Ischemia deteriorates spike encoding at cortical GABAergic neurons and cerebellar Purkinje cells by increasing the intracellular Ca2+

Brain Research Bulletin 131 (2017) 55–61

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Ischemia deteriorates spike encoding at cortical GABAergic neurons and cerebellar Purkinje cells by increasing the intracellular Ca2+ Li Huang a,1 , Chun Wang b,1 , Rongjing Ge a , Hong Ni a , Shidi Zhao a,∗ a b

Department of Pathophysiology, Bengbu Medical College, Bengbu, Anhui 233030, China Department of Endocrinology, The Second Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233040, China

a r t i c l e

i n f o

Article history: Received 27 December 2016 Accepted 10 March 2017 Available online 14 March 2017 Keywords: Ischemia GABAergic neuron Purkinje cells Whole-cell recording Action potential Intracellular Ca2+

a b s t r a c t GABAergic neurons play a critical role in the central nervous system, such as well-organized behaviors. The ischemic cell death is presumably initiated by neuronal excitotoxicity resulted from the dysfunction of GABAergic neurons. It is not clear how ischemia influences different types of GABAergic neurons and whether intracellular Ca2+ plays a key role in the ischemic excitotoxicity. We have investigated this issue at cortical GABAergic neurons and cerebellar Purkinje cells by whole-cell recording in mouse brain slices, and the roles of intracellular Ca2+ are examined by BABTA infusion. Compare with the data from a group of control, ischemia causes by lowering purfusion rate lowers spike encoding at cortical GABAergic neurons and enhances encoding ability at cerebellar Purkinje cells. These differential effects of ischemia on spike encoding are mechanistically associated with the changes in the refractory periods and threshold potentials of sequential spikes. These ischemia-induced dysfunction of spike encoding at two types of GABAergic cells are prevented by BABTA infusion. Therefore, the ischemia destabilizes the spike encoding of GABAergic cells via raising intracellular Ca2+ . Our findings indicate that ischemia preferentially causes the dysfunction of spike encoding at GABAergic neurons by the up-regulation of intracellular Ca2+ level, which leads to neuronal excitotoxicity. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Sequential action potentials at neurons are one type of essential neural codes, and the different spike patterns encode various messages to control the well-organized behaviors and cognitions (Chen et al., 2008; Franke et al., 2015; Kipiani, 2009; Yu et al., 2011; Toups et al., 2012; Wang et al., 2008). The ischemic neuronal death is presumably initiated by neuronal excitotoxicity in the early phase in brain, which is believed to be related to an increase in glutamate release, intracellular calcium and free radicals, as well as a deficit of ␥-aminobutyric acid (GABA) neurotransmission (Ray et al., 2014; Choi, 1988; Lipton, 1999; Kuebler et al., 2015; Zahra et al., 2015; Welsh et al., 2002). Previous studies showed that the changes of excitatory potential were related to the neuronal excitotoxicity (Olney and Sharpe, 1969). It is not clear whether the neuronal spike patterns of different neurons have the different changes during the

∗ Corresponding author at: Department of Physiology, Bengbu Medical College. 2600 Donghai Street, Longzihu District, Bengbu, Anhui 233030, China. E-mail address: [email protected] (S. Zhao). 1 These authors contributed this work equally. http://dx.doi.org/10.1016/j.brainresbull.2017.03.005 0361-9230/© 2017 Elsevier Inc. All rights reserved.

early stage of ischemia. If it is a case, what changes do they have, and what are the mechanisms underlying? GABAergic inhibitory neurons play an important role in maintaining the functional homeostasis of the brain (Lu et al., 2014; Lopez-Pigozzi et al., 2016; Wang and Kelly, 2001; Wang et al., 2009; Stringer et al., 2016), and it is well known that they are vulnerable to pathological factors, especially for ischemia and epilepsy (Wang, 2003; Wang et al., 2015; Zhao et al., 2008; Huang et al., 2010; de Lanerolle et al., 2011). There are two kinds of GABAergic neurons in brain, interneurons in cerebral cortex and Purkinje cells in cerebellum. The morphology and spike patterns of them are complete different (Lu et al., 2014; Kelsch et al., 2014; Kitamura and Kano, 2013). Furthermore, Ca2+ signals are differently distributed in these two kinds of neurons (Qi et al., 2009). As ischemic alternations of these two types of GABAergic neurons are not well studied, here, we focus on investigating the different dysfunctions of GABAergic neurons which are affected during ischemia, and the roles of intracellular Ca2+ in GABAergic neurons injury. Does ischemia influence spike encoding differently? If the rise of Ca2+ is required for the ischemia-induced overexcitation? To address these issues, we induced ischemia by reducing the perfusion rate to brain slices, and investigate the changes of the spike encoding during the early ischemia. Also, we loaded BAPTA(a

