Summary
Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells.
Graphical Abstract
Highlights
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Activity-dependent node of Ranvier Ca2+ influx in Purkinje cell axons
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Cav and KCa3.1 channels required for axonal spike propagation
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Nodal KCa3.1 channels provide repolarizing drive to sustain axonal spike propagation
Functional connectivity between brain regions relies on long-range signaling by myelinated axons. Gründemann and Clark show that local, activity-dependent calcium influx at nodes of Ranvier recruits calcium-activated potassium channels (KCa3.1) that drive repolarization and sustain node excitability, providing a pivotal mechanism to secure spike propagation along Purkinje cell axons.
Introduction
Understanding information transmission within neuronal circuits, and the factors underlying long-range axonal signaling malfunction, relies on identifying the axonal ion channels that are key regulators of node of Ranvier (NoR) excitability. NoRs are highly specialized zones in myelinated axons containing high densities of voltage-gated sodium channels (Navs) and associated cytoskeletal complexes, creating active zones essential for saltatory conduction of action potentials (APs) (Debanne et al., 2011). The repolarizing currents required to sustain Nav availability at NoRs for reliable AP transmission is less clear cut, particularly in the mammalian brain. Both low- and high-voltage-activated potassium (K+) channel subunits (Kv7 and Kv3.1/3) have been anatomically localized to NoRs (Devaux et al., 2003, 2004; Pan et al., 2006), and although their presence can vary between brain regions and axon types (Debanne et al., 2011; Devaux et al., 2003, 2004), recent experiments provide direct evidence that Kv7 channels stabilize NoR membrane potential (Vm) in cortical L5 pyramidal cells (Battefeld et al., 2014) consistent with findings in peripheral nerve (Schwarz et al., 2006). The influence of delayed rectifier (DR) K+ channels (Kv1.1 and Kv1.2), which are widely observed in the juxtaparanodal (JP) zone (Devaux et al., 2003; Ogawa et al., 2010; Rasband, 2010; Zhou et al., 1998), has, however, been difficult to assess without demyelination (Röper and Schwarz, 1989; Wilson and Chiu, 1990), and whether local Vm becomes sufficiently depolarized to recruit JP Kv1 channels during saltatory conduction is disputed (Arancibia-Carcamo and Attwell, 2014). Voltage-gated Ca2+ channels (Cavs), which could provide an additional source of depolarization as well as gating Ca2+-dependent processes including recruitment of Ca2+-dependent K+ channels (KCa), have been described in central myelinated axons and shown to influence excitability at the axon initial segment (AIS) (Bender and Trussell, 2009; Bender et al., 2010; Yu et al., 2010). However, although Cavs have been proposed to influence NoR formation during development (Alix et al., 2008), their presence at mature NoRs in the brain is not established (Zhang et al., 2006).
Purkinje cells (PCs) in the cerebellum fire at high rates, both spontaneously and in response to synaptic input, and thus require fast recovery of sodium channels at their NORs. We have obtained direct evidence that, rather than solely relying on voltage-gated potassium channels, activity-dependent, spatially localized Ca2+ influx at NoRs of PC axons recruits an intermediate-type KCa (IK, or KCa3.1) to provide a node-specific repolarizing conductance crucial for axonal spike propagation.
