneurons in rat CA1 hippocampal region using specific agonist choline and selective antagonist ML A of ?7-nAChR to detect membrane current and membrane potential,including change of APs discharge frequency,whereby the electrophysiological functional expression of a7-nAChR in pyramidal neurons and interneurons were detected.3.Through multi-valve perfusion system,pyramidal neurons and interneurons were transiently treated with various concentrations of A?1-42 and were further detected changes of membrane current and membrane potential.The effect of selective a7-nAChR antagonist MLA on changes of membrane current and membrane potential induced by A?1-42 was also explored in these 2 kinds of cells.4.Pyramidal neurons and interneurons in rat CA1 hippocampal region were perfused with relative low dosage of A?1-42?0.1?1?10nM?for various time ranges?1?3?5?7?9min?.By using whole-cell current clamp technique,gradual enhancement of depolarization,changes of increment step current injection-induced membrane potential as well as APs were recorded in these 2 kinds of cells while the effect of A?1-42 on neuronal excitability during the time ranges were also observed in these cells.5.Hippocampal slices were perfused with InM A?1-42 for 30min to detect the long time effects of A?1-42 on gradual enhancement of depolarization,changes of increment step current injection-induced membrane potential as well as APs were recorded in these 2 kinds of cells.Meanwhile,MLA,AP5?DNQX and bicuculline were employed to antagonize a7-nAChR?NMDAR?AMPAR and GABAAR respectively to further explore the mechanism of effects of A?1-42 on these neurons.6.Pyramidal neurons and interneurons were perfused with InM A?1-42.Under monitor of whole-cell current clamp,the neurons were injected continuously increasing clamping current?-100 pA-100 pA?to induce burst of Aps as results of gradual depolarization in the cells.The effects of A?1-42 on the resting potential and threshold potential of hippocampal CA1 pyramidal neurons and interneurons were observed.7.By using slice patch clamp and in voltage clamp mode,the effect of InM A?1-42 perfusion on 100mM choline-induced membrane current in pyramidal neurons and interneurons were tested.In current clamp mode,changes of neuronal excitability triggered by 0.1 mM choline after A?1-42 treatment were recorded in pyramidal neurons and interneurons.Changes of neuronal excitability induced by A?1-42,A?1-42 with choline and choline alone were compared in these neurons.Results:1.There existed distinct difference between pyramidal neurons and interneurons in rat CA1 hippocampal region,including different sites in rat CA1,cell size,cell shape,morphology of axons and dendrites as well as distribution trend.By employing slice patch clamp,we also observed different electrophysiological characteristics in these neurons,such as resting membrane potential,threshold potential,membrane resistance,action potential frequency and amplitude.The two kinds of neurons could be compared and identified by morphological feature combined with patch-clamp techniques.2.In voltage clamp mode,specific a7-nAChR agonist choline could trigger cell membrane inward current in a concentration dependent mode in pyramidal neurons and interneurons in rat CA1 hippocampal region,and in current clamp mode,choline could excite pyramidal neurons and interneurons in a concentration dependent mode,while selective a7-nAChR antagonist ML A could statistically suppress cell membrane inward current and frequency of APs discharge in these neurons.It suggested that there have clear electrophysiological functional expression of a7-nAChR both in pyramidal neurons and interneurons in rat CA1 hippocampal region.3.High concentration of A?1-42 could directly trigger membrane inward current in pyramidal neurons resulting in hyperactivity in the same neurons,while this effect could be partially suppressed by MLA.However,A?1-42 could neither induce membrane inward current nor increase the neuronal excitability in interneurons.4.Perfusion of various concentration of A?1-42?0.1?1?10nM?,corresponding steps of clamp current injection,low concentration of A?1-42 induced increment of excitability in pyramidal neurons.With the more concentration of A?1-42 added,the more bursted APs were detected during indicated unit time in pyramidal neurons.MLA could attenuate the increment of excitability triggered by A?1-42 in pyramidal neurons.While NMDAR,AMPAR,GABAAR was antagonized by AP5,DNQX,bicuculline respectively,there had no statistically effects of these drugs on excitability changes triggered by A?1-42.5.Perfusion of various concentration of A?1-42.?0.1?1?10nM?,corresponding steps of clamp current injection,A?1-42 suppressed cellular excitability in interneurons.With the more concentration of A?1-42 added,the fewer discharge APs were detected during indicated unit time in interneeurons.MLA could attenuate the suppression of excitability induced by A?1-42 in interneurons.While NMD AR,AMPAR,GAB AAR was antagonized by AP5,DNQX,bicuculline respectively,there had no statistically effect of these drugs on excitability changes triggered by A?1-42.6.During various indicated time points,low concentration of A?1-42 perfusion always could clearly increase cellular excitability in pyramidal neurons and suppress cellular excitability in interneurons.7.Chronic action of A?1-42 could decrease threshold potential in pyramidal neurons,which attenuated the difference between threshold potential and resting potential and increased the cellular excitability.However,in interneurons,chronic action of A?1-42 could decrease both threshold potential and resting potential.Notably,the decrease of resting potential was even more striking than threshold potential.Meanwhile,the difference between threshold potential and resting potential was found to be elevated and the cellular excitability was recorded to be attenuated.8.Chronic perfusion of InM A?1-42 could suppress 100mM choline-induced membrane current in pyramidal neurons and interneurons.Perfusion of 0.1 mM choline could statistically increase cellular excitability in pyramidal neurons and interneurons.However,chronic perfusion of InM A?1-42 could suppress choline-triggered cellular excitability in pyramidal neurons and intrnmeurons.Choline treatment attenuates the effects of A?1-42 on the excitability of pyramidal neurons and the inhibition of interneurons in rat CA1 hippocampal region.Conclusions:1.A?1-42 enhances the excitability of pyramidal neurons and inhibits the excitability of interneurons in CA1 hippocampal region,suggesting that A?1-42 promotes neuronal network dysfunction in CA1 hippocampal region and produces neuropathic excitotoxicity effects.2.A?1-42 exerts neuropathic excitotoxicity by at least partial activation of a7-nAChR.3.A?1-42?interaction with choline has interactive inhibition effects on a7-nAChR,suggesting that enhancing the activation of a7-nAChR by cholinergic neurotransmitters in vivo could attenuate the excitotoxicity of A?1-42 to neurons.This provides potentially new therapeutic targets and new clues for treatment of AD. |
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