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chelator Ca2+ ) into neurons via recording pipettes and measured their spike encoding under BAPTA infusion before and after the ischemia in cortical GABAergic neurons and cerebellar Purkinje cells by whole-cell recordings in mouse brain slices. If the infusion of BAPTA prevents ischemia-induced overexcitation in brain slices, the up-regulation of cytoplasm Ca2+ would presumably be involved in ischemia of GABAergic neurons injury.

2. Methods and materials 2.1. Brain slices and neurons Cortical and cerebellar slices (400 ␮m) were prepared from FVBTg(Gad GFP)4570Swn/J mice (Jackson Lab, Bar Harbor, ME 04609, USA) at postnatal days 15–22. Mice were anesthetized by injecting chloral hydrate (300 mg/kg) and then decapitated with a guillotine. Brain slices were sectioned with a vibratome in oxygenated (95% O2 and 5% CO2 ) ice-cold artificial cerebrospinal fluid (ACSF) (mM:124 NaCl, 26 NaHCO3 , 1.2 NaH2 PO4 , 3 KCl, 0.5CaCl2 , 4 MgSO4 , 10 dextrose, and 5 HEPES, pH 7.35). The slices were held in (95% O2 and 5% CO2 ) ACSF (mM:124 NaCl, 26 NaHCO3 , 1.2 NaH2 PO4 , 3 KCl, 2.4 CaCl2 , 1.3 MgSO4 , 10 dextrose, and 5 HEPES, pH 7.35) at 25 ◦ C for 1–2 h before experiments. A slice was then transferred to a submersion chamber (Warner RC-26G) that was perfused at 2 ml/min with oxygenated ACSF at 31 ◦ C for whole-cell recording (Wang and Kelly, 2001; Wang et al., 2009; Qi et al., 2009; Guan et al., 2006; Chen et al., 2006). Chemical reagents were from Sigma. The procedures were approved by Institutional Animal Care and Use Committee in Anhui, China. Cortical GABAergic neurons for whole-cell recording in layer II and IV of sensory cortex were selected under fluorescent microscope (Nikon, FN-E600) for our study. These neurons showed a round or ovarylike soma and tree branch-like dendrites, and their morphology could be seen with an excitation wavelength at 488 nm. GABAergic Purkinje cells in cerebellum cortex were selected based on the morphology under DIC microscope (Nikon, FN-E600), and the criteria (Lu et al., 2014; Zhao et al., 2008; Guan et al., 2006) for detail. These neurons demonstrated the typical properties of interneurons, such as fast-spiking and less adaptation in spike amplitude and frequency (Lu et al., 2014; Wang and Kelly, 2001; Wang et al., 2009; Zhao et al., 2008; Kelsch et al., 2014; Kitamura and Kano, 2013; Guan et al., 2006). 2.2. In vitro ischemia To simulate artery occlusion and intracranial anastomotic circulation under in vivo ischemic stroke, we reduced the perfusion rate to cortical and cerebellar slices from 2 ml/min to 0.2 ml/min for 6 min to induce ischemia (Huang et al., 2010). We measured the intrinsic properties of sequential spikes of cortical GABAergic neurons and Purkinje cells under control and after ischemia. 2.3. Whole-cell recording Electrical signals were recorded under current-clamp model by using an Axoclamp-200B amplifier with pClamp 10.3 (Axon Instrument Inc., Foster CA, USA) for data acquisition and analysis. The output bandwidth of amplifier was 3 kHz. Spike patterns were evoked by depolarization current pulses, in which the amplitude and duration were based on the aim of experiments. Standard solution were filled into pipettes for whole-cell recordings that contained (mM) 150 K-gluconate, 5 NaCl, 5 HEPES, 0.4 EGTA, 4 Mg-ATP, 0.5 Tris-GTP, and 5 phosphocreatine (pH was adjusted to 7.4 by 2 M KOH). Fresh pipette solution was filtered with centrifuge fil-