Results
Potassium Channels at Nodes of Ranvier
Using simultaneous somatic and axonal patch-clamp recordings, local pharmacology, and two-photon Ca2+ imaging of cerebellar PC axons, we directly investigated which ion channels are engaged at NoRs during AP propagation. We visualized PC axons in cerebellar slices by dye filling via the somatic recording pipette and recorded axonal APs downstream of NORs (see the Experimental Procedures; Figure 1A) identified by virtue of their presence at axonal branchpoints (Clark et al., 2005). APs are securely transmitted by PC axons at high firing rates, with failures occurring above ≈250 Hz (Khaliq and Raman, 2005; Monsivais et al., 2005). This propagation reliability is retained across axonal branchpoints, with equal limiting frequency in both the main projection axon (257 ± 17 Hz; also Monsivais et al., 2005) and in recurrent axon collaterals (Figures 1B and 1C, 253 ± 14 Hz, 0.05% differentially propagated spikes, n = 6 cells, see also Foust et al., 2010). We used local application (Figure S2A) of various ion channel antagonists to test their impact on AP propagation at NoRs in spontaneously firing PCs (firing rates 20–80 Hz). TTX (10 μM) completely blocked AP propagation, confirming the presence of a NoR at branchpoints (Figure S1D; see also Khaliq and Raman, 2005). In contrast, application of TEA at a concentration (10 mM) that should block a wide variety of K+ channels including Kv1, Kv3, and Kv7 types (Grissmer et al., 1994; Hadley et al., 2000) did not affect AP propagation, having no impact on axonal capacitive current amplitude, firing rate, or conduction velocity (Figures S1B and S1C). This lack of effect was similar at high firing rates, and the limiting frequency for spike propagation was unchanged (Figure S1D). These results are unexpected given that TEA-sensitive K+-channels are thought to contribute to axonal AP repolarization (Devaux et al., 2003; Hille, 1967; Röper and Schwarz, 1989), to stabilize nodal Vm as well as preventing antidromic spike reflection (Goldstein and Rall, 1974), and Kv3.3 subunits have been localized to PC axons (Chang et al., 2007). Potential explanations for these results might be that first, TEA-sensitive K+ channels are absent from PC axons and/or their NoRs and repolarization is mediated by an alternative mechanism. Second, they may be present but at insufficient density to be primary regulators of node excitability. Third, TEA-sensitive K+ channels might be located behind tight myelin junctions in the juxtaparanodes (JPs) (Wang et al., 1993) and contribute to repolarization of the axonal membrane while being inaccessible to TEA, as observed in peripheral nerve (Chiu and Ritchie 1980; Kocsis and Waxman 1980) (however, see Mierzwa et al., 2010). Fourth, these channels may be present in the JPs but weakly activated by the limited Vm changes that are calculated to occur in healthy myelinated axon segments (Arancibia-Carcamo and Attwell, 2014).
Recently, an intermediate-type KCa conductance (IK, KCa3.1), not previously observed in the CNS and with a lower sensitivity to TEA (IC50 = 24 mM) (Wei et al., 2005), was described in cerebellar PCs and shown to contribute to postsynaptic integration (Engbers et al., 2012). We examined whether IK might provide an alternative mechanism for regulating excitability at NoRs and found that local application of the selective IK channel antagonist TRAM-34 (Engbers et al., 2012; Wulff et al., 2000) to axonal branchpoints led to a concentration-dependent block of axonal spike propagation during spontaneous firing (20–80 Hz; Figures 1D–1G), with no observed dependence on firing rate (R = 0.1). Clotrimazole (1 μM), another IK-selective blocker, had similar effects (Figure 1G), while apamin (1 μM) and iberiotoxin (1 μM), which block small (SK) and large (BK) KCa channels, respectively, had no impact (98% ± 0.44% [n = 5] and 97.3% ± 1.2% [n = 4] of control axonal spike amplitude). IK gating depends solely on intracellular [Ca2+] (Kd: 0.1–0.3 μM) (Wei et al., 2005; Joiner et al., 1997), and, since both local removal of extracellular Ca2+ and application of Ni2+ (100 μM) similarly suppressed action potential propagation (Figures 1D–1G), the activation of IK is likely initiated by Ca2+ influx via Cavs. We confirmed that PC axons are immunopositive for the KCa3.1 subunit, the sole molecular entity that constitutes the IK channel, but found, surprisingly, that no hotspots were seen at NoRs and that KCa3.1 labeling was instead relatively uniform along the axon (Figure 2).
Activity-Dependent Ca2+ Influx at Nodes of Ranvier
Activity-dependent Ca2+ influx has been observed in optic nerve (Lev-Ram and Grinvald, 1987; Zhang et al., 2006), but the resulting Ca2+ transients are spatially uniform along both NoRs and internodes (Zhang et al., 2006), and the route of Ca2+ entry is uncertain. As yet, there is no direct evidence for Ca2+ influx localized to NoRs in the brain.
