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Research Article: New Research | Neuronal Excitability
Sodium Channel-Dependent and -Independent Mechanisms Underlying Axonal Afterdepolarization at Mouse Hippocampal Mossy Fibers 1
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Shunsuke Ohura and Haruyuki Kamiya 1
Department of Neurobiology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
DOI: 10.1523/ENEURO.0254-18.2018 Received: 29 June 2018 Revised: 25 July 2018 Accepted: 26 July 2018 Published: 9 August 2018
Author contributions: H.K. designed the experiments. S.O. performed the experiments. H.K. performed the numerical simulation. S.O. and H.K. performed data analysis. H.K. wrote the manuscript. All the authors approved the final version of the manuscript. Funding: http://doi.org/10.13039/501100001691Japan Society for the Promotion of Science (JSPS) KAKENHI 18K06514
Conflict of Interest: The authors report no conflict of interest. This work was supported by JSPS KAKENHI grant number 16K15177 to HK. Correspondence should be addressed to Haruyuki Kamiya, MD, PhD, Department of Neurobiology, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan. Phone: 81-11-706-5027; Fax: 81-11-706-7863; E-mail:
[email protected] Cite as: eNeuro 2018; 10.1523/ENEURO.0254-18.2018 Alerts: Sign up at eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published.
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 Ohura and Kamiya This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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Sodium channel-dependent and -independent mechanisms underlying
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axonal afterdepolarization at mouse hippocampal mossy fibers
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Shunsuke Ohura1 and Haruyuki Kamiya1
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1Department
of Neurobiology, Hokkaido University Graduate School of
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Medicine, Sapporo, 060-8638, Japan
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Abbreviated title: Components of afterdepolarization in axon terminal
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Correspondence should be addressed to::
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Haruyuki Kamiya, M.D., Ph.D.
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Department of Neurobiology, Hokkaido University Graduate School of
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Medicine, Sapporo, 060-8638, Japan
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Phone: 81-11-706-5027
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Fax: 81-11-706-7863
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E-mail:
[email protected]
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Number of Figures: 6 Figures
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Number of Tables: 0 Table
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Number of Multimedia: 0 multimedia
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Number of words in Abstract: 250 words
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Number of words in Significance statement: 117 words
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Number of words in Introduction: 623 words
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Number of words in Discussion: 1522 words
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Acknowledgements: This work was supported by JSPS KAKENHI grant number 16K15177 to
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HK.
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Conflict of interest: The authors report no conflict of interest
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Author contributions:
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H.K. designed the experiments. S.O. performed the experiments. H.K.
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performed the numerical simulation. S.O. and H.K. performed data analysis.
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H.K. wrote the manuscript. All the authors approved the final version of the
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manuscript.
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Abstract Action potentials propagating along axons are often followed by prolonged
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afterdepolarization (ADP) lasting for several tens of milliseconds. Axonal
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ADP is thought to be an important factor in modulating the fidelity of spike
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propagation during repetitive firings. However, the mechanism as well as the
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functional significance of axonal ADP remain unclear, partly due to
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inaccessibility to small structures of axon for direct electrophysiological
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recordings. Here, we examined the ionic and electrical mechanisms
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underlying axonal ADP using whole-bouton recording from mossy fiber
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terminals in mice hippocampal slices. ADP following axonal action potentials
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was strongly enhanced by focal application of veratridine, an inhibitor of Na+
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channel inactivation. On the contrary, tetrodotoxin (TTX) partly suppressed
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ADP, suggesting that a Na+ channel-dependent component is involved in
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axonal ADP. The remaining TTX-resistant Na+ channel-independent
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component represents slow capacitive discharge reflecting the shape and
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electrical properties of the axonal membrane. We also addressed the
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functional impact of axonal ADP on presynaptic function. In paired-pulse
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stimuli, we found that axonal ADP minimally affected the peak height of
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subsequent action potentials, although the rising phase of action potentials
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was slightly slowed, possibly due to steady-state inactivation of Na+ channels
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by prolonged depolarization. Voltage clamp analysis of Ca2+ current elicited
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by action potential waveform commands revealed that axonal ADP assists
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short-term facilitation of Ca2+ entry into the presynaptic terminals. Taken
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together, axonal ADP maintains reliable firing during repetitive stimuli and
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plays important roles in the fine-tuning of short-term plasticity of
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transmitter release by modulating Ca2+ entry into presynaptic terminals.