ters (0.1 ␮m) before the use. Its osmolarity was 295–305 mOsmol and resistance was 5–6 M. Neuronal intrinsic properties in our studies included the threshold potentials (Vts) of firing spikes and absolute refractory periods (ARP) after each spike. The threshold potentials were an initiating voltage of spike rising phase, which were presented as the gap between the resting membrane potential (Vr) and threshold potential (Vts) (Lu et al., 2014; Zhao et al., 2008; Chen et al., 2006; Huang et al., 2015). The use of Vts-Vr, instead of Vts alone, was because that the values of Vts and Vr vary among the CNS neurons (Lu et al., 2014); Vts-Vr was an energy barrier to raise Vr toward Vts; and Vts-Vr represented how synaptic inputs easily drive neurons to fire spikes. The ARP of sequential spikes were measured by injecting two depolarization current pulses (3 ms in duration and 5% above threshold in intensity) into the neurons (Fig. 2). We changed the inter-pulse intervals of the two pulses, and let pulses one induced spike at 100% firing probability while pulses two induced a subsequent spike at 50% probability. The duration between spike one and two was define as ARP (Qi et al., 2009; Huang et al., 2015). Spike capacity was represented as inter-spike interval (ISI) (Qi et al., 2009; Huang et al., 2015). Data were analyzed if the recorded cortical GABAergic neurons had the resting membrane potentials negatively more than −60 mV and Purkinje cells more than −55 mV. The criteria for the acceptation of each experiment also included less than 5% changes in resting membrane potential, spike amplitude, and input resistance throughout all the experiments. Vts, ARP, and ISI were presented as mean ± se. The comparisons between groups were done by t-test.

3. Results 3.1. Ischemia impairs spike encoding at cortical and cerebellar GABAergic neurons The changes of ischemia on the functions of GABAergic neurons in cortical slices and Purkinje cells in cerebellar slices were examined by whole-cell current clamp. Inter-spike intervals (ISI, an index of spike capacity) were measured under control and after reducing perfusion rate by evoking action potentials with depolarization currents (>100 ms). The influences of ischemia on spike capacity of GABAergic neurons at cortical and cerebellar slices are showed in Fig. 1. Fig. 1A and B illustrates the changes during ischemia on sequential spikes at cortical GABAergic neurons. Ischemia (blue line) decreases the number of spikes in a given time (Fig. 1A). The values for ISI1-2 up to ISI4-5 under control (filled symbols, Fig. 1B) are 6.01 ± 0.22, 8.04 ± 0.24, 10.11 ± 0.38 and 11.97 ± 0.52 ms; and those under ischemia (open symbols, Fig. 1B) are 8.64 ± 0.25, 10.93 ± 0.39, 13.14 ± 0.50 and 15.01 ± 0.47 ms. ISI values for corresponding spikes under these two conditions are statistically different (**p < 0.01, n = 15). Thus, the ischemia attenuates capacity of spike at cortical GABAergic neurons. Fig. 1C and D shows the changes during ischemia on sequential spikes at cerebellar GABAergic Purkinje cells. Ischemia increases the number of spikes in a given time (Fig. 1C). The values for ISI1-2 up to ISI4-5 under control (filled symbols, Fig. 1D) are 11.39 ± 0.39, 13.09 ± 0.39, 14.21 ± 0.28 and 15.80 ± 0.27 ms under the control; and those under ischemia (open symbols, Fig. 1D) are 9.57 ± 0.29, 11.28 ± 0.28, 12.85 ± 0.22 and 14.04 ± 0.30 ms during ischemia. ISI values for corresponding spikes under these two conditions are statistically different (**p < 0.01, n = 15). Thus, the ischemia enhances spike capacity at cerebellar Purkinje cells. We then studied the mechanisms underlying these ischemic changes. In terms of biophysical aspects, we examined spike refractory periods and threshold potentials during the ischemia.

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Fig. 1. Ischemia reduces spike encoding at cortical GABAergic neurons and enhances spike encoding at cerebellar Purkinje cells in brain slices. (A) The superimposed waveforms of sequential spikes are evoked by depolarization pulses 6 min after reducing perfusion rate (blue trace) vs. control (red trace) at cortical GABAergic neurons. (B) Inter-spike intervals (ISI) of sequential spikes under control (filled symbols) and ischemia (open symbols) at cortical GABAergic neurons. (C) The superimposed waveforms of sequential spikes are evoked by depolarization pulses during ischemia (blue trcce) vs. control (red trace) at cerebellar Purkinje cells. (D) Inter-spike intervals (ISI) of sequential spikes under control (filled symbols) and ischemia(open symbols) at cerebellar Purkinje cells. (For interpretation of the references to colour/color in this figure legend, the reader is referred to the web version of this article.)

