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Firing Pattern Modulation Through SK Channel Current Increase Underlies Neuronal Survival in an Organotypic Slice Model of Parkinson’s Disease

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Abstract

Dopaminergic (DA) neurons in substantia nigra pars compacta (SNc) are vulnerable to excitotoxicity in Parkinson’s disease (PD). Neurotoxic stimuli may alter the firing patterns of DA neurons. However, whether firing pattern change underlies neurotoxic stress-induced death of DA neurons remains unknown. In this study, we established long-term cultures of SNc organotypic slices and used this model to evaluate the neurotoxic effects on firing mode and DA neuronal viability following chronic treatment with neurotoxin 6-hydroxydopamine (6-OHDA). Using whole-cell patch clamp to explore the intrinsic membrane properties and firing mode, we showed that chronic exposure to 6-OHDA raised the resting membrane potential of SNc DA neurons and altered their firing pattern, causing it to switch from a regular rhythmic pacemaking firing to an irregular bursting. This firing pattern change correlated with increased death of SNc DA neurons. The 6-OHDA-induced firing pattern change correlated with an increase in the activity of the small conductance calcium-activated potassium channel (SK channel) and with an increase in both the level and activity of protein phosphatase 2A (PP2A). Activation of the SK channel by its agonist 1-EBIO attenuated 6-OHDA-induced firing irregularity and death, while the SK channel antagonist apamin exacerbated the toxic effects of 6-OHDA. Thus, SK channel current is a substantial element in sustaining the SNc DA neuronal rhythmic pacemaking and homeostasis and perturbing SK channel activity underlies 6-OHDA-induced neurotoxicity.

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Acknowledgments

The authors wish to thank Zixu Mao from the Department of Pharmacology, Emory University School of Medicine for his advice and critical reading of the manuscript. The authors appreciate the work by MAXC (maxchelator.stanford.edu) in calculating calcium concentration. This work was supported by National Basic Research Program of China grant (973 Program, grant no. 2011CB510002) and National Natural Science Foundation of China grant (grant no. 31371400) to Qian Yang.

Conflict of Interest

The authors have no conflict of interest related to this study.

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Authors and Affiliations

  1. Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, 710038, China

    Yuan Wang, Liang Qu, Xue-Lian Wang, Li Gao, Zhen-Zhen Li, Guo-Dong Gao & Qian Yang

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  1. Yuan Wang
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Correspondence to Qian Yang.

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Yuan Wang, Liang Qu, and Xue-Lian Wang contributed equally to this study.

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Fig. 1

The viability of PC12 cells following 6-OHDA treatment and modulation of SK channel activity. A. Effects of modulation of SK channel activity on 6-OHDA-induced death. A representative image of TUNEL assay of PC12 cells treated with 6-OHDA (40 μM, 18 hrs) with or without 1-EBIO (10 μM, 18 hrs) or apamin (100 nM, 18 hrs) cotreatment (green: TUNEL staining; blue: DAPI; scale bar: 100 μm). B. Percentages of cells that are TUNEL positive following 6-OHDA treatment. PC12 cells were treated as described in A and scored for TUNEL positive percentage (averages are given as mean ± S.D). C. Quantification of PC12 cell viability following 6-OHDA treatment. PC12 cell viability was assessed by MTT analysis following treatment as described in A. Bars are mean ± S.D. (one-way ANOVA with Newman-Keuls Multiple Comparison Test: *P < 0.01 vs. control; # P < 0.05 vs. 6-OHDA) (GIF 79 kb)

High resolution image (TIFF 750 kb)

Fig. 2

The effect of SK channel on 6-OHDA toxicity revealed by immunohistochemistry analysis. A Fluorescent images of TH (green) in cultured ventral mesencephalic slice: a control group, b 6-OHDA (25 μM), c 6-OHDA with Apamin (100 nM), d 6-OHDA with 1-EBIO (10 nM). Scare bar = 25 μm. B Quantitative analysis of the number of TH positive cells in different groups. Bars are mean ± S.D. (one-way ANOVA with Newman-Keuls Multiple Comparison Test: *P < 0.05 vs. control, # P < 0.05 vs. 6-OHDA) (GIF 356 kb)

High resolution image (TIFF 33381 kb)

Fig. 3

Plots of Ca2+ channel current versus time in SNc neurons, showing the increase of current amplitude induced by 0.5 mM 6-OHDA exposure (n = 8) (GIF 48 kb)

High resolution image (TIFF 17702 kb)

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Wang, Y., Qu, L., Wang, XL. et al. Firing Pattern Modulation Through SK Channel Current Increase Underlies Neuronal Survival in an Organotypic Slice Model of Parkinson’s Disease. Mol Neurobiol 51, 424–436 (2015). https://doi.org/10.1007/s12035-014-8728-3

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