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The role of axonal voltage-gated potassium channels in tDCS

Open AccessPublished:May 28, 2022DOI:https://doi.org/10.1016/j.brs.2022.05.019

      Highlights

      • tDCS is a neurostimulation technique that its underlying mechanism is unclear.
      • Potassium conductance plays a crucial role in DCS-induced neuromodulation.
      • Potassium Kv1 channels are involved in DCS modulation of sEPSCs and AP waveform.
      • We demonstrate that inhibition of Kv1 channels occludes the impact of DCS.

      Abstract

      Background

      Transcranial direct current stimulation (tDCS) is a non-invasive sub-threshold stimulation, widely accepted for its amelioration of distinct neuropsychiatric disorders. The weak electric field of tDCS modulates the activity of cortical neurons, which in turn modifies brain functioning. However, the underlying mechanisms for that are not fully understood.
      Objective/Hypothesis: Previous studies demonstrated that the axons are the most sensitive subcellular compartment for tDCS-induced polarization. Moreover, it was posited that DCS-induced axonal polarization is amplified by modifying the conductance of ionic channels. We posit that voltage-gated potassium-channels that are highly expressed in axons play a crucial role in DCS-induced modulation of cortical neurons functioning.

      Methods

      We examined the involvement of voltage-gated potassium-channels in the active modulation of spontaneous vesicle release by DCS. For that, we measured spontaneous excitatory postsynaptic currents (sEPSCs) from layer-V motor cortex during DCS application, while co-applying distinct voltage-gated potassium-channels blockers. Moreover, we examined the role of Kv1 potassium channels in DCS-induced modulation of action potential waveform at axon terminals by recording action potentials at terminal axon blebs during DCS application while locally inhibiting the Kv1 potassium-channels.

      Results

      We demonstrated that inhibiting voltage-gated potassium-channels occluded the DCS-induced modulation of subthreshold presynaptic vesicle release. Moreover, we showed that inhibiting Kv1 voltage-gated potassium-channels also occluded the DCS-induced modulation of action potential waveform at axon terminals.

      Conclusion

      We suggest that DCS-induced depolarization inactivates the Kv1 potassium channels thus reducing potassium conductance, which amplifies axonal depolarization, subsequently enhancing the presynaptic component of synaptic transmission. Whereas DCS-induced hyperpolarization induces opposite effects.