3.2. Ischemia impairs the functions of spike encoding at cortical and cerebellar GABAergic neurons via affecting ARP and Vts As the spike capability is controlled by ARP and Vts mediated by voltage-gated sodium channels (VGSC) (Wang and Kelly, 2001; Zhao et al., 2008), the influences of ischemia to spike encoding at cortical and cerebellar GABAergic neurons are likely due to the effect on ARP and Vts. The effects of ischemia on ARPs at GABAergic neurons are showed in Fig. 2. Compare with control group (red lines, Fig. 2A), ischemia prolongs ARP (blue lines, Fig. 2A). The values of ARP for spikes one to four are 3.60 ± 0.07, 4.03 ± 0.06, 4.29 ± 0.10 and 4.49 ± 0.11 ms under control at cortical GABAergic neurons (filled symbols, Fig. 2B), and 4.16 ± 0.10, 4.75 ± 0.12, 5.11 ± 0.14 and 5.44 ± 0.18 ms under ischemia (open symbols, Fig. 2B). ARP values for corresponding spikes under these two conditions are statistically different (**p < 0.01, n = 15). At cerebellar GABAergic Purkinje cells, ARPs appears shorter under ischemia (blue lines, Fig. 2C) than control (red lines, Fig. 2C). The values of ARP for spikes one to four are 4.28 ± 0.13, 4.38 ± 0.17, 5.96 ± 0.18 and 6.58± 0.16 ms under the control (filled symbols in Fig. 2D), and 3.93 ± 0.17, 4.53 ± 0.13, 4.98 ± 0.13 and 5.14 ± 0.18 ms under ischemia (open symbols, Fig. 2D). ARP values for corresponding spikes under these two conditions are statistically different (*p < 0.05, **p < 0.01, n = 15). Thus, the ischemia prolongs ARPs of

action potentials at cortical GABAergic neurons, and shortens ARPs at cerebellar Purkinje cells. The effects of ischemia on Vts at GABAergic neurons are showed in Fig. 3. Fig. 3A shows the influences during ischemia of Vts at cortical GABAergic neurons. Ischemia raises Vts (blue line) compared to control (red line). Fig. 3B shows that Vts-Vr values for spikes one to five are 29.57 ± 0.79, 34.53 ± 1.24, 35.27 ± 1.03, 35.98 ± 0.79 and 37.08 ± 0.77 mV under control (filled symbols); and its values are 33.81 ± 0.59, 37.19 ± 0.61, 38.08 ± 0.73, 38.81 ± 0.55 and 39.33 ± 0.65 mV under the ischemia (open symbols). Vts-Vr values for corresponding spikes under these two conditions are different (*p < 0.05, **p < 0.01, n = 15). At cerebellar Purkinje cells, Vts appears lower under ischemia (blue lines, Fig. 3C) than control (red lines, Fig. 3C). Vts-Vr values for spikes one to five are 23.94 ± 1.29, 24.71 ± 1.23, 25.43 ± 1.37, 25.52 ± 1.40 and 25.76 ± 1.45 mV under the control (filled symbols, Fig. 3D); and its values are 19.77 ± 1.18, 20.47 ± 1.14, 20.50 ± 1.18, 20.66 ± 1.22 and 20.96 ± 1.23 mV during the ischemia (open symbols, Fig. 3D). Vts-Vr values for corresponding spikes under these two conditions are different (*p < 0.05, n = 15). Thus, the threshold potentials of action potentials are raised at cortical GABAergic neurons, and lowered at cerebellar Purkinje cells during ischemia. In summary, ischemia increased ARPs and Vts at cortical GABAergic neurons and decreased ARPs and Vts at cerebellar Purkinje neurons, which lead to differential effect on spike capacity. In