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Significance statement Axonal action potentials are often followed by depolarizing or
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hyperpolarizing afterpotentials. This study illuminated the mechanisms of
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ADP in the hippocampal mossy fibers, where morphological as well as
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biophysical data were accumulated. We found that slow activating Na+
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channels are partly involved in ADP. Capacitive components also
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substantially contribute to ADP, suggesting that axonal shape and electrical
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properties are optimized for high-fidelity propagation during repetitive
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stimuli. We also tested the roles of ADP in the activity-dependent tuning of
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the presynaptic Ca2+ current. Action potential-driven Ca2+ entry into the
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axon terminals was facilitated by paired stimuli, possibly due to Ca2+ current
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facilitation by ADP. Therefore, ADP contributes to fine-tuning of transmitter
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release and ensures high-fidelity spiking of axons.
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Introduction The propagation of action potentials along axons is a fundamental
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process in the nervous system to reliably carry neuronal information to the
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target cells (Debanne et al., 2011; Kole and Stuart, 2012). Mechanisms
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enabling ultrafast and reliable spike signaling were studied extensively in
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various preparations of the central nervous system, including myelinated
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and unmyelinated axons. However, the mechanisms and functional
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consequences of axonal ADP, which often follows axonal action potentials
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and lasts for several tens of milliseconds, remain to be elucidated. Thus far,
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few studies have directly addressed the mechanisms for the generation of
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axonal ADP in the central nervous system, except for the calyx of Held axon
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terminals (Borst et al., 1995; Kim et al., 2010), which enable direct
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electrophysiological recording from the large axon terminals.
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As local application of the Na+ channel blocker tetrodotoxin (TTX)
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minimally affected ADP recorded from the calyx of Held axon terminals, the
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slow capacitive discharge of axonal membrane has been implicated in the
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generation of axonal ADP (Borst et al., 1995), as suggested in a previous
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study on lizard myelinated motor axons using intra-axonal recordings
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(Barrett and Barrett, 1982).
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In this context, it should be noted that ADP has been demonstrated to
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exhibit clear dependency on the initial membrane potentials. Upon
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depolarization of the axonal membrane, the amplitude of ADP decreased and
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occasionally reversed in polarity (Begum et al., 2016; Sierksma and Borst,
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2017). This indicated that ADP not only consists of passive component, but
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also incorporates some active component due to the activation of
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voltage-dependent conductance.
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On the other hand, ADP was found to be largely suppressed by the
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application of low-concentration TTX to the same calyx of Held axon
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terminals (Kim et al., 2010). In this report, the author raised the possibility
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that certain subtypes of voltage-dependent Na+ channels mediate ADP. Na+
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channels are functionally classified into at least three distinct subtypes: the
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transient-type (INaT), persistent-type (INaP), and resurgent-type (INaR).
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Involvement of slowly activating resurgent Na+ current (INaR) in axonal ADP
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was suggested in the study by Kim et al. (2010) because dialysis of a small
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peptide fragment of the β4 subunit of Na+ channels, which is an essential
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molecular component for INaR, selectively enhanced ADP. As the reason for
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the controversial conclusion of these studies (Borst et al., 1995; Kim et al.,
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2010) remains unclear, thorough investigation is needed to clarify the
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mechanisms underlying axonal ADP in the central nervous system.
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The functional significance of axonal ADP must also be addressed. It was
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widely considered that ADP lowers the threshold of subsequent action
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potentials, thereby enhancing the fidelity of spiking during high frequency
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neuronal activity (Barrett and Barrett, 1982). Moreover, the prolonged time
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course of ADP was suggested to improve fine-tuning of presynaptic functions,
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such as action potential-driven Ca2+ entry and subsequent transmitter
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release, by affecting the voltage-dependent conductance shaping axonal
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action potentials such as Na+ and K+ channels. However, detailed analysis of
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the axon terminals of calyx of Held revealed that ADP minimally affects Ca2+
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currents by balancing and cancelling out changes in the driving force and
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gating of voltage-dependent Ca2+ channels (Clarke et al., 2016).
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In this study, we examined the mechanisms underlying axonal ADP at
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hippocampal mossy fibers, where it was reported that prominent ADP
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follows axonal action potentials (Geiger and Jonas, 2000). Whole-cell
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recording from large axon terminals of hippocampal mossy fibers in
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combination with numerical simulation based on a realistic model of
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hippocampal mossy fibers (Engel and Jonas, 2005) was adopted to examine
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the ionic and electrical mechanisms underlying axonal ADP. We also closely
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investigated the influence of axonal ADP on action potential waveforms and
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Ca2+ current in presynaptic terminals in order to evaluate its functions for
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the activity-dependent tuning of presynaptic Ca2+ entry and subsequent
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transmitter release.
