Neuroscience Letters 580 (2014) 62–67

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Persistent sodium currents contribute to A␤1-42 -induced hyperexcitation of hippocampal CA1 pyramidal neurons Shuan-cheng Ren a , Peng-zhi Chen a , Hui-hui Jiang a , Ze Mi a , Fenglian Xu b,∗ , Bo Hu a , Jun Zhang a , Zhi-ru Zhu a,∗∗ a b

Department of Physiology, Third Military Medical University, Chongqing 400038, PR China Department of Physiology and Pharmacology, The Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta T2N 4N1, Canada

h i g h l i g h t s • • • •

We investigated the effects of soluble A␤1-42 on neuronal excitability. Soluble A␤1-42 increased the mean frequency of hippocampal spontaneous discharges. Soluble A␤1-42 also increased the amplitude of persistent sodium current. Riluzole inhibited the A␤1-42 -induced neuronal hyperexcitation.

a r t i c l e

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Article history: Received 5 March 2014 Received in revised form 3 July 2014 Accepted 25 July 2014 Available online 4 August 2014 Keywords: Alzheimer’s disease Amyloid ␤ Persistent sodium current Hyperexcitation Riluzole

a b s t r a c t Patients with Alzheimer’s disease (AD) have elevated incidence of epilepsy. Moreover, neuronal hyperexcitation occurs in transgenic mouse models overexpressing amyloid precursor protein and its pathogenic product, amyloid ␤ protein (A␤). However, the cellular mechanisms of how A␤ causes neuronal hyperexcitation are largely unknown. We hypothesize that the persistent sodium current (INaP ), a subthreshold sodium current that can increase neuronal excitability, may in part account for the A␤-induced neuronal hyperexcitation. The present study was designed to evaluate the involvement of INaP in A␤-induced hyperexcitation of hippocampal CA1 pyramidal neurons using a whole-cell patch-clamp recording technique. Our results showed that bath application of soluble A␤1-42 increased neuronal excitability in a concentration-dependent manner. Soluble A␤1-42 also increased the amplitude of INaP without significantly affecting its activation properties. In the presence of riluzole (RLZ), an antagonist of INaP , the A␤1-42 -induced neuronal hyperexcitation and INaP augmentation were significantly inhibited. These findings suggest that soluble A␤1-42 may induce neuronal hyperexcitation by increasing the amplitude of INaP and that RLZ can inhibit the A␤1-42 -induced abnormal neuronal activity. © 2014 Published by Elsevier Ireland Ltd.

1. Introduction Alzheimer’s disease (AD), the most common type of dementia, is characterized by progressive memory impairment and cognitive decline. Excessive accumulation of amyloid ␤ (A␤) is thought to be a causal factor in producing the cognitive deficits [7]. It is still

∗ Corresponding author at: Department of Physiology and Pharmacology, The Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta T2N 4N1, Canada. Tel.: +1 403 220 3775. ∗∗ Corresponding author at: Department of Physiology, Third Military Medical University, Gaotanyan Street 30, Chongqing 400038, PR China. Tel.: +86 23 68752248; fax: +86 23 68752248. E-mail addresses: [email protected] (F. Xu), [email protected] (Z.-r. Zhu). http://dx.doi.org/10.1016/j.neulet.2014.07.050 0304-3940/© 2014 Published by Elsevier Ireland Ltd.

unclear how A␤ accumulation leads to impaired memory and cognitive function. Recent studies indicated that A␤-related neuronal hyperexcitation and aberrant network activity may contribute to the cognitive deficits in AD [16]. Mouse models of AD that have elevated levels of A␤ exhibit altered neuronal activity, spontaneous seizures and epileptiform discharges [14], which further contribute to memory impairments and cognitive deficits [23]. In addition, blocking A␤-induced epileptiform discharges can ameliorate cognitive decline and behavior dysfunction in transgenic AD mouse models [18]. Accumulating evidence further suggests that soluble A␤, rather than amyloid plaques, correlates well with the severity of cognitive decline and crucially leads to malfunction of neurons [19]. The hippocampal region of the AD transgenic mouse has an increased proportion of hyperactive neurons prior to the formation of A␤ plaques. Moreover, extracellular application of soluble