Using two-photon Ca2+-imaging, we investigated whether the spatial distribution of calcium signals in PC axons might confer NoR specificity of IK recruitment. We detected prominent activity-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) at NoRs (identified by their location at branchpoints) and at the AIS (see Bender and Trussell, 2009) during trains of evoked APs (Figures 3A–3C; 204 ± 21 Hz). Ca2+ signals were restricted to approximately 5 μm from the center of NoRs (Figures 3C and 3D), and there were no detectable changes in internodal [Ca2+]i (Figure 3B), consistent with the lack of effect on spike propagation of 0 mM Ca2+, 10 mM BAPTA application to the internodes (Figure S2B). [Ca2+]i at NoRs required axonal APs, were suppressed when spontaneous firing was arrested by somatic hyperpolarization (Figure 3G), and could not be driven by somatic depolarization when spikes were blocked with bath-applied TTX (Figures 3E and 3F). Increases in [Ca2+]i were spike rate dependent (Figure S3), slow, and cumulative and lead to sustained [Ca2+]i during continuous activity (Figures 3G and 3H). [Ca2+]i increase was also suppressed by removal of extracellular Ca2+ (16% ± 6% of control n = 4; Figure 3I) and bath application of mibefradil (Figures 3I and 5 μM, 43% ± 14% of control, p = 0.013) but was not sensitive to agatoxin IVa, which blocked Ca2+ transients in PC synaptic boutons as expected (Hillman et al., 1991) (Figures 3I; 9% ± 3% of control, p < 0.0001). This implies that T-type and not P-type CaVs are the primary source of Ca2+ entry at NoRs. Additionally, although prior depolarization of the soma reduced [Ca2+]i at the AIS, AP-triggered nodal Ca2+ signals were unaffected (Figures S3C and S3D), demonstrating that, in PCs, nodal [Ca2+]i is independent of somatic Vm.
KCa3.1/IK Current Is Sufficient to Sustain Node Excitability
To understand the major impact of IK block on axonal AP propagation, we used a multicompartmental model of a PC (Clark et al., 2005) and tested whether IK, as the sole repolarizing conductance at the NoRs, can support reliable propagation of APs in a continuously firing axon (see the Experimental Procedures for details). At a minimum IK density of 0.05 S/cm2, with an associated T-type CaV with a maximum permeability of 0.002 cm/s (Anwar et al., 2012) and an accompanying, low-density, juxtaparanodal DR K+ conductance (0.002 S/cm2, Clark et al., 2005) to mimic the Kv1.1/Kv1.2 channels commonly observed in immunohistochemical studies (Rasband 2010), AP propagation along the model axon was highly reliable. Complete removal of the IK conductance alone caused NoR depolarization, trapping of sodium channels in inactivated and blocked (Khaliq et al., 2003) states, and AP propagation block (Figure 4A). IK may therefore act to stabilize the NoR Vm during continuous firing and application of the IK channel inhibitor TRAM-34 (Figure 1) could cause depolarization block. Our observed lack of effect of TEA application to NoRs (Figure S1) implies that other K+ channel types previously shown to be present in PC axons (Kv3.1, Chang et al., 2007) and NoRs (Kv7.3, Pan et al., 2006) may not, in the absence of IK, be sufficient to support AP propagation. A direct test of the hypothesis that IK stabilizes nodal Vm requires quantitative measurements of axonal Vm during IK block. Since PC NoRs are too small for patch-clamp recording and voltage-sensitive dye methods cannot be used to detect absolute changes in Vm, we used the AIS as a proxy for NoRs while recording spontaneous firing at the soma, presuming that some IK might be present there, as suggested by our immunohistochemistry data (Figure 2). During TRAM-34 application to the AIS, we observed a reduction in somatic firing rate of 25% ± 5.2% (p < 0.001, n = 15 cells, Figure 4C), accompanied by a small somatic depolarization (1.5 ± 0.2 mV, p < 0.0001) and an increase in AP threshold (1.8 ± 0.16 mV, p < 0.0001). In a minority of cells (4 / 15), TRAM-34 application to the AIS caused sufficient depolarization (ΔVm = 2.56 ± 1.28 mV, mean Vm = −48.4 ± 1.9 mV) to reversibly but completely block firing in some trials. Reduction of firing rate correlated with initial firing rate (R = 0.53, p < 0.05) so that cells with higher baseline rates showed a smaller reduction in spike rate on TRAM-34 application. This may reflect the presence of rate-dependent recruitment of BK and SK currents shown to regulate PC somatic Vm during spontaneous firing (Raman and Bean, 1999). Analysis of the second derivative of the somatic AP revealed a reduction in the component driven by current flow from the AIS (Figure 4D) as well as the local somatic component of the action potential during TRAM-34 application, indicating a reduced recruitment of sodium current at both locations.