      Keywords

      1. Introduction

      Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation procedure that is currently used in humans for copious indications [
      • Lefaucheur J.-P.
      • et al.
      Evidence-based guidelines on the therapeutic use of transcranial direct current stimulation (tDCS).
      ], and is gaining increasing popularity. In principle, tDCS delivers a weak subthreshold current across the cortical region via strategically placed electrodes. Several clinical studies have illustrated that this weak electric field alters the activity [
      • George M.S.
      • Aston-Jones G.
      Noninvasive techniques for probing neurocircuitry and treating illness: vagus nerve stimulation (VNS), transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS).
      ,
      • Zaghi S.
      • Acar M.
      • Hultgren B.
      • Boggio P.S.
      • Fregni F.
      Noninvasive brain stimulation with low-intensity electrical currents: putative mechanisms of action for direct and alternating current stimulation.
      ] and excitability [
      • Nitsche M.A.
      • Paulus W.
      Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation.
      ,
      • Fregni F.
      • Boggio P.S.
      • Nitsche M.
      • Bermpohl F.
      • Antal A.
      • Feredoes E.
      • Marcolin M.A.
      • Rigonatti S.P.
      • Silva M.T.A.
      • Paulus W.
      • Pascual-Leone A.
      Anodal transcranial direct current stimulation of prefrontal cortex enhances working memory.
      ,
      • Antal A.
      • Fischer T.
      • Saiote C.
      • Miller R.
      • Chaieb L.
      • Wang D.J.J.
      • Plessow F.
      • Paulus W.
      • Kirschbaum C.
      Transcranial electrical stimulation modifies the neuronal response to psychosocial stress exposure.
      ] of cortical neurons. Furthermore, in humans and rodents, this modulation of cortical activity gives rise to cognitive and memory enhancement [
      • George M.S.
      • Aston-Jones G.
      Noninvasive techniques for probing neurocircuitry and treating illness: vagus nerve stimulation (VNS), transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS).
      ,
      • Zaghi S.
      • Acar M.
      • Hultgren B.
      • Boggio P.S.
      • Fregni F.
      Noninvasive brain stimulation with low-intensity electrical currents: putative mechanisms of action for direct and alternating current stimulation.
      ,
      • Nitsche M.A.
      • Paulus W.
      Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation.
      ]. Despite scrupulous studies [
      • Nitsche M.A.
      • Paulus W.
      Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation.
      ,
      • Liebetanz D.
      • Nitsche M.A.
      • Tergau F.
      • Paulus W.
      Pharmacological approach to the mechanisms of transcranial DC-stimulation-induced after-effects of human motor cortex excitability.
      ,
      • Nitsche M.A.
      • Fricke K.
      • Henschke U.
      • Schlitterlau A.
      • Liebetanz D.
      • Lang N.
      • Henning S.
      • Tergau F.
      • Paulus W.
      Pharmacological modulation of cortical excitability shifts induced by transcranial direct current stimulation in humans.
      ,
      • Nitsche M.
      • Seeber A.
      • Frommann K.
      • Klein C.C.
      • Rochford C.
      • Nitsche M.
      • Fricke K.
      • Liebetanz D.
      • Lang N.
      • Antal A.
      • Paulus W.
      • Tergau F.
      Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex.
      ,
      • Nitsche M.A.
      • Doemkes S.
      • Karakose T.
      • Antal A.
      • Liebetanz D.
      • Lang N.
      • Tergau F.
      • Paulus W.
      Shaping the effects of transcranial direct current stimulation of the human motor cortex.
      ], the underlying cellular mechanisms of tDCS are still not fully clear, which in turn diminishes the reliability of its use for therapeutic applications. It is accepted that tDCS differentially polarizes the subcellular compartments of cortical neurons in an orientation-dependent manner, and that the axons are the most sensitive sub-cellular structure for the external field due to their elongated nature, and maximal polarization is saturated towards their terminals [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ,
      • Kabakov A.Y.
      • Muller P a
      • Pascual-Leone A.
      • Jensen F.E.
      • Rotenberg A.
      Contribution of axonal orientation to pathway-dependent modulation of excitatory transmission by direct current stimulation in isolated rat hippocampus.
      ,
      • Rahman A.
      • Reato D.
      • Arlotti M.
      • Gasca F.
      • Datta A.
      • Parra L.C.
      • Bikson M.
      Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.
      ,
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ]. It was postulated that tDCS-induced axonal polarization consists of a passive membrane component [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ], amplified by an active component that is dependent on altering the conductance of ionic channels in the polarization process [
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ,
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ]. Small membrane polarizations were shown to be sufficient for inducing calcium influx that alters the baseline calcium concentration [
      • Awatramani G.B.
      • Price G.D.
      • Trussell L.O.
      Modulation of transmitter release by presynaptic resting potential and background calcium levels.
      ], and such modulations of calcium concentrations modify the vesicle release from presynaptic terminals [
      • Augustine G.J.
      How does calcium trigger neurotransmitter release?.
      ,
      • Rozov A.
      • Burnashev N.
      • Sakmann B.
      • Neher E.
      Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.
      ,
      • Catterall W.A.
      • Few A.P.
      calcium channel regulation and presynaptic plasticity.
      ,
      • Südhof T.C.
      Calcium control of neurotransmitter release.
      ]. Taken together, we predicted that the active amplification of axonal polarization by altering ionic conductances culminates in modulating the presynaptic release of vesicles. Coinciding with this prediction, a recent study showed that anodal-DCS enhances the spontaneous excitatory postsynaptic currents (sEPSC) frequency, while cathodal-DCS diminishes the frequency of sEPSC in cortical neurons. Furthermore, it showed that sodium currents play a role in this presynaptic vesicle release modulation due to the alteration of their sodium conductance, which actively amplifies the axonal polarization [
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ]. However, besides sodium channels, axons are also highly enriched with various potassium channels, and these play a significant role in the modulation of axonal functionality, and in the maintenance of the membrane potential [
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      The open pore conformation of potassium channels.
      ,
      • Sansom M.S.P.
      • Shrivastava I.H.
      • Bright J.N.
      • Tate J.
      • Capener C.E.
      • Biggin P.C.
      Potassium channels: structures, models, simulations.
      ,
      • Yellen G.
      The voltage-gated potassium channels and their relatives.
      ,
      • MacKinnon R.
      Potassium channels.
      ]. However, the diversity of potassium channels is much wider and complex in comparison to that of sodium channels. Distinct types of voltage-gated potassium channels react differently to membrane potential alterations [
      • Kuang Q.
      • Purhonen P.
      • Hebert H.
      Structure of potassium channels.
      ], and many of them might modify axonal functioning due to tDCS. Being voltage sensitive in nature, voltage-gated potassium channels open in response to depolarization (which assists repolarization and after hyperpolarization) [
      • Yellen G.
      The voltage-gated potassium channels and their relatives.
      ,
      • Harvey A.L.
      Recent studies on dendrotoxins and potassium ion channels.
      ]. However, some potassium channels undergo inactivation during sustained depolarization, moving from an open conformation to a closed one. This reduction in potassium conductance, further depolarizes the axonal compartment, enhancing calcium influx [
      • Rozov A.
      • Burnashev N.
      • Sakmann B.
      • Neher E.
      Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.
      ,
      • Nimmervoll B.
      • Flucher B.E.
      • Obermair G.J.
      Dominance of P/Q-type calcium channels in depolarization-induced presynaptic FM dye release in cultured hippocampal neurons.
      ], which may promote vesicle release from the presynaptic compartment [
      • Augustine G.J.
      How does calcium trigger neurotransmitter release?.
      ,
      • Rozov A.
      • Burnashev N.
      • Sakmann B.
      • Neher E.
      Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.
      ,
      • Catterall W.A.
      • Few A.P.
      calcium channel regulation and presynaptic plasticity.
      ,
      • Südhof T.C.
      Calcium control of neurotransmitter release.
      ]. And indeed, other studies showed that during depolarization, the inactivation of potassium channels supports the facilitation of neurotransmitter release from axon terminals [
      • Byrne J.H.
      • Kandel E.R.
      Presynaptic facilitation revisited: state and time dependence.
      ,
      • Meir A.
      • Ginsburg S.
      • Butkevich A.
      • Kachalsky S.G.
      • Kaiserman I.
      • Ahdut R.
      • Demirgoren S.
      • Rahamimoff R.
      Ion channels in presynaptic nerve terminals and control of transmitter release.
      ,
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Cho I.H.
      • Panzera L.C.
      • Chin M.
      • Alpizar S.A.
      • Olveda G.E.
      • Hill R.A.
      • Hoppa M.B.
      The potassium channel subunit Kvβ1 serves as a major control point for synaptic facilitation.
      ]. In addition, this inactivation of potassium channels also modulates the action potential (AP) waveform at axon terminals [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Kole M.H.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Goldberg E.M.
      • Clark B.D.
      • Zagha E.
      • Nahmani M.
      • Erisir A.
      • Rudy B.
      K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons.
      ]. Therefore, we hypothesized that the DCS-induced variations in potassium channel conductance play a role in the active modulation of axonal polarization and in the subsequent modification of spontaneous presynaptic vesicle release, and in modifying the waveform of action potentials (APs). Noteworthy, the modulation of presynaptic vesicle release dynamics has a critical role in maintaining neuronal circuitry [
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ], and modulation of AP morphology alters its strength analogically [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Kole M.H.
      • Stuart G.J.
      Signal processing in the axon initial segment.
      ]. Both of these effects are critical for the computation performed by neural circuits. For this, we performed a range of electrophysiological experiments while utilizing various potassium channel blockers during DCS application.

      2. Materials and methods

      2.1 Animals

      The subjects of the experiments were all male C57Bl/6 mice. For postsynaptic events, mice were 2 months old, and for axon bleb patching experiments, mice were 19–24 days old. Axon blebs are the artificial structures created through slicing; patchable only while myelin is in absentia [
      • Hu W.
      • Shu Y.
      Axonal bleb recording.
      ]. Hence, these experiments are carried out only on pups in their developmental stage (P19–24), before myelin is developed. In our experiments, wild-type C57Bl/6 mice were bred in the animal facility with 12 h light-dark cycle. Water and food were available ad-libitum. Housing, handling, and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the University of Haifa animal ethics committee.

      2.2 Slices production for electrophysiology

      Slices were produced by cutting with a Campden vibratome 7000smz-2 that has a Zero Z technology to minimize Z-axis deflection. Animals were cervically dislocated and its brain is taken immediately to harvest cortical coronal slices (300 μm) using Campden Vibratome 7000 smz2 (Campden Instruments, UK) in the fluff of frozen slicing solution (in mM): 110 Sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 0.5 CaCl2, 7 MgCl2, 5 Glucose. Slices were kept for incubation at 34.5 °C for ∼1.5 h in artificial cerebrospinal fluid (ACSF) containing in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 d-glucose, 2 CaCl2, and 1 MgCl2. An additional recovery period of 30 min at room temperature was given inside the electrophysiology chamber in ACSF (2 mL/min).