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A␤1-42 induces hyperactivity of hippocampal CA1 neurons in wildtype mice [2]. These findings link A␤ to neuronal hyperexcitation, aberrant network activity and cognitive impairment. However, the underlying cellular mechanisms and pathways that mediate A␤induced neuronal hyperexcitation are poorly understood. Voltage-gated sodium channels control neuronal excitability by initiating and propagating action potentials. Persistent sodium current (INaP ), a slow inactivating component of TTX-sensitive sodium current, is important for regulating neuronal excitability [4,6]. As a low-voltage-activated current, INaP depolarizes membrane potential toward the threshold for action potential initiation. Despite its small magnitude compared with the peak of transient sodium current (INaT ), INaP has important effects on neuronal functions, including generating subthreshold oscillatory activity, amplifying synaptic potentials, and facilitating repetitive firing patterns [26]. INaP keeps the membrane depolarized longer to facilitate epileptic firings; thus, it is involved in both acquired and genetically determined epilepsy [20]. In animal models of temporal lobe epilepsy, a particular type of epilepsy that exhibits both the memory impairment and the hippocampal neuropathology that occur in AD, increased INaP has been found in neurons with elevated excitability [8]. Furthermore, an antiepileptic drug that blocks INaP has been demonstrated to be effective in reducing epileptiform discharges in a mouse model of AD [28]. Thus, it is tempting to speculate that INaP may participate in A␤-induced neuronal hyperexcitation and epileptiform neuronal activity in AD. In the present study, we first investigated the effects of soluble A␤1-42 on the excitability of hippocampal CA1 pyramidal neurons. We then examined the alteration of INaP in the presence of soluble A␤1-42 . Lastly, we tested the effect of riluzole (RLZ), an inhibitor of INaP [22], on A␤1-42 -induced abnormal neuronal activity in hippocampal neurons. 2. Materials and methods 2.1. Soluble Aˇ1-42 preparations All reagents were obtained from Sigma-Aldrich, USA. Soluble A␤1-42 was prepared as described previously [10]. In brief, A␤1-42 was first dissolved in hexafluoro-2-propanol (HFIP) and aliquoted. HFIP was then removed by evaporation under vacuum, and the resulting clear peptide films were stored at −20 ◦ C. Prior to use, an aliquot of A␤1-42 peptide film was dissolved in anhydrous dimethyl sulfoxide (DMSO) and then added to ice-cold artificial cerebral spinal fluid (ACSF) to obtain a working concentration of 100 ␮M. This solution was then incubated at 4 ◦ C for 24 h without any disturbance and then centrifuged to obtain the supernatant. The supernatant included the soluble A␤1-42 preparation. Previous studies by western blotting and atomic force microscopy have demonstrated that the major species was A␤1-42 monomer and also included the trimer, tetramer, and, to a lesser extent, the dimer [10,11]. These A␤1-42 preparations were diluted in ACSF for immediate use, and the final concentration of DMSO was always ≤0.1%. 2.2. Slice preparation Sprague-Dawley rats (P12-14) were used in this study. Animals were obtained from the Laboratory Animal Center at the Third Military Medical University in China, and all protocols and procedures were approved by the University Animal Care and Use Committee. Rats at this age range stably shows datable levels of INaP , but without significant fluctuation of INaP conductance [13]. After halothane anesthesia, animals were decapitated, and the brain was removed quickly. The brain was subsequently submerged in cold ACSF containing (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO3 , 1.25 KH2 PO4 , 1.2