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Fig. 2. Ischemia prolongs ARP at cortical GABAergic neurons and shortens ARP at cerebellar Purkinje cells in brain slices. (A) The superimposed waveforms show ARP measured by changing the inter-pulse interval of depolarization currents (3 ms) under the control (red trace) vs. ischemia (blue trace) at cortical GABAergic neurons. (B) The comparisons of ARP of sequential spikes under the control (filled symbols) vs. ischemia (open symbols) at cortical GABAergic neurons. (C) The superimposed waveforms show ARP measured by changing the inter-pulse interval of depolarization currents under the control (red trace) vs. ischemia (blue trace) at cerebellar GABAergic Purkinje cells. (D) The comparisons of ARP of sequential spikes under the control (filled symbols) vs. ishemia (open symbols) at cerebellar Purkinje cells. (For interpretation of the references to colour/color in this figure legend, the reader is referred to the web version of this article.)

terms of the different Ca2+ signals were distributed in these two kinds of neurons (Qi et al., 2009), we examined the roles of cytoplasm Ca2+ in the ischemia-induced changes of GABAergic neurons by lowering intracellular Ca2+ level. If lowering cytoplasm Ca2+ prevents their ischemic changes, Ca2+ must be required in the ischemic pathway. 3.3. Cytoplasm Ca2+ is essential to ischemic changes of GABAergic neurons To examine if the rise of cytoplasm Ca2+ was required in the ischemic pathway of GABAergic neurons, we loaded BAPTA (a chelator of Ca2+ ) into the recorded cells via recording pipettes, and measured the spike encoding under BAPTA infusion before and after ischemia. The changes in spike capacity of GABAergic neurons under control (filled symbols) and ischemia (open symbols) with the infusion of BAPTA are illustrated in Fig. 4. The values for ISI1-2 up to ISI4-5 under control are 6.89 ± 0.21, 8.92 ± 0.21, 11.46 ± 0.32 and 12.78 ± 0.41 ms; and those under ischemia are 6.95 ± 0.22, 9.19 ± 0.26, 11.93 ± 0.27 and 13.12 ± 0.23 ms (Fig. 4A). At cerebellar GABAergic Purkinje cells, the values for ISI1-2 up to ISI4-5 under control are 11.51 ± 0.39, 13.47 ± 0.38, 14.51 ± 0.35 and 15.22 ± 0.37 ms; and those under ischemia are 11.03 ± 0.38, 12.97 ± 0.39, 14.00 ± 0.35 and 14.83 ± 0.37 ms (Fig. 4B). ISI values for corresponding spikes under these two conditions are not statistically different (# p > 0.05, n = 12). The preventive effect of BAPTA

on ischemic changes indicates that the increase of cytoplasm Ca2+ is essential during ischemia of GABAergic neurons. 4. Discussion Our studies on the changes of GABAergic neurons during the early ischemia demonstrated that ischemia impaired the intrinsic excitability of the neurons, such as spike capacity. We measured the changes of spike capacity of GABAergic neurons during the early ischemia. Our finding, the early ischemia decreased the ability of spike encoding at cortical GABAergic neurons and increased spike encoding at cerebellar Purkinje cells (Fig. 1). Such ischemic changes in spike capacity were associated with the VGSC-mediated threshold potential and absolute refractory periods (Fig. 2 and 3). We found that ischemia prolonged the threshold potentials and absolute refractory periods parameters at cortical GABAergic neurons and shortened these at cerebellar GABAergic Purkinje cells. Therefore, our finding suggested that the therapeutic strategy to protect ischemic stroke at different areas may different. Neuronal excitotoxicity in the early phase of ischemia presumably caused neuronal cell death (Choi, 1988). It had been found that GABAergic neurons were vulnerable to pathological factors, especially for ischemia and epilepsy (Wang, 2003; Wang et al., 2015; Zhao et al., 2008; Huang et al., 2010; de Lanerolle et al., 2011). Ischemic dysfunction of GABAergic neurons was initiated by neuronal excitotoxicity. In terms of the mechanisms underlying

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Fig. 3. Ischemia elevates Vts at cortical GABAergic neurons and lowers Vts at cerebellar Purkinje cells in brain slices. (A) The superimposed waveforms show Vts measured during burst spikes evoked by depolarization currents under control (red trace) vs. ischemia (blue trace) at cortical GABAergic neurons. (B) The comparisons of the Vts of sequential spikes under control (filled symbols) vs. ischemia (open symbols) at cortical GABAergic neurons. (C) The superimposed waveforms show Vts measured during burst spikes evoked by depolarization currents under the control (red trace) vs. ischemia (blue trace) at cerebellar Purkinje cells. (D) The comparisons of the Vts of sequential spikes under the control (filled symbols) vs. ischemia (open symbols) at cerebellar Purkinje cells. (For interpretation of the references to colour/color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. The infusion of BAPTA into cortical GABAergic neurons and cerebellar Purkinje cells prevents ischemia-induced overexcitation in brain slices. (A) The comparisons of ISI for sequential spikes under control (filled symbols) and ischemia (open symbols) while infusing BAPTA at cortical GABAergic neurons. (B) Inter-spike intervals (ISI) of sequential spikes under control (filled symbols) and ischemia (open symbols) while infusing BAPTA at cerebellar Purkinje cells.