      2.3 Electrophysiology

      The whole-cell rig microscope was an Olympus BX51-WI, with 60X objective with a NA = 1.0 and an oil condenser with a NA = 1.4. The camera for DIC imaging was DAGE-MTI IR-1000E. Electrophysiological recordings were acquired using Multiclamp 700B amplifier with Digidata 1440 digitizer from Molecular devices, run by pclamp10 software.
      Slices were kept 30 min in an electrophysiology rig for recovery while carboxygenated with 95% O2 + 5% CO2. Cortical regions of brain slices were observed using a 60X objective with NA = 1.0 mounted on a whole-cell rig microscope (BX51-WI, Olympus, Center Valley, PA). The imaging was captured with a charge-coupled device camera IR-1000E (Dage MTI, Michigan City, IN). Electrophysiological recordings were amplified using Multiclamp 700B and were digitized using Digidata 1440 (molecular devices, Sunnyvale, CA), ran by pclamp10 software. Whole-cell recordings were done according to standard procedure. Borosilicate glass pipettes (3–5 MΩ for soma, 7–9 MΩ for axon) were pulled using a pipette puller (P-1000; Sutter Instruments, Navato, CA) and filled with a K-gluconate based internal solution (in mM): 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl2, 4 Na2ATP, 0.5 TrisGTP, 14 phosphocreatine, Alexa-488 (100 μM) osmolarity 290 mOsm and pH = 7.3. For the synaptic current measurements, EPSCs were recorded in voltage-clamp from the somas of layer-5 pyramidal cells at the region of the M1 motor cortex, at a holding potential of −70 mV using the same K-Gluconate based internal solution used for current-clamp recording. Noteworthy, the voltage clamp procedure does not affect the presynaptic component that comes from neurons whose axons are connecting to the recorded cell. These neruons, which their incoming connecting axons convey their input to the recorded cell via the presynaptic boutons vary in their resting membrane potential, and are not affected at all by the voltage clamp procedure itself, but only by the DCS. The method for measuring active intrinsic properties from an AP was based on previous studies with 150 pA current is applied for a brief duration of 10 ms [
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ]. Membrane potential acceleration to 30 V/s point determined AP threshold. Potassium channel inhibitor (DTX) is dissolved in artificial cerebrospinal fluid (aCSF) inside a glass pipette (resistance ∼1 MOhm) connected to a picospritzer-III (Parker). The puff is applied in front of the bleb relative to the direction of the aCSF laminar inflow (>2 mL/min, 2 μM), so the inhibitor flows past the bleb without coming in contact with potential recording sites located proximal to the origin of the flow. The inhibitor was washed out from the potential recording sites and not recycled to the aCSF reservoir. A low puff pressure (10 kPa) is used to avoid mechanical impact on the bleb. All recordings were low-pass filtered at 10 kHz and sampled at 50 kHz. Series resistance was 90% compensated. Series resistance, input resistance, and membrane capacitance were monitored during the entire experiment. Data exclusion criteria were based on changes in the above parameters of more than 15% from the baseline. All analyses were performed using Clampfit 10 (Molecular Devices, Sunnyvale CA, USA). The experiments were performed at constant 21 °C room temperature.

      2.4 DC field application

      The uniform DC electric field is generated by a linear stimulus isolation unit (LSIU-02, Cygnus Technology, PA, USA) connected to the cathode and anode, which were two parallel thick (2 mm) Ag–AgCl wires (783500, AM-System, WA, USA). The wires were positioned parallel to the contour of the outer cortical border at the site of the recorded cells. The applied voltage was modified before each experiment to obtain a homogenous and uniform electric field (EF) of 5 V/m in the y-axis which is diagonal to the wires, and there was no variability in the x-axis along the wires or in the z-axis along the depth of the slice. DCS stimulation contained three distinct polarization conditions; depolarizing, hyperpolarizing, and no-polarizing. The use of a higher electric field intensity (5 V/m) in our ex-vivo DCS experiments compared to the <1 V/m used in tDCS in humans was chosen in order to compensate for the suboptimal conditions of the ex-vivo DCS experiment. The ex-vivo DCS in brain slices derived from young mice brains is predicted to generate a much smaller axon terminal polarization relative to the axon terminal polarization generated by tDCS in humans. Terminal polarization is positively dependent both on the measure of the axon's length constant (λ), and on the axon length (up to 4λ) [
      • Rahman A.
      • Reato D.
      • Arlotti M.
      • Gasca F.
      • Datta A.
      • Parra L.C.
      • Bikson M.
      Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.
      ]. In the herein experiments axons are thinner and non-myelinated, thus having a much shorter length constant (λ) than in humans, and the distance of the patched axon bleb is ∼200–300 μm, much shorter than the distance of axons and their terminals in humans. Nonetheless, the exact relations between the mouse ex-vivo and human in-vivo conditions are not fully known and so is the required level of compensation.

      2.5 Experimental design and statistical analysis

      The electrophysiological data analysis was done with Clampfit 10 (Molecular Devices, Sunnyvale CA, USA). Statistical analyses were performed using GraphPad Prism 7 by paired t-test and one-way repeated measures ANOVA followed by Bonferroni's multiple comparisons tests. We also performed one-sample t-tests to compare the statistical variation of data from zero. Results are displayed as mean ± SD in graphs. Significance was set at p < 0.05 using two-tailed t-tests or ANOVAs wherever necessary and significance is represented as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Cell numbers (n) and animal numbers (N), are added in the figure legends.

