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 .
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 ( rper 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 ca channels ( cavs ) , which could provide an additional source of depolarization as well as gating ca - dependent processes including recruitment of ca - 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 ca 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 .
using simultaneous somatic and axonal patch - clamp recordings , local pharmacology , and two - photon ca 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 . ,
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 2080  hz ) .
ttx ( 10  m ) 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 ;
, 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 ; rper 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 ( 2080  hz ; figures 1d1 g ) , with no observed dependence on firing rate ( r  = 0.1 ) .
clotrimazole ( 1  m ) , another ik - selective blocker , had similar effects ( figure  1 g ) , while apamin ( 1  m ) and iberiotoxin ( 1  m ) , 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 [ ca ] ( kd : 0.10.3  m ) ( wei et  al . , 2005 ;
1997 ) , and , since both local removal of extracellular ca and application of ni ( 100  m ) similarly suppressed action potential propagation ( figures 1d1 g ) , the activation of ik is likely initiated by ca 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 ca influx has been observed in optic nerve  ( lev - ram and grinvald , 1987 ; zhang et  al . , 2006 ) , but the resulting ca transients are spatially uniform along both nors and internodes ( zhang et  al . , 2006 ) , and the route of ca entry is uncertain .
as yet , there is no direct evidence for ca influx localized to nors in the brain . using two - photon ca - 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 ca concentration ( [ ca]i ) at nors ( identified by their location at branchpoints ) and at the ais ( see bender and trussell , 2009 ) during trains of evoked aps ( figures 3a3c ; 204  21  hz ) .
ca signals were restricted to approximately 5  m from the center of nors ( figures 3c and 3d ) , and there were no detectable changes in internodal [ ca]i ( figure  3b ) , consistent with the lack of effect on spike propagation of 0  mm ca , 10  mm bapta application to the internodes ( figure  s2b ) . [ ca]i at nors required axonal aps , were suppressed when spontaneous firing was arrested by somatic hyperpolarization ( figure  3 g ) , and could not be driven by somatic depolarization when spikes were blocked with bath - applied ttx ( figures 3e and 3f ) .
increases in [ ca]i were spike rate dependent ( figure  s3 ) , slow , and cumulative and lead to sustained [ ca]i during continuous activity ( figures 3 g and 3h ) .
[ ca]i increase was also suppressed by removal of extracellular ca ( 16%  6% of control
n  = 4 ; figure  3i ) and bath application of mibefradil ( figures 3i and 5  m , 43%  14% of control , p  = 0.013 ) but was not sensitive to agatoxin iva , which blocked ca 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 ca entry at nors .
additionally , although prior depolarization of the soma reduced [ ca]i at the ais , ap - triggered nodal ca signals were unaffected ( figures s3c and s3d ) , demonstrating that , in pcs , nodal [ ca]i is independent of somatic vm . 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 / cm , 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 / cm , 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 can not 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 ( vm  = 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 .
kca channels have been implicated in control of excitability in both unmyelinated ( lscher 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 ca 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 ni and the suppression of the ca 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 ca entry .
this might be of advantage since , due to the higher affinity of ik for ca ( wei et  al . , 2005 )
, a small ca 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 ca influx , it could be recruited and sustained at levels controlled by mean spike rate .
its localized recruitment by ca may also be amplified by ca - induced ca release ( llano et  al .
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 [ ca]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 ca entry at these sites .
besides its role in axonal electrogenesis , nodal ca influx could also be important in regulating nor structure and functional properties .
for example , activity - dependent local ca 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 ) .
ca 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 ca signals could provide a  readout of neuronal circuit activity , which could result in ca - dependent axonal plasticity beyond the ais ( grubb and burrone , 2010 ; grndemann and husser , 2010 ; kole , 2011 ; kuba et  al .
, 2010 ) . in providing direct evidence for activity - dependent ca entry at nors and its recruitment of ik ,
our findings show that ca may play both short- and long - term roles in regulating axonal excitability , crucial for long - range signaling in neuronal circuits .
methods used for preparing and recording from pcs and pc axons in cerebellar slices ( 200250  m ) from p18p43 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 34c  1c .
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 .
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 ( 0.510 psi , parker ) .
30  m alexa fluor 488 or 594 was included in the solution to help adjust pressure and pulse duration to target localized drug ejection .
two - photon ca 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  m oregon green 488 bapta-1 ( ogb-1 , invitrogen ) replaced egta in the pipette solution , which also contained 50  m 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 were digitized ( itc-18 , instrutech , heka ) at 50100 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 ( 520 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 ca - sensitive fluorescence ( ogb-1 , f / f ) were calculated as raw fluorescence fraw minus baseline fluorescence f0 normalized to the background ( fb ) subtracted baseline fluorescence ( yasuda et  al . , 2004 ) :
data are displayed as raw ogb-1 fluorescence values or relative changes in ogb-1 fluorescence normalized to the alexa fluor 594 red fluorescence ( f / r , figure  s3 ) .
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 ) .
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 m 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 ) .
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 ca 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 simulations were run for 500  ms , and values shown in figure  4 were measured at the center of the compartment .
 summaryfunctional 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 . 