ischemia-induced pathological changes in brains, previous studies indicated the roles of cellular metabolic changes, such as a malfunction of glutamate transporter, a glutamate-dependent elevation of cytoplasmic Ca2+ and free radicals (Ray et al., 2014; Choi, 1988;

Lipton, 1999; Kuebler et al., 2015; Zahra et al., 2015; Welsh et al., 2002). These data did not show what types of neuronal cells and underlying mechanisms leading to neuronal excitotoxicity, such

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as cortical GABAergic neurons and cerebellar Purkinje cells, were involved in these processes. Why did ischemia lead to the dysfunction of spike encoding at central GABAergic neurons? Ischemia regulated the refractory period and threshold potential of action potentials, and in turn influenced spike encoding at GABAergic neurons (Figs. 1–3). These results further supported a notion that refractory periods and threshold potentials navigated neuronal encoding, which was concluded by examining a correlation between these parameters and spike encoding when raising the intensity of excitatory inputs, changing after hyperpolarization, and tracing postnatal development (Lu et al., 2014; Qi et al., 2009; Guan et al., 2006; Chen et al., 2006). Ischemia may lead to cytoplism Ca2+ overload (Zhao et al., 2008; Huang et al., 2010), and it had been reported that the level of Ca2+ activated signals, such as calcineurin and CaMK-II were different in these two types of GABAergic neurons (Qi et al., 2009). The ratios of CaM-KII to calcineurin were higher in cortical GABAergic neurons than cerebellar Purkinje cells. Moreover, ischemic Ca2+ overload activated calcineurin and CaMK-II (Shioda et al., 2007). CaMK-II phosphorylated VGSCs to upper ARP and Vts in cortical GABAergic neurons. On the other hand, calcineurin dephosphorylated VGSCs to lower ARP and Vts in cerebellar Purkinje cells. Therefore, we hypothesized that the alterantive of spike encoding induced by ischemia mainly via Ca2+ overload in cortical and cerebellar GABAergic neurons. If this was the case, the reducing of cytoplasm Ca2+ may protect the ischemic-injury of GABAergic neurons. By loading BAPTA (a chelator of Ca2+ ) into the recorded cells via recording pipettes, we further found that the spike encoding had no significant changes under BAPTA infusion during the early phase of ischemia at these two kinds of neurons, these results indicated that the increase of cytoplasm Ca2+ was essential during ischemia of GABAergic neurons. Our results and postulations were supported by a report that VGSCs were phosphorylated and inactivated by protein kinase C (Klapal et al., 2016), and the distribution of VGSCs in cerebellar Purkinje cells and cortical GABAergic neurons was different. Cortical GABAergic neurons mainly included Nav1.2 and Nav1.1, but cerebellar Purkinje cells included Nav1.1 (Klapal et al., 2016). We supposed the differences between these two types of GABAergic neurons was resulted from the kinetics of VGSCs were different. And we were designing the experiments to test this hypothesis in detail. Conflict of interest The authors declare no potential conflicts of interest with respect to the anthorship and/or publication of this article. Author contributions Conceived and designed the experiments: LH. Contributed this work equally: LH and CW. Performed the experiments: LH and CW. Analyzed the data: LH,CW,RJG, HN and SDZ. Contributed reagents/materials/analysis tools: LH, CW, RJG, HN and SDZ. Wrote the paper: LH. All authors have read and approved final version. Acknowledgments We thank Dr. Jinhui Wang for critical reading before submission. This study is granted by Natural Science Foundation of China (31500836) to RJG, Anhui Natural Science Foundation (1308085QH147) to LH and (1408085MH185) to SDZ, as well as Natural Science Foundation of Bengbu Medical College (BYKY201622ZD) to LH and (BYKY201635ZD) to CW.

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