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MgSO4 , 2 CaCl2 and 10 dextrose, bubbled with 95% O2 –5% CO2 , pH 7.4. The brain was blocked, and an oscillating tissue slicer (Leica, VT1000, Wetzlar, Germany) was used to cut 400 ␮m-thick horizontal sections. Slices were initially incubated for a minimum of 90 min at room temperature (22–24 ◦ C) in ACSF and were then transferred to and submerged in a recording chamber, where they were perfused continuously with carbogen buffered ACSF. 2.3. Whole-cell patch-clamp recordings Whole-cell patch-clamp recordings were obtained from cell bodies of CA1 pyramidal neurons in the rat hippocampus. The cell bodies of these neurons were found using an upright microscope equipped with Leica differential interference contrast optics, a 40× water immersion objective, and an infrared video imaging camera. Data acquisition was conducted with EPC10 amplifiers (HEKA Elektronik, Lambrecht/Pfalz, Germany). The signal was stored for off-line analysis with Pulse/Pulse fit v.8.74 (HEKA Elektronik) and Igor Pro v.4.03 (WaveMatrics). Pipettes (4–8 M) for whole-cell recordings were pulled on a horizontal micropipette puller (P97, Sutter Instrument) from filamented capillary glass and were filled with a pipette solution containing (in mM) 145 K-gluconate, 0.5 EGTA, 2 MgCl2 , 5 HEPES, 5 K-ATP, and 0.4 Na-GTP, pH 7.4, 290–295 mOsm. Liquid junction potential was calculated to be −10 mV for our intrapipette solution and membrane voltages were corrected off-line. To record INaP , recording electrodes were filled with an internal solution containing (in mM) 110 CsCl, 5 NaCl, 3 MgCl2 , 1 CaCl2 , 3 EGTA and 40 HEPES, pH 7.4. The bath solution consisted of (in mM) 100 NaCl, 40 TEA-Cl, 3 KCl, 1 MgCl2 , 1 CaCl2 , 10 D-glucose, 10 HEPES, 1 BaCl2 , 1 CsCl, 2 4-AP and 0.1 CdCl2 , bubbled with 95% O2 -5% CO2 , pH 7.4. After seal formation and cell membrane rupturing, capacitance currents were minimized using the amplifier circuitry. Series resistance was compensated by 80% and was continually monitored throughout the experiment. Neurons were discarded if the series resistance changed by more than 15%. Under a holding potential of −60 mV, INaP was recorded in CA1 pyramidal neurons by applying a 3 s depolarization ramp current from −80 to 0 mV. To isolate INaP , current responses to depolarizing voltage ramps were recorded in the absence and presence of TTX (Tetrodotoxin, 1 ␮M) and then subtracted. To account for any differences in cell size, all current recordings were normalized to whole cell capacitance to get current density. Conductance–voltage (G–V) relationships of INap were calculated from G = I/(V − Vrev ), where I is the recorded INaP measured at potential V and Vrev is the Na reversal potential calculated from the Nernst equation. Normalized activation curves were fitted to Boltzmann relationships in the form G/Gmax = 1/{1 + exp[(V1/2 + V)/␬]}, where Gmax is the maximal peak conductance, G is the peak conductance at each test voltage, V1/2 is the voltage at which half-maximal activation is reached, and ␬ is the slope factor. 2.4. Data analysis Statistical analysis was made using statistical analysis software Origin 8.0 (Microcal, Inc, Northampton, MA, USA) and SPSS 13.0 (IBM, New York, NY). The values were presented as the mean ± S.E.M. Differences in the mean values among groups were analyzed using Student’s t-test and one-way ANOVA. Values of P < 0.05 were considered significant. 3. Results 3.1. Soluble Aˇ1-42 induced hyperexcitation of hippocampal CA1 pyramidal neurons

at

Previous studies have demonstrated that soluble A␤ nanomolar concentrations induced abnormal synaptic

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Fig. 1. A␤1-42 induces hyperexcitation of hippocampal CA1 pyramidal neurons. (A) Pyramidal neurons in the hippocampal CA1 region. (B) Electrophysiological characteristics of pyramid neurons. (C–E) Representative traces showing that application of A␤1-42 (applied during the bar) leads to alterations in spontaneous neuronal firing and resting membrane potential. Lower traces are enlarged to show change in membrane potentials. (F) Bar graph of pyramidal neuron firing frequency in different concentrations of soluble A␤1-42 .