Discussion
KCa channels have been implicated in control of excitability in both unmyelinated (Lüscher et al., 1996) and myelinated (Lev-Ram and Grinvald, 1987) mammalian axons, although the evidence for the latter is indirect and the physiological impact unknown. Our results provide the first direct evidence for local, activity-dependent Cav-mediated Ca2+ influx at NoRs and for the recruitment of an intermediate KCa current (IK, KCa3.1) that is an essential component for spike propagation. Our model indicates that IK alone can sustain the Nav availability required for propagation security, although it is conceivable that other conductances might also play a role, for example, in setting the nodal Vm in the absence of firing. The sensitivity of AP conduction to low concentrations of Ni2+ and the suppression of the Ca2+ transients by mibefradil is consistent with the involvement of a T-type Cav, as seen in association with dendritic IK in PCs (Engbers et al., 2012). During sustained firing, this channel type is expected to be largely inactivated but could provide a small window current for Ca2+ entry. This might be of advantage since, due to the higher affinity of IK for Ca2+ (Wei et al., 2005), a small Ca2+ influx can be effective but less expensive for an energetically demanding cellular compartment. IK may provide advantages over other K+ channel types in axons that continuously transmit APs, often at high rates. IK lacks intrinsic voltage dependence and does not inactivate, but, by virtue of the activity dependence of Ca2+ influx, it could be recruited and sustained at levels controlled by mean spike rate. Its localized recruitment by Ca2+ may also be amplified by Ca2+-induced Ca2+ release (Llano et al., 1994). It has recently been shown that PCs also express BK channels under the myelin in the paranode region (Hirono et al., 2015). These channels appear to become functionally relevant at firing frequencies above 100 Hz, suggesting that they may act as a complementary partner to IK under conditions when [Ca2+]i is sufficient for their activation. In the absence of fast voltage-gated K+ currents, APs at NoRs may also be broader, as generated by the model, than the exceptionally narrow somatic APs in PCs, potentially further facilitating Ca2+ entry at these sites. Besides its role in axonal electrogenesis, nodal Ca2+ influx could also be important in regulating NoR structure and functional properties. For example, activity-dependent local Ca2+ levels might be crucial to target and maintain Cavs (Forti et al., 2000) and Navs (Hund et al., 2010) at NoRs (Wang et al., 2007) and to preserve or modify nodal architecture and myelin distribution (Alix et al., 2008; Einheber et al., 1997). Ca2+ signaling is thought to underlie the activity-dependent changes in mitochondrial motility observed in mammalian axons (Chiu, 2011), notably in PCs (Ohno et al., 2011). Importantly, given their spike frequency dependence, NoR Ca2+ signals could provide a readout of neuronal circuit activity, which could result in Ca2+-dependent axonal plasticity beyond the AIS (Grubb and Burrone, 2010; Gründemann and Häusser, 2010; Kole, 2011; Kuba et al., 2010). In providing direct evidence for activity-dependent Ca2+ entry at NoRs and its recruitment of IK, our findings show that Ca2+ may play both short- and long-term roles in regulating axonal excitability, crucial for long-range signaling in neuronal circuits.
Experimental Procedures
Electrophysiological Recordings
Methods used for preparing and recording from PCs and PC axons in cerebellar slices (200–250 μm) from P18–P43 C57Bl/6 mice were as previously described (Monsivais et al., 2005) and carried out under institutional and national approval. PC axons visualized by dye-filling and AP-associated cell-attached axonal capacitive currents were recorded in voltage clamp mode at 34°C ± 1°C. Amplitude of capacitive currents reflects the rate of rise of the axonal action potential and therefore pharmacological reduction in the amplitude reflects reduced sodium channel availability.
Drug Application
Drugs were diluted in ACSF and bath applied or diluted in HEPES-buffered or standard ACSF and pressure-applied locally via patch electrodes using a Picospritzer (0.5–10 psi, Parker). 30 μM Alexa Fluor 488 or 594 was included in the solution to help adjust pressure and pulse duration to target localized drug ejection. Ca2+ was replaced by Mg2+ in 0 mM Ca2+ solutions.
