| BackgrandParkinson’s disease (PD) is the second most common neurodegenerative disorder incentral nerve system. The neuropathological features of PD are the loss of SNc DAneurons and the formation of Lewy body. But the mechanism of PD is not clear. Currentstudies show that PD is strongly associated with aging, gene, environmental toxin and etc.In our country, there are over1.8million PD patients. The incidence of PD is expected torise dramatically worldwide in the next25years with the extension of life expectancy byimproved health care. Thus, Parkinson’s disease has become a major problem in the fieldof neurological diseases.Dopaminergic neurons in Substantia nigra are the important part of the basalganglia.In recent studies, it is reported that dopaminergic neurons in SNc are vulnerable toexcitotoxicity, while dopaminergic neurons in VTA are spared. And some studies havefound that the specific L-type calcium channel antagonists protect against neurotoxinswhich cause the loss of dopaminergic neurons in SNc. Therefore, we can speculate that the distribution of ion channels and firing pattern involve in the degeneration of SNc DAneurons, which may play an important role in the pathogenesis and progression ofParkinson’s disease. Several studies reveal that calcium channels are related to themechanism of PD, but it is not well studied so far.SNc DA neurons have an unusual physiological phenotype. Unlike the vast majorityof neurons in the brain, SNc DA neurons are autonomously active, generating actionpotentials regularly (2–4Hz). There are several subtypes of calcium channels which openat relatively hyperpolarized potentials, driving the membrane potential to spike threshold.In physiological condition, calcium entry from those channels lead to the fundamentaloxidative stress of intracellular mitochondria. However, the mechanism of the change offiring pattern and the feature of membrane is still unknown.Organotypic brain slice cultures provide a unique platform for the investigation of theneurodegenerative disorders with prominent advantages. Cultured slices preserve theintact tissue cytoarchitecture and functional local synaptic circuitry with robust neuronalactivities. Cultured Slises are easy to be manipulated and observed in the changingextracellular environment, and it can be used to mimic pathologic process of Parkinson’sdisease in vitro.AimThis paper aims to observe the6-OHDA induced changes of electrophysiologicalproperties in SNc DA neurons. This study attempt to investigate the neuronal excitabilityand the transformation of firing patternunder6-OHDA induced oxidative stress, and itsmechanism about ion channels such as the voltage-dependent calcium channels and smallconductance calcium-activated potassium channel. Furthermore, we try to explore theroles of SK channel in transformation of firing pattern. We also investigate the protectiverole of SK channel on6-OHDA induced neuronal toxicity by electrophysiological and cellbiological approaches. And it may play an important role on the treatment of Parkinson’sdisease.MethodsIn this study, we use acute isolated brain slices and organotypic mesencephalic cultured slices to investigate the effect of6-OHDA on SNc DA neurons. Neuronalexcitability, firing pattern and the action potential rates are recorded by whole cell patchrecordings to observe the short-term or long-term effect of6-OHDA exposure.Furthermore, the activity of VGCCs and SK channel are measured by voltage clamp toverify whether6-OHDA affects the firing pattern by those channels such as VGCCs andSK channel.There are two parts in this study. In the first part, we observe the acute effect of6-OHDA by electrophysiological approaches. In current clamp model， we record themembrane properties of SNc DA neuron, including ISI analysis and CV. In voltage clampmodel, we isolate the L-type, N-type and other types of calcium current with the specificcalcium channel blocker, comparing the proportion of whole calcium current in6-OHDAgroup and control group.IAHPwas elicited by a brief depolarization pulse.In the second part, we established a long-term culture of SNc organotypic slice andused this model to evaluate the neurotoxic effects on firing mode. LDH assay shows theviability of cultured slices. We use the immunohistochemistry technology to observe themorphological feature of dopaminergic neurons which express tyrosine hydroxylase inorganotypic slice. Using whole-cell patch clamp was used to explore the intrinsicmembrane properties and firing pattern with the chronic6-OHDA treatment. With theanalysis of autocorrelogram, we attempt to verify whether SK channel was involved in theswitching of firing pattern after6-OHDA application. Using TUNEL test, we evaluate theeffect of SK channel agonist or antagonist on6-OHDA induced neuronal death.Results1. The acute effect of6-OHDA on dopaminergic neurons in SNc1.16-OHDA inhibits the neuronal excitability, reduces the firing rate of SNc DAneurons,which has a dose-dependent manner. The coefficient of variation in6-OHDAgroup（CV=0.435±0.354） is larger than control group（CV=0.058±0.009）（P=0.037）1.2The rates of depolarization in action potential are slow down by the6-OHDA exposurewith the phase plot analysis. 1.36-OHDA enhances the whole current of voltage gated calcium channels, and theproportion of N-type calcium channel current increases under oxidative stress.1.4With18hours6-OHDA exposure, the peak amplitude of mAHP was significantlyincreased to177.53±4.96pA(149.12±4.11%of the control, P <0.01).2. Firing pattern modulation through SK channel current increase underlies neuronalsurvival in an organotypic mesencephalic slice model2.1The viability and features of dopaminergic neurons in organotypic mesencephalic sliceculture are similar to the neurons of acute isolated slice. LDH assay showed the neurons inthe slices were in relatively normal metabolic state during DIV7to DIV21.2.2With18hours6-OHDA treatment,the resting membrane potential was alteredsignificantly, comparing to the control group (P<0.01).2.3The spike trajectory reduced after6-OHDA application, whereas the section of AHPwas relatively larger than the control group.6-OHDAreduced the rate of action potential.2.4With SK channel antagonist (Apamin) application, the display of irregular-burstybecome more frequent. On the contrary,1-EBIO palys the opposite effect with6-OHDAexposure.2.56-OHDA treatment increases67.13±8.45%of the Apamin-sensitive mAHP currentcomparing with control group. Higher EGTA concentration (3mM or5mM) in the internalsolution led to a clear and dose dependent decrease in the amplitudes of ImAHP with orwithout6-OHDA.2.6The activation of SK channel resist6-OHDA induced neuronal death, while Apaminenhance the6-OHDA induced neuronal death.ConclusionPacemaking is the feature of SNc DA neurons. In this study, our results show that6-OHDA changes the firing pattern of DA neurons from pacemaking to irregular bursting.This change in firing pattern is caused by the increasing level of calcium entry and SKcurrent in SNc DA neurons in organotypic slices. Increasing the activity of SK channelcurrent attenuates the toxic effects of6-OHDA. Thus, our studies verify the activity of SKchannel plays an important role in6-OHDA-induced neurotoxicity. |
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