      3. Results

      3.1 Potassium channel activity is vital for DCS-induced modulation of spontaneous vesicle release

      In our previous study, we showed that DCS across the cortical region significantly altered the sEPSCs frequency; Anodal-DCS increased the sEPSCs frequency, while cathodal-DCS decreased sEPSCs frequency. To examine the role of potassium channels in modulating the spontaneous vesicle release by DCS, we recorded sEPSCs from layer-V pyramidal neurons of the motor cortex during application of different DCS conditions, while blocking voltage dependent potassium channel blocker using TEA 1 μM that was applied in the bath (Fig. 1A and B). Under TEA application, neither anodal-DCS nor cathodal-DCS induced any significant variation in the mean sEPSC frequency in comparison to no-DCS condition [F(1.48, 30.99) = 1.16, p = 0.31 in RM-ANOVA; t(21) = 0.52, p > 0.99 and t(21) = 1.25, p = 0.68 in posthoc Bonferroni corrected comparison between anodal to no-DCS and cathodal to no-DCS, respectively] (Fig. 1D). Likewise, the cumulative distributions of the inter-event intervals under anodal-DCS and cathodal-DCS were also comparable to the no-DCS condition [D = 0.04, p > 0.99 and D = 0.09, p = 0.13 in K–S between anodal-DCS to no-DCS and between cathodal to no-DCS, respectively] (Fig. 1E). Like for sEPSCs frequency, when TEA was administered, DCS did not modulate sEPSCs amplitudes, and the mean sEPSC amplitudes under anodal and cathodal-DCS were similar to those under no-DCS condition [F(1.74, 36.55) = 1.81, p = 0.18 in RM-ANOVA; t(21) = 0.92, p > 0.99 and t(21) = 2.00, p = 0.18 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 1F). Likewise, when TEA was applied, the cumulative distributions of the sEPSCs amplitudes in the three different DCS conditions (anodal, no-DCS, and cathodal) were all comparable [D = 0.00, p > 0.99 in K–S for both between anodal and no-DCS and between cathodal and no-DCS] (Fig. 1G). Taken together, it is evident that potassium channels are critical for any DCS-induced modulation of sEPSCs, which suggests a role for potassium channels in the mechanism by which tDCS works.
      Fig. 1
      Fig. 1Inhibiting potassium channels with TEA application prevents DCS-induced modulation of spontaneous vesicle release in pyramidal neurons.
      A. Illustration of the experimental setup. Whole-cell recordings of layer-V pyramidal neurons in the M1 motor cortex were performed during DCS (5 V/m) administration, while TEA was applied in the bath.
      B. Illustration of the orientation of the DCS electrical field in relation to the anatomy of the recorded layer-V cortical pyramidal neurons. In the illustration the anodal administration is displayed.
      C. Sample traces of the recoded sEPSCs during anodal DCS (red), cathodal DCS (blue) and no-DCS (grey) conditions when TEA was applied.
      D. Anodal and cathodal-DCS do not affect sEPSC frequency when TEA is applied concomitantly [F(1.48, 30.99) = 1.16, p = 0.31 in RM-ANOVA; t(21) = 0.52, p > 0.99 and t(21) = 1.25, p = 0.68 in posthoc Bonferroni corrected comparison between anodal to no-DCS and cathodal to no-DCS, respectively]. Data are presented as Mean ± SD.
      E. Anodal and cathodal-DCS do not shift the cumulative distribution curves of sEPSCs inter-event intervals, when TEA is applied in the bath [D = 0.04, p > 0.99 and D = 0.09, p = 0.13 in K–S between anodal-DCS to no-DCS and between cathodal to no-DCS, respectively].
      F. Neither anodal nor cathodal-DCS altered the amplitudes of sEPSCs in the presence of TEA [F(1.74, 36.55) = 1.81, p = 0.18 in RM-ANOVA; t(21) = 0.92, p > 0.99 and t(21) = 2.00, p = 0.18 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively]. Data are presented as Mean ± SD.
      G. Cumulative distributions of sEPSCs amplitudes were unaltered by either anodal or cathodal DCS application, when TEA was applied in the bath [D = 0.00, p > 0.99 in K–S for both between anodal and no-DCS and between cathodal and no-DCS].
      N = 6 mice n = 22 neurons for all panels. ns = non significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

      3.2 Kv1 channels control DCS induced synaptic vesicle release modulation

      As aforementioned, the potassium channels are essential for DCS-induced modulation of sEPSCs. Potassium channels are the most diverse group of ionic channels with varieties of subgroups [
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      The open pore conformation of potassium channels.
      ,
      • Sansom M.S.P.
      • Shrivastava I.H.
      • Bright J.N.
      • Tate J.
      • Capener C.E.
      • Biggin P.C.
      Potassium channels: structures, models, simulations.
      ,
      • Yellen G.
      The voltage-gated potassium channels and their relatives.
      ,
      • MacKinnon R.
      Potassium channels.
      ]. Wherein, Kv1 channels form a subgroup that is abundant in axon terminals and well known for their interaction with synaptic vesicle release by indirectly affecting intracellular calcium levels [
      • Harvey A.L.
      Recent studies on dendrotoxins and potassium ion channels.
      ,
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Harvey A.L.
      • Robertson B.
      Dendrotoxins: structure-activity relationships and effects on potassium ion channels.
      ]. Previous studies showed that subthreshold depolarization inactivates Kv1 channels, which induces widening the AP morphology at the axon [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ], and facilitates vesicle release [
      • Byrne J.H.
      • Kandel E.R.
      Presynaptic facilitation revisited: state and time dependence.
      ,
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ]. Therefore, to examine the Kv1 channels in particular, with regard to their role in DCS-induced modulation of sEPSCs, we repeated the abovementioned experiment with α-DTX instead of TEA (Fig. 2A and B). While TEA inhibits a wide array of potassium channels, α-DTX inhibits selectively Kv1.1, Kv1.2, Kv1.6. Similar to the aforementioned results with TEA administration, bath application of α-DTX 500 nM blocked the DCS-induced sEPSC modulations. Anodal and cathodal DCS, in the presence of α-DTX, did not alter the sEPSCs frequencies, and both were comparable to the sEPSCs frequency under no-DCS condition [F(1.95, 44.94) = 2.7, p = 0.08 in RM-ANOVA; t(23) = 1.89, p = 0.22 and t(23) = 0.19, p > 0.99 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 2D). Likewise, the cumulative distributions of the interevent intervals were also similar between anodal and cathodal-DCS to that of the no-DCS condition [D = 0.09, p = 0.15, and D = 0.02, p > 0.99 in K–S between anodal to no-DCS and cathodal to no-DCS, respectively] (Fig. 2E). Similar to the effects on sEPSCs frequency, α-DTX inhibited also the DCS-induced variation of sEPSCs amplitude. Neither anodal nor cathodal-DCS induced any variation in the mean sEPSC amplitudes compared to the no-DCS condition, when α-DTX was present in the bath [F(1.74, 40.05) = 0.74, p = 0.47 in RM-ANOVA; t(23) = 0.16, p > 0.99 and t(27) = 0.84, p > 0.99 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 2F). Also, the cumulative distributions of the sEPSCs amplitudes under the three DCS conditions were comparable in the presence of α-DTX [D = 0.00, p > 0.99 in K–S for both between anodal to no-DCS and cathodal to no-DCS] (Fig. 2G). These results demonstrate that even Kv1 channels alone are vital for the effects that tDCS bear on the presynaptic vesicle release mechanism.
      Fig. 2
      Fig. 2Inhibiting Kv1 potassium channels with αDTX application prevents DCS-induced modulation of spontaneous vesicle release in pyramidal neurons.
      A. Illustration of the experimental setup. Whole-cell recordings of layer-V pyramidal neurons in the M1 motor cortex were performed during DCS (5 V/m) administration, while αDTX was applied in the bath.
      B. Illustration of the orientation of the DCS electrical field in relation to the anatomy of the recorded layer-V cortical pyramidal neurons. In the illustration the anodal administration is displayed.
      C. Sample traces of the recoded sEPSCs during anodal DCS (red), cathodal DCS (blue) and no-DCS (grey) conditions when αDTX was applied.
      D. Anodal and cathodal-DCS do not affect sEPSC frequency when αDTX is applied concomitantly [F(1.95, 44.94) = 2.7, p = 0.08 in RM-ANOVA; t(23) = 1.89, p = 0.22 and t(23) = 0.19, p > 0.99 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively]. Data are presented as Mean ± SD.
      E. Anodal and cathodal-DCS do not shift the cumulative distribution curves of sEPSCs inter-event intervals, when αDTX is applied in the bath [D = 0.09, p = 0.15, and D = 0.02, p > 0.99 in K–S between anodal to no-DCS and cathodal to no-DCS, respectively].
      F. Neither anodal nor cathodal-DCS altered the amplitudes of sEPSCs in the presence of αDTX [F(1.74, 40.05) = 0.74, p = 0.47 in RM-ANOVA; t(23) = 0.16, p > 0.99 and t(27) = 0.84, p > 0.99 in posthoc Bonferroni corrected comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively]. Data are presented as Mean ± SD.
      G. Cumulative distributions of sEPSCs amplitudes were unaltered by either anodal or cathodal DCS application, when αDTX was applied in the bath [D = 0.00, p > 0.99 in K–S for both between anodal to no-DCS and cathodal to no-DCS].
      N = 6 mice n = 24 neurons for all panels. ns = non significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