transmission, neuronal excitability [11] and network activity [12,15]. We first explored the effects of nanomolar concentrations soluble A␤1-42 on hippocampal CA1 pyramidal neurons. CA1 pyramidal neurons were identified according to their pyramidal-shaped cell bodies with long apical dendrites (Fig. 1A). These neurons fired regular spike patterns and exhibited obvious “sag” characteristics (Fig. 1B). The effects of A␤1-42 on CA1 neuron excitability were initially evaluated by recording membrane potentials and action potentials. After establishing a stable baseline of membrane potentials at least 5 min after formation of the whole-cell configuration, soluble A␤1-42 was applied by superfusion to CA1 neurons for a duration of 1–2 min. This application led to a depolarization and an increase in firing frequency, which was concentration-dependent (Fig. 1C–F). A␤1-42 at 1 nM resulted in minimal membrane voltage change (Fig. 1C). A␤1-42 at 100 nM facilitated neuronal firing for a short time (Fig. 1D), whereas 300 nM A␤1-42 increased neuronal

firing frequency for a prolonged duration (Fig. 1E). A␤1-42 at concentrations of 1, 10, 100 and 300 nM caused a mean firing frequency (2–3 min after A␤1-42 application) of 0.42 ± 0.28 Hz, 0.71 ± 0.34 Hz, 1.33 ± 0.59 Hz, and 5.75 ± 0.97 Hz, respectively (Fig. 1F, n = 12 for each group). At 300 nM, A␤1-42 facilitated firing relative to the 1 nM A␤1-42 group (Fig. 1F, **P < 0.01). Collectively, these results indicated that under our experimental conditions, 300 nM soluble A␤1-42 significantly induced neuronal hyperexcitation. Therefore, 300 nM A␤1-42 was used in the remaining experiments. 3.2. Aˇ1-42 increased INaP of hippocampal CA1 pyramidal neurons We next sought to examine whether soluble A␤1-42 -induced hyperactivity of CA1 neurons involves alteration of INaP , as INaP has been shown to be abundant in these neurons [6]. We used a TTX subtraction protocol in conjunction with a depolarizing voltage

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Fig. 2. A␤1-42 increases INaP of hippocampal CA1 pyramidal neurons. (A) Traces of INaP (bottom) induced by increasing applied voltage (top) in a pyramidal neuron. Inset shows current traces before and after TTX addition. (B) Bar graph showing that RLZ inhibits the peak current densities of INaP . (C) Traces of INaP in CA1 neurons in control, A␤1-42 , A␤1-42 + RLZ and wash groups. (D) Bar graph showing A␤1-42 increases the average peak current density of INaP , which is inhibited by RLZ. (E) I–V relationship of INaP in CA1 neurons from control, A␤1-42 , A␤1-42 + RLZ and wash groups (*p < 0.05, **p < 0.01, A␤1-42 vs. control group. # p < 0.05, ## p < 0.01, A␤1-42 + RLZ vs. A␤1-42 group). (F) Voltage-dependent activation of INaP in CA1 neurons.

ramp. The subtracted current trace reflects the current–voltage relationship of INaP . INaP was induced at potentials of −60 to −50 mV and reached a peak at −38 mV (Fig. 2A). After dividing peak current amplitude by cell capacitance to calculate INaP density, RLZ at 10 ␮M significantly reduced the peak INaP current density (Fig. 2A and B, control: −4.30 ± 0.45 pA/pF, RLZ: −0.89 ± 0.17 pA/pF, n = 6 for each group, P < 0.01). This reduction of INaP was partially recovered following washout in all tested neurons (Fig. 2A and B, wash: −1.72 ± 0.41 pA/pF, n = 6). These results suggested that the inward sodium current was due to INaP . Treatment with vehicle (6.7 × 10−3 % DMSO, equal to the concentration of DMSO at 300 nM soluble A␤1-42 ) did not have significantly different effects on INaP (Fig. 2A and B, vehicle: −4.29 ± 0.46 pA/pF, n = 6, P > 0.05). We then examined the effects of A␤1-42 on INaP . The average peak value of the INaP current density was significantly increased by application of 300 nM A␤1-42 (Fig. 2C and D, control: −3.86 ± 0.89 pA/pF, A␤1-42 : −9.47 ± 0.92 pA/pF, n = 6 for each group, P < 0.01). The increased INaP was inhibited by RLZ (Fig. 2C and D, A␤1-42 + RLZ: −0.60 ± 0.23 pA/pF, n = 6, P < 0.01). The inhibited INaP was partially recovered following washout.