Two-Photon Ca2+ Imaging
Two-photon Ca2+ imaging and simultaneous electrophysiological recordings were performed using a custom-built dual galvanometer-based laser-scanning microscope (Prairie Technologies). A Ti:sapphire pulsed laser (MaiTai, Spectra Physics) tuned to 810 nm was used for two-photon excitation. 200 μM Oregon green 488 BAPTA-1 (OGB-1, Invitrogen) replaced EGTA in the pipette solution, which also contained 50 μM Alexa Fluor 594 hydrazide (Sigma-Aldrich) to visualize morphology. PCs were dialyzed for at least 15 min after establishing whole-cell mode before imaging.
Data Acquisition and Analysis
Data were digitized (ITC-18, InstruTECH, Heka) at 50–100 kHz and acquired using AxoGraph X (http://www.axographx.com/). Analysis was performed using custom-written routines in MATLAB (MathWorks) or IGOR Pro 6 (Wavemetrics) in combination with Neuromatic (http://www.thinkrandom.com/).
Two-photon imaging data were acquired with custom-written MATLAB software either as line scans (2 ms/line, 500 Hz) or as XYT frame scans for a chosen region of interest (5–20 frames/s), digitized with a BNC-2090 board (National Instruments), and analyzed with custom-written scripts in MATLAB, ImageJ, and IGOR Pro 6. Line scan and XYT frame-scan data were filtered using a boxcar running average or 2D-averaging filter, respectively. Relative changes in Ca2+-sensitive fluorescence (OGB-1, ΔF/F) were calculated as raw fluorescence Fraw minus baseline fluorescence F0 normalized to the background (FB) subtracted baseline fluorescence (Yasuda et al., 2004): ΔF / F = Fraw – F0 / F0 – FB. In some cases, data are displayed as raw OGB-1 fluorescence values or relative changes in OGB-1 fluorescence normalized to the Alexa Fluor 594 red fluorescence (ΔF/R, Figure S3). Line scans are presented as averages of three consecutive trials.
Data are shown as mean ± SEM. Significance is tested using Student’s t test unless otherwise stated, and a p value <0.05 is regarded as significantly different (InStat, GraphPad Software).
Immunohistochemistry and Confocal Microscopy
Mice were perfused with ice-cold PBS for 1 min and then with 4% paraformaldehyde (PFA) in PBS (10 min). Brains were dissected and 80 μm slices of cerebellar vermis were cut using a vibratome (Leica). Slices were washed in PBS and blocked in 10% goat serum for 2 hr before 48 hr antibody incubation in 2% goat serum (KCa3.1, mouse monoclonal, Santa Cruz Biotechnology, Calbindin D-28k, rabbit, Swant) followed by second antibody incubation for 24 hr (633 goat anti-rabbit, 488 as well as 633 goat anti-mouse, Invitrogen). Confocal image stacks were acquired using a 63× objective (NA 1.4, z-step: 130 nm, LSM700, Zeiss). High-magnification images of branchpoints were deconvolved with Huygens Software (SVI).
Multicompartmental Modeling
AP propagation along PC axons was simulated in NEURON using a previously published multicompartmental PC model (Clark et al., 2005), which included a resurgent sodium conductance typical of PCs (Khaliq et al., 2003). Additionally, at NoRs the model included a low-threshold Ca2+ conductance (CaVT, 0.002 cm/s) (Anwar et al., 2012) as well as an intermediate type KCa (IK, KCa3.1). Nodal KCa3.1 was based on a previously published BK conductance (Solinas et al., 2007), and its kinetics were adjusted to a four-state model to match previously published experimental recordings of IK (Bailey et al., 2010; Hirschberg et al., 1998). The nodal KCa3.1 current reversal potential was set to −80 mV. The simulations were run for 500 ms, and values shown in Figure 4 were measured at the center of the compartment.
Author Contributions
J.G. and B.A.C. planned and performed experiments and wrote the manuscript.
Acknowledgments
We thank Tiago Branco for discussion, technical advice, and two-photon acquisition software; Christoph Schmidt-Hieber and Arnd Roth for critical discussion; and Michael Häusser for discussion, support, and comments on the manuscript. Work was supported by grants from the Wellcome Trust (WT094077), the European Research Council (AdG 250345), and the Gatsby Charitable Foundation (GAT2919). J.G. was supported by the Wellcome Trust 4 year PhD Programme and an EMBO and Marie Curie Actions postdoctoral fellowship and is currently an Ambizione fellow of the Swiss National Science Foundation.
Published: September 3, 2015
Footnotes
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Supplemental Information includes three figures and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.08.022.
Supplemental Information
References
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