      3.3 Kv1 channels control DCS-induced modulation of action potential waveform

      As demonstrated above, potassium channels, in particular Kv1 type channels, are involved in the DCS-induced modulation of spontaneous vesicle release. However, another important facet of Kv1 type potassium channels is their role in modulating the AP waveform, thus altering its postsynaptic effect [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Kole M.H.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Goldberg E.M.
      • Clark B.D.
      • Zagha E.
      • Nahmani M.
      • Erisir A.
      • Rudy B.
      K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons.
      ]. Subthreshold depolarization inactivates Kv1 channels, which widens the AP waveform and lowers its amplitude [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Goldberg E.M.
      • Clark B.D.
      • Zagha E.
      • Nahmani M.
      • Erisir A.
      • Rudy B.
      K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons.
      ]. The widening of the AP strengthens the postsynaptic response by enhancing the axonal calcium influx [
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ]. Given that the DCS-induced polarization is prominent in the axon terminals, we posited that anodal-DCS application inactivates the potassium channels located at the axon terminals widening the AP waveforms. For this, we performed current-clamp whole-cell recording from terminal axon blebs under different DCS conditions (anodal, cathodal, and no-DCS), while the DC field orientation is parallel to the axons for achieving maximal axonal polarization (Fig. 3A and B). This experiment demonstrated that both cathodal and anodal DCS significantly affect the AP half-width (F(1.584, 36.44) = 30.25, p < 0.0001 in RM-ANOVA), so anodal-DCS increases the AP half-width, while cathodal-DCS decreases the AP half-width, when compared to no-DCS condition [t(23) = 5.03, p < 0.001 and t(23) = 4.04, p < 0.01 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 3C and D). Furthermore, both cathodal and anodal DCS significantly affect the AP amplitude (F(1.289, 29.65) = 13.86, p < 0.001 in RM-ANOVA), as anodal-DCS reduces the AP amplitude, while cathodal-DCS enlarges the AP amplitude, when compared to no-DCS condition [t(23) = 2.80, p < 0.05 and t(23) = 3.62, p < 0.01 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 3C,E). Next, in order to examine the role of Kv1 channels in DCS-induced modulation of the AP waveform, we performed the same experiment of measuring the AP from the terminal axon blebs during DCS application, but this time it was while blocking Kv1 channels, using a local puff of α-DTX (2 μM) (Fig. 3C). As predicted, a local application of α-DTX puff occluded all DCS-induced modulations of the AP waveform (F(1.898, 34.17) = 1.55, p = 0.23 in RM-ANOVA), and the half-widths of the APs under anodal and cathodal DCS were comparable to no-DCS conditions [t(18) = 0.39, p > 0.99 and t(18) = 1.55, p = 0.41 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 3C,F). Similarly, the DCS effects on the APs amplitudes were also completely occluded by Kv1 inhibition (F(1.226, 22.06) = 1.23, p = 0.29 in RM-ANOVA), and became similar for anodal, cathodal and no-DCS conditions [t(18) = 1.50, p = 0.45 and t(18) = 0.25, p > 0.99 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively] (Fig. 3C,G). Taken together, these results suggest that DCS application affects AP morphology by altering the portion of the inactivated potassium Kv1 channels at the axon terminals, which further support the role of Kv1 channels in DCS effects.
      Fig. 3
      Fig. 3Inhibiting axonal Kv1 potassium channels with a local puff of α−DTX prevents DCS-induced modulation of AP waveform.
      A. Illustration of the experimental setup. Whole-cell recordings of terminal axon blebs were performed during DCS (5 V/m) administration, while α−DTX was locally puffed to the axon blebs.
      B. Illustration of the orientation of the DCS electrical field in relation to the anatomy of the recorded layer-V axons. In the illustration, the anodal administration is displayed.
      C. Sample traces of action potentials recorded from terminal axon blebs during anodal DCS (red), cathodal DCS (blue), and no-DCS (grey) are displayed.
      D. Anodal DCS application significantly enhances the half-width of the AP in comparison to no-DCS condition [t(23) = 5.03, p < 0.001 in Bonferroni corrected post hoc comparison], whereas cathodal DCS application significantly reduces the half-width of the action potential waveform in comparison to the no-DCS condition [t(23) = 4.04, p < 0.01 in Bonferroni corrected post hoc comparison]. N = 6 mice n = 24. Data are presented as Mean ± SD.
      E. Anodal DCS application reduces the AP amplitude compared to no-DCS condition [t(23) = 2.80, p < 0.05 in Bonferroni corrected post hoc comparison], while cathodal DCS application elevates the AP amplitude in comparison to no-DCS condition [t(23) = 3.62, p < 0.01 in Bonferroni corrected post hoc comparison]. N = 6 mice n = 24. Data are presented as Mean ± SD.
      F. The AP half-width is unaltered by either anodal or cathodal DCS application when α−DTX is locally puffed on the terminal axon blebs [t(18) = 0.39, p > 0.99 and t(18) = 1.55, p = 0.41 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively]. N = 6 mice n = 19. Data are presented as Mean ± SD. ns = non significant.
      G. Action potential amplitudes were unaltered by either anodal or cathodal DCS application when α−DTX was locally puffed to the axon blebs [t(18) = 1.50, p = 0.45 and t(18) = 0.25, p > 0.99 in Bonferroni corrected post hoc comparisons between anodal to no-DCS and between cathodal to no-DCS, respectively]. N = 6 mice n = 19. Data are presented as Mean ± SD. ns = non significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