Voltage-dependent activation is a physiologically important characteristic of ion channels that directly influences the excitability of neurons. I–V curves showed that the current densities of INaP were increased by application of 300 nM A␤1-42 , and this increase was eliminated by administration of 10 ␮M RLZ (Fig. 2E, n = 6). The voltage dependence of the activation of INaP was calculated and fitted with the Boltzmann equation as shown in Fig. 2F. The activation curve was slightly shifted leftward in the A␤1-42 group compared to the control group but was not significant (Fig. 2F, control: V1/2 = −46.81 mV,  = 3.36 mV A␤1-42 : V1/2 =−47.24 mV,  = 3.38 mV. P > 0.05, n = 6). These results showed that soluble A␤1-42 significantly increased the amplitude of INaP without altering its activation properties. 3.3. Inhibitory effects of RLZ on the Aˇ1-42 -induced hyperexcitation Based on the above findings showing that increased INaP contributed to A␤1-42 -induced hyperexcitation of hippocampal neurons, we next tested the ability of RLZ to prevent

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Fig. 3. RLZ inhibits A␤1-42 -induced neuronal hyperexcitation in hippocampal neurons. (A) A␤-induced neuronal hyperexcitation is significantly inhibited in the presence of RLZ (upper panels). Lower panels are expansions of the original traces at the marked times (indicated by arrows). (B) Bar graph showing that RLZ significantly reduced A␤1-42 -induced increases in mean firing frequency. (C) Neuronal responses to depolarizing step current pulses (0.2 nA) in control, A␤1-42 and A␤1-42 + RLZ groups.

hyperexcitation. A␤1-42 at 300 nM was first applied to induce hyperactivity in neurons, and then, 10 ␮M RLZ was added in the continued presence of A␤1-42 . We found that the robust firing of action potentials induced by A␤1-42 was gradually diminished and completely abolished by the application of RLZ for approximately 5 min (Fig. 3A), further indicating the involvement of INaP in A␤1-42 induced changes in hippocampal neuronal excitability. Meanwhile, RLZ prevented the A␤1-42 -induced increase in average firing frequency (Fig. 3B, control: 0.09 ± 0.04 Hz, A␤1-42 : 6.14 ± 0.77 Hz, A␤1-42 + RLZ: 0.99 ± 0.45 Hz, n = 6 for each group, **P < 0.01). Although RLZ largely abolished spontaneous repetitive firing, a single action potential was evoked at the initial phase of the depolarizing current pulse (Fig. 3C), indicating that these CA1 neurons were still capable of generating action potentials in the presence of RLZ. 4. Discussion Our results showed that soluble A␤1-42 increased neuronal excitability and the peak amplitude of INaP in hippocampal CA1 pyramidal neurons in brain slices. Furthermore, RLZ significantly decreased INaP and reversed neuronal hyperactivity induced by A␤1-42 . 4.1. Effects of Aˇ on neuronal excitability There is increasing evidence suggesting that the accumulation of A␤ can induce changes in neuronal excitability and network activity [15]; however, those findings are controversial. Low dose A␤1-42 (1 nM) has been found to inhibit neuronal excitability in the rat prefrontal cortex, whereas prolonged application of high-dose A␤1-42 (500 nM) induces the opposite effects [24]. A␤1-42 and A␤25-35