      4. Discussion

      The accepted principle for the cellular effects of tDCS is that it induces differential polarization in the subcellular neuronal compartments. In comparison to other compartments, axon terminals are the most sensitive in their response to the external electric field [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ,
      • Rahman A.
      • Reato D.
      • Arlotti M.
      • Gasca F.
      • Datta A.
      • Parra L.C.
      • Bikson M.
      Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.
      ,
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ]. The axon dynamics was previously modeled using cable equation and written in terms of passive polarization of axons [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ]. However, the theoretically predicted passive polarization alone is small to induce substantial modulation of neural functioning leading to cognition and clinical effects [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ,
      • Rahman A.
      • Reato D.
      • Arlotti M.
      • Gasca F.
      • Datta A.
      • Parra L.C.
      • Bikson M.
      Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.
      ]. However, experimental data from recorded from axons show that polarization is higher than the theoretical predictions [
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ,
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ]. This disparity is due to the active involvement of ionic channels during the polarization process, such as the sodium channels and calcium channels, which were also shown to affect axonal polarization and synaptic vesicle release [
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ,
      • Vasu S.O.
      • Kaphzan H.
      Calcium channels control tDCS-induced spontaneous vesicle release from axon terminals.
      ]. In the herein study, we demonstrate that along with the sodium channels and calcium channels, potassium channels also play a vital role in the modulation of synaptic vesicle release by DCS.
      Potassium channels are crucial for the maintenance of membrane potential, neuronal connectivity, and propagation of the AP [
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      The open pore conformation of potassium channels.
      ,
      • Sansom M.S.P.
      • Shrivastava I.H.
      • Bright J.N.
      • Tate J.
      • Capener C.E.
      • Biggin P.C.
      Potassium channels: structures, models, simulations.
      ,
      • Yellen G.
      The voltage-gated potassium channels and their relatives.
      ,
      • MacKinnon R.
      Potassium channels.
      ]. Unlike other ionic channels, most voltage-gated potassium channels present in axon terminals have a low threshold nature and they respond to subthreshold depolarization [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ,
      • Dodson P.D.
      • Forsythe I.D.
      Presynaptic K+ channels: electrifying regulators of synaptic terminal excitability.
      ]. Even though potassium channels are diverse in nature, the majority of potassium channels are voltage sensitive and they open in response to depolarization. Thus, they prevent aberrant neuronal activity by restricting depolarization in axons [
      • Yellen G.
      The voltage-gated potassium channels and their relatives.
      ,
      • MacKinnon R.
      Potassium channels.
      ]. Voltage gated potassium channels are also known for their interaction in AP wave form and subsequent modulation of synaptic vesicle release inorder to fine tune the cortical circuits [
      • Kuang Q.
      • Purhonen P.
      • Hebert H.
      Structure of potassium channels.
      ,
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ]. Some of the potassium channels that are prevalent in axons belong to the Kv1 family. The Kv1 channel family is highly diverse and contains eight α-subunits which are Kv1.1–1.8. Among these, Kv1.1, Kv1.2, and Kv1.6 are highly expressed in cortical neurons [
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ,
      • Wang W.
      • Kim H.J.
      • Lv P.
      • Tempel B.
      • Yamoah E.N.
      Association of the Kv1 family of K+ channels and their functional blueprint in the properties of auditory neurons as revealed by genetic and functional analyses.
      ,
      • Pérez-García M.T.
      • Cidad P.
      • López-López J.R.
      The secret life of ion channels: Kv1.3 potassium channels and proliferation.
      ,
      • Feria Pliego J.A.
      • Pedroarena C.M.
      Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons.
      ]Pliego and Pedroarena, 2020). Wherein, Kv1.4 is fast inactivating A-type potassium channels, while most of the other Kv1 channels (Kv1.1–1.3, Kv1.5-1.8) are slowly inactivating delayed rectifier types. These Kv1 channels are inactivated during depolarization and facilitate vesicle release from axon terminals [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ]. DCS application was shown to modulate vesicles release in the cortical neurons; While anodal DCS enhances the frequency and amplitude of sEPSCs, cathodal DCS reduces their frequency and amplitudes [
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ]. The enhancement in sEPSC frequency due to the anodal-DCS-induced depolarization of the axons is attributed to the subsequent increase in the baseline calcium concentrations [
      • Awatramani G.B.
      • Price G.D.
      • Trussell L.O.
      Modulation of transmitter release by presynaptic resting potential and background calcium levels.
      ], which further augments the vesicle release from the presynaptic compartment [
      • Augustine G.J.
      How does calcium trigger neurotransmitter release?.
      ,
      • Rozov A.
      • Burnashev N.
      • Sakmann B.
      • Neher E.
      Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.
      ,
      • Catterall W.A.
      • Few A.P.
      calcium channel regulation and presynaptic plasticity.
      ,
      • Südhof T.C.
      Calcium control of neurotransmitter release.
      ]. The cathodal DCS hyperpolarizes the axonal compartment, which in turn reduces the calcium concentration in the axon terminal, thus decreasing vesicle release. The effects of DCS on the axonal potassium conductance are very complex. Axons contain several types of potassium channels, and amongst the voltage-dependent potassium channels, some inactivate in response to prolonged depolarization, like the Kv1 (KCNA) family, and some do not inactivate like the Kv7 (KCNQ) family. This makes it difficult to estimate reliably the overall effect of DCS-induced polarization on the general potassium conductance. Therefore, it is hard to understand the role of potassium channels in DCS. However, the results clearly show that inhibiting most voltage-dependent potassium channels by application of TEA inhibited any DCS-induced alterations of sEPSCs frequencies and amplitudes, and these were similar to the no-DCS condition. This clearly indicates a role for potassium conductance in the modulation of synaptic release by DCS.
      Within the voltage-sensitive potassium channels, the Kv1-type channels activate and inactivate in the subthreshold region, and are vital for vesicle release [
      • Byrne J.H.
      • Kandel E.R.
      Presynaptic facilitation revisited: state and time dependence.
      ,
      • Meir A.
      • Ginsburg S.
      • Butkevich A.
      • Kachalsky S.G.
      • Kaiserman I.
      • Ahdut R.
      • Demirgoren S.
      • Rahamimoff R.
      Ion channels in presynaptic nerve terminals and control of transmitter release.
      ,
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Cho I.H.
      • Panzera L.C.
      • Chin M.
      • Alpizar S.A.
      • Olveda G.E.
      • Hill R.A.
      • Hoppa M.B.
      The potassium channel subunit Kvβ1 serves as a major control point for synaptic facilitation.
      ]. Evidence shows that they are actively involved in the subthreshold dynamics [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ], and their abundance in the distal end of the axon makes them strong participants in DCS-induced vesicle release modulation. Inactivation of the Kv1 channels due to subthreshold depolarization facilitates presynaptic release [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ]. This may occur via enhancing the depolarization of the presynaptic compartment, which increases the calcium concentration, and that in turn increases the dynamics of vesicles’ spontaneous exocytosis. Besides the effects of Kv1 channels on membrane potential, they also affect synaptic vesicle release as they directly interact with synaptotagmin, a calcium sensor that plays a crucial role in spontaneous vesicle release from the presynaptic boutons [
      • Fili O.
      • Michaelevski I.
      • Bledi Y.
      • Chikvashvili D.
      • Singer-Lahat D.
      • Boshwitz H.
      • Linial M.
      • Lotan I.
      Direct interaction of a brain voltage-gated K+ channel with syntaxin 1A: functional impact on channel gating.
      ]. Here we demonstrated that inhibiting Kv1 channels using α-DTX application occluded the ability of DCS to modulate synaptic vesicle release similar to TEA. These results distinctly emphasize the role of Kv1 channels in DCS-induced modulation of synaptic vesicles release, but the mechanism for their involvement is not completely clear. It is possible that DCS-induced membrane polarization modifies the potassium channel conductance, which culminates in modulating the vesicles release dynamics. Moreover, as previously suggested by modeling, the mere modification of the potassium conductance by the initial membrane polarization further enhances the resultant polarization [
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ]. For example, inactivation of Kv1 channels due to the depolarization induced by anodal-DCS would prompt further depolarization due to the reduction in overall potassium conductance, and result in enhancing presynaptic calcium levels, thus facilitating vesicle release. Hence, it is possible that in our experiment, inhibition of Kv1 channels with TEA or DTX, occludes the impact of anodal-DCS to enhance depolarization by Kv1 inactivation, thus preventing the further elevation of intracellular calcium levels and facilitating vesicle release compare to the no-DCS condition. It is known that DTX and TEA in themselves induce depolarization of the resting potential [
      • Feria Pliego J.A.
      • Pedroarena C.M.
      Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons.
      ,
      • Barrett E.F.
      • Morita K.
      • Scappaticci K.A.
      Effects of tetraethylammonium on the depolarizing after-potential and passive properties of lizard myelinated axons.
      ]. This suggested mechanism aligns with the experimental results. Comparisons of the previously published raw data of sEPSCs frequencies with no drug [
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ] to the sEPSCs frequencies under DTX administration show that DTX application in itself enhances the sEPSCs frequency, and is comparable to the sEPSCs frequency under anodal-DCS without any drug application (Fig. S1). A similar mechanism, but in the opposite direction, might explain the effects of cathodal-DCS application. Hyperpolarization that is induced by Cathodal-DCS can possibly enhance itself by two parallel active mechanisms. First, as previously shown, hyperpolarization increases Kir channels conductance [
      • Hibino H.
      • Inanobe A.
      • Furutani K.
      • Murakami S.
      • Findlay I.
      • Kurachi Y.
      Inwardly rectifying potassium channels: their structure, function, and physiological roles.
      ], which will further enhance hyperpolarization. Second, Kv1 channels have a window current due to their activation-inactivation properties. Therefore, slight hyperpolarization in a limited range can enhance Kv1 conductance, also resulting in further enhancement of hyperpolarization [
      • Wang W.
      • Kim H.J.
      • Lv P.
      • Tempel B.
      • Yamoah E.N.
      Association of the Kv1 family of K+ channels and their functional blueprint in the properties of auditory neurons as revealed by genetic and functional analyses.
      ,
      • Pérez-García M.T.
      • Cidad P.
      • López-López J.R.
      The secret life of ion channels: Kv1.3 potassium channels and proliferation.
      ,
      • Storm J.F.
      Temporal integration by a slowly inactivating K+ current in hippocampal neurons.
      ,
      • Chen S.-H.
      • Fu S.-J.
      • Huang J.-J.
      • Tang C.-Y.
      The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.
      ,
      • Hasan S.
      • Bove C.
      • Silvestri G.
      • Mantuano E.
      • Modoni A.
      • Veneziano L.
      • Macchioni L.
      • Hunter T.
      • Hunter G.
      • Pessia M.
      • D'Adamo M.C.
      A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia.
      ,
      • Chou S.-M.
      • Li K.-X.
      • Huang M.-Y.
      • Chen C.
      • Lin King Y.-H.
      • Li G.G.
      • Zhou W.
      • Teo C.F.
      • Jan Y.N.
      • Jan L.Y.
      • Yang S.-B.
      Kv1.1 channels regulate early postnatal neurogenesis in mouse hippocampus via the TrkB signaling pathway.
      ]. The enhancement of hyperpolarization by increasing either Kv1 or Kir channels will further lower the resting calcium levels, subsequently reducing presynaptic vesicle release.
      An additional support for the involvement of axonal potassium Kv1 channels in DCS effects can be observed in examining their critical role in determining the AP morphology and the subsequent subthreshold neuromodulation [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Kole M.H.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Shu Y.
      • Duque A.
      • Yu Y.
      • Haider B.
      • McCormick D a
      Properties of action-potential initiation in neocortical pyramidal cells: evidence from whole cell axon recordings.
      ,
      • Kole M.H.P.
      • Ilschner S.U.
      • Kampa B.M.
      • Williams S.R.
      • Ruben P.C.
      • Stuart G.J.
      Action potential generation requires a high sodium channel density in the axon initial segment.
      ,
      • Alle H.
      • Geiger J.R.
      Analog signalling in mammalian cortical axons.
      ], as shown by the effects of DCS-induced polarization on the postsynaptic response due to modulating the release of the quantal content from the presynaptic compartment. These studies showed that beyond the digital component of the AP, as an all or none phenomenon, there is an important analog component to the AP that is its morphology (waveform), which modulates the synaptic transmission, subsequently affecting the postsynaptic reponse. This analogous component of the AP waveform is governed by the level of subthreshold potential of the axon upon the AP is conducted [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Alle H.
      • Geiger J.R.
      Analog signalling in mammalian cortical axons.
      ,
      • Debanne D.
      • Bialowas A.
      • Rama S.
      What are the mechanisms for analogue and digital signalling in the brain?.
      ,
      • Brunner J.
      • Szabadics J.
      Analogue modulation of back-propagating action potentials enables dendritic hybrid signalling.
      ]. Given that the spike-dependent neurotransmitter release is highly dependent on presynaptic calcium concentration, the relationship between calcium and neurotransmitter release is extremely nonlinear (a power-law relationship of 3rd to 5th between calcium concentration and exocytosis) [
      • Augustine G.J.
      How does calcium trigger neurotransmitter release?.
      ,
      • Catterall W.A.
      • Few A.P.
      calcium channel regulation and presynaptic plasticity.
      ,
      • Südhof T.C.
      Calcium control of neurotransmitter release.
      ]. Hence, even a small variation in the AP waveform (widening) amplifies the neurotransmitter release [
      • Alle H.
      • Geiger J.R.P.
      Combined analog and action potential coding in hippocampal mossy fibers.
      ,
      • Clark B.
      • Häusser M.
      Neural coding: hybrid analog and digital signalling in axons.