fibrils, but not oligomers, reduce neuronal resting membrane potentials, which in turn promote neuronal hyperexcitability and trigger progressive epilepsy [12]. In the present study, we demonstrated that acute application of soluble A␤1-42 led to increased firing frequency of CA1 pyramidal neurons in a dose-dependent manner (Fig. 1C–F). Together, these studies suggest that pathologically relevant levels of A␤ [15] may exert effects on neuronal excitability in a time-dependent [11], concentration-dependent, and form-dependent manner. Our findings are consistent with previous studies showing that local application of A␤1-42 oligomers induces an excitatory effect on hippocampal CA1 pyramidal neurons in vivo [2]. Our results further support a previous in vitro study that A␤1-42 oligomers significantly induce inward currents, leading to increased firing of action potential in hippocampal neurons [1]. However, regarding selective neuronal vulnerabilities in AD, A␤ may exert different effects on different brain regions. In support of this idea, Yun et al. [27] reported that oligomeric A␤1-42 decreased neuronal excitability in the mouse dentate gyrus, whereas our present study showed that A␤1-42 increased excitability of hippocampal CA1 pyramidal neurons. Considering the complex effects that A␤ has on neuronal excitability, it seems plausible that in the progression of AD, various forms of A␤ may act differentially on different types of neurons and in various manners to produce the overall abnormal network activity. 4.2. Potential roles of INaP in AD pathogenesis Persistent sodium current, a consequence of single Nav1.6 channel opening [3], contributes significantly to shaping the repetitive firing discharges of CA1 neurons [17]. Increases in the expression levels of Nav1.6 accompanied by elevated INaP amplitude

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have been proven to cause neuronal hyperactivity [9]. Our present data showed that soluble A␤1-42 increased INaP amplitude without affecting its activation properties (Fig. 2C–F). What is the cellular mechanism underlying the A␤-induced alterations of INaP ? Considering the relatively short time course used in our study, the change in INaP amplitude is unlikely to be a result of increases in the expression of Nav1.6 subunits or reductions by degradation of the channel proteins. Instead, soluble A␤1-42 may act directly and/or indirectly to interfere with the function of Nav1.6 subunits, thus leading to increases in INaP amplitude. In addition, lowering extracellular calcium concentration has been found to up-regulate INaP in hippocampal CA1 pyramidal neurons [21]. Although the role of calcium was not investigated in the present study, it is possible that calcium-dependent pathways likely contribute to A␤-induced INaP increases, as disturbance in calcium homeostasis has been linked to almost all the brain pathological process observed in AD [25]. Further studies are required to investigate whether calcium participates in A␤-induced changes of INaP . In the present study, acute exposure (1–2 min) to soluble A␤1-42 increased INaP density (Fig. 2C–E), accompanied by neuronal hyperactivity (Fig. 1C–F). Driscoll et al. [5] reported that increased synaptic excitation decreased Nav1.6 expression and subsequently reduced sodium current density and neuronal excitability through homeostatic mechanisms in rat visual cortex pyramidal neurons. Because changes in neuronal activity modulate expression of voltage-gated channels, we suspected that chronic exposure (i.e., hours to days) of A␤1-42 may induce a reduction in INaP density. Consistent with our reasoning, a previous study demonstrated that AD mouse models with inhibitory synaptic impairments and aberrant network activity had reduced levels of Nav1.6 in the parietal cortex [23], which may indicate a decrease of INaP . In conclusion, INaP contributes to A␤1-42 -induced hyperexcitation of hippocampal CA1 pyramidal neurons, and RLZ may potentially have protective effects against A␤1-42 neuronal toxicity. Acknowledgments This work was supported by grants from the National Natural Foundation of China (31100759), the Scientific Foundation of Chongqing (CSTC2011BB5039) to Dr. Zhu. We also acknowledge the support by a grant from the Natural Sciences and Engineering Research Council of Canada (10001964) to Dr. Xu. References [1] E. Alberdi, M.V. Sánchez-Gómez, F. Cavaliere, A. Pérez-Samartín, J.L. Zugaza, R. Trullas, M. Domercq, C. Matute, Amyloid ␤ oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors, Cell Calcium 47 (2010) 264–272. [2] M.A. Busche, X. Chen, H.A. Henning, J. Reichwald, M. Staufenbiel, B. Sakmann, A. Konnerth, Critical role of soluble amyloid-␤ for early hippocampal hyperactivity in a mouse model of Alzheimer’s disease, Proc Natl Acad Sci 109 (2012) 8740–8745. [3] A. Chatelier, J. Zhao, P. Bois, M. Chahine, Biophysical characterisation of the persistent sodium current of the Nav1.6 neuronal sodium channel: a singlechannel analysis, Pflugers Archiv: Eur J Physiol 460 (2010) 77–86. [4] W.E. Crill, Persistent sodium current in mammalian central neurons, Annu Rev Physiol 58 (1996) 349–362.

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