      ,
      • Debanne D.
      • Bialowas A.
      • Rama S.
      What are the mechanisms for analogue and digital signalling in the brain?.
      ] leading to a significant change in the subsequent measured postsynaptic response. Ionic channel dynamics determines and regulates the waveform of the AP and a small variation in the axonal ionic channel dynamic can alter the AP morphology. Nonetheless, it is well established that the axon terminals are the most sensitive subcellular compartment to external electrical field application both by passive polarization in accordance with the cable equation [
      • Arlotti M.
      • Rahman A.
      • Minhas P.
      • Bikson M.
      Axon terminal polarization induced by weak uniform DC electric fields: a modeling study.
      ,
      • Kabakov A.Y.
      • Muller P a
      • Pascual-Leone A.
      • Jensen F.E.
      • Rotenberg A.
      Contribution of axonal orientation to pathway-dependent modulation of excitatory transmission by direct current stimulation in isolated rat hippocampus.
      ,
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ,
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ], and by active involvement of ionic channels, which further enhances the polarization of the axon terminals [
      • Chakraborty D.
      • Truong D.Q.
      • Bikson M.
      • Kaphzan H.
      Neuromodulation of axon terminals.
      ,
      • Vasu S.O.
      • Kaphzan H.
      The role of sodium channels in direct current stimulation—axonal perspective.
      ,
      • Vasu S.O.
      • Kaphzan H.
      Calcium channels control tDCS-induced spontaneous vesicle release from axon terminals.
      ]. Interestingly, the same ionic channels dynamics determine the initiation, waveform, and propagation of the APs [
      • MacKinnon R.
      Potassium channels.
      ,
      • Coleman S.K.
      • Newcombe J.
      • Pryke J.
      • Dolly J.O.
      Subunit composition of Kv1 channels in human CNS.
      ]. Amongst these ion channels, potassium channels, in particular Kv1 type, are crucial in determining the AP waveform morphology [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Kole M.H.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Goldberg E.M.
      • Clark B.D.
      • Zagha E.
      • Nahmani M.
      • Erisir A.
      • Rudy B.
      K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons.
      ]. Subthreshold depolarization inactivates the potassium Kv1 channels, subsequently increasing the width of the AP, especially along the axons where Kv1 channels are abundant [
      • Kole M.H.P.
      • Letzkus J.J.
      • Stuart G.J.
      Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.
      ,
      • Wang W.
      • Kim H.J.
      • Lv P.
      • Tempel B.
      • Yamoah E.N.
      Association of the Kv1 family of K+ channels and their functional blueprint in the properties of auditory neurons as revealed by genetic and functional analyses.
      ,
      • Coleman S.K.
      • Newcombe J.
      • Pryke J.
      • Dolly J.O.
      Subunit composition of Kv1 channels in human CNS.
      ]. This effect institutes semi-analog properties to the APs, and such AP widening was shown to enhance the postsynaptic response [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ] and alter the synaptic plasticity [
      • Taschenberger H.
      • Von Gersdorff H.
      Fine-tuning an auditory synapse for speed and fidelity: developmental changes in presynaptic waveform, EPSC kinetics, and synaptic plasticity.
      ,
      • Hoppa M.B.
      • Gouzer G.
      • Armbruster M.
      • Ryan T.A.
      Control and plasticity of the presynaptic action potential waveform at small CNS nerve terminals.
      ]. Our results demonstrate that DCS-induced modulation of AP follows a similar pattern where anodal DCS application enhances the AP width, whereas cathodal DCS application reduces the width of the AP. This role of Kv1 channels is supported by demonstrating Kv1 inhibition, using α-DTX completely occluded the DCS effects. Moreover, it is evident that not only that α-DTX abolished any DCS-induced modulations of the AP waveform, but it also in itself strongly increased the AP width regardless of the DCS stimulation, even with cathodal-DCS or without any DCS, and showed a similar effect to anodal-DCS. This finding coincides with the aforementioned observation of sEPSC frequency enhancement by α-DTX application that was comparable for all DCS conditions (cathodal, anodal and no-DCS). The fact that α-DTX occludes the physiological effects of DCS on both the intrinsic excitability level (AP waveform) and the extrinsic excitability level (spontaneous vesicle release rate), strengthens the premise that DCS conveys its effects, at least in part, by modulating axonal potassium Kv1 channels.
      To conclude, the herein study show for the first time that the modulation of axonal voltage-dependent potassium channels conductance is one of the pivotal underlying mechanisms by which DCS conveys its effects. Anodal-DCS inactivates Kv1 channels, thus facilitating presynaptic vesicle release that is crucial for building and maintaining neural circuits [
      • Zhang H.-Y.
      • Sillar K.T.
      Report short-term memory of motor network performance via activity-dependent potentiation of Na +/K + pump function.
      ] [
      • Catterall W.A.
      • Few A.P.
      calcium channel regulation and presynaptic plasticity.
      ,
      • Pellerin L.
      • Magistretti P.J.
      Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization (glutamate rnsporter/Na+/K+-ATPase/2-deoxyglucose/positron-embsson tomography/magnetic resonance imaging).
      ,
      • Lorincz A.
      • Nusser Z.
      Molecular identity of dendritic voltage-gated sodium channels.
      ,
      • Kavalali E.T.
      The mechanisms and functions of spontaneous neurotransmitter release.
      ,
      • Andreae L.C.
      • Burrone J.
      The role of spontaneous neurotransmission in synapse and circuit development.
      ], and widens the AP waveform instigating a stronger postsynaptic response [
      • Shu Y.
      • Hasenstaub A.
      • Duque A.
      • Yu Y.
      • McCormick D a
      Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.
      ,
      • Debanne D.
      • Campanac E.
      • Bialowas A.
      • Carlier E.
      • Alcaraz G.
      Axon physiology.
      ], which also strengthen neural connectivity. On the other hand, cathodal-DCS increases potassium conductance, which reduces the presynaptic vesicle release rate and narrows the AP waveform, both are presumed to weaken neural connectivity. By projecting these findings to tDCS, it can be assumed that tDCS relay some of its therapeutic and cognitive effects in humans by modifying the conductance of axonal potassium channels leading to altering neuronal circuitry. Altogether, knowing how ionic channels and their active modulation play a role in enhancing tDCS effects better explains how does this relatively mild electrical field is able to significantly modify over time cortical excitability and interneuronal connectivity, and what carries its non-linear qualities. Such understanding has the potential to aid in developing pharmacological augmentation strategies that will improve the efficacy of tDCS by utilizing drugs that will specifically manipulate distinct relevant voltage-dependent channels in a refined and targeted manner, so tDCS effects could be enhanced.

      CRediT authorship contribution statement

      Sreerag Othayoth Vasu: Formal analysis, Methodology, Investigation, Data acquisition, Writing – original draft, Writing – review & editing. Hanoch Kaphzan: Conceptualization, Funding acquisition, Supervision, Formal analysis, Methodology, Writing – original draft, Writing – review & editing.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      The work was supported by the Israel Science Foundation Grant No. 248/20 ( HK ).

      Appendix ASupplementary data

      The following is the Supplementary data to this article:

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