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Materials and Methods
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Animals and slice preparations C57BL/6J mice of either sex were used in this study, and were treated
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according to the guidelines for the care and use of laboratory animals of
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Hokkaido University. Transverse hippocampal slices of 300 μm thickness
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were prepared from p14-29 mice (number of animals = 46) as described
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previously (Kamiya, 2012; Ohura and Kamiya, 2018). Animals were
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anesthetized with ether, and the brain was dissected in an ice-cold sucrose
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solution containing the following: 40 mM NaCl, 25 mM NaHCO3, 10 mM
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glucose, 150 mM sucrose, 4 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, and
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7 mM MgCl2. Transverse slices were cut using a VT1200S microslicer (Leica
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Biosystems, Germany). The slices were then exchanged in a NMDG-HEPES
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recovery solution containing the following: 93 mM NMDG, 2.5 mM KCl, 1.2
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mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM
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Na-ascorbate, 2 mM Thiourea, 3 mM Na-pyruvate, 10 mM MgSO4, and 0.5
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mM CaCl2, and incubated for no longer than 15 min at 30-32°C (Ting et al.,
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2014). Then the solution was exchanged with standard artificial
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cerebro-spinal fluid (ACSF) containing the following: 127 mM NaCl, 1.5 mM
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KCl, 1.2 mM KH2PO4, 26 mM NaHCO3, 10 mM glucose, 2.4 mM CaCl2, and
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1.3 mM MgSO4, and the slices were kept in an interface-type chamber
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saturated with 95% O2, and 5% CO2. The slices were incubated in the ACSF
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at room temperature for at least 1 hr before experiments.
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Electrophysiology Mossy fiber boutons (MFBs) were visually identified under a microscope
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with IR-DIC optics (BX-51 WI, Olympus, Tokyo, Japan), as reported
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previously (Ohura and Kamiya, 2018; see also Geiger and Jonas, 2000; Alle
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and Geiger, 2006). Slices were continuously perfused at approximately 2
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ml/min with ACSF. In addition, the slice surface of the recording site was
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locally perfused with the drug-containing solution at approximately 0.2
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ml/min though a flow pipe with a 250-μm open-tip diameter connected to an
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electromagnetic valve system (Valve Bank, Automate Scientific, Barkley, CA).
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All recordings were made at room temperature (25 ± 1°C) with a patch clamp
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amplifier (MultiClamp700B, Molecular Devices, Sunnyvale, CA). Patch
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pipettes (typically 8-14 MΩ electrode resistance) were made from borosilicate
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glass with a microelectrode puller (Sutter P-97, Sutter Instruments, Novato,
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CA). Ca2+-free ACSF (an equal concentration of Mg2+ was replaced for Ca2+; 0
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mM CaCl2 and 3.7 mM MgSO4) was perfused in the bath and focally applied
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to minimize the synaptic input from the surrounding cells. In whole-cell
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current clamp recordings, the patch pipettes were filled with an intracellular
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solution containing the following: 140 mM K-gluconate, 10 mM KCl, 0.2 mM
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EGTA, 2 mM MgATP, and 10 mM HEPES, adjusted to pH 7.2. Electrical
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stimuli for 200 μs were given every 10 s at the granule cell layer of the
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dentate gyrus, except for in experiments shown in Fig. 3E-I, in which stimuli
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were delivered every 30 s. The liquid junction potential was estimated as -15
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mV using pCLAMP10 software and compensated for the holding potential.
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The series resistance and electrode capacitance were compensated before
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measurement. The recordings were adopted only when the resting
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membrane potentials were between -60 and -85 mV immediately after the
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break-in. The membrane potential was set to -80 mV manually by applying a
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small holding current if necessary (Geiger and Jonas, 2000). Input resistance
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was continuously monitored by injecting a hyperpolarizing current pulse (-10
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pA, 300 ms) in each sweep. MFBs with input resistance larger than 800 MΩ
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and series resistance lower than 70 MΩ were used for later analyses. The
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data were excluded if the series resistance changed by more than 20% of the
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initial value during the recording. In voltage-clamp experiments, pipettes
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were filled with an internal solution containing the following: 145 mM CsCl,
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2 mM MgCl2, 2 mM Na2ATP, 0.3 mM NaGTP, 5 mM Na2-phosphocreatinine,
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10 mM HEPES, and 10 mM EGTA, adjusted to pH 7.2. To record the Ca2+
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current, ACSF containing 1 μM tetrodotoxin (TTX), 20 mM
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tetraethylammonium chloride (TEA), and 5 mM 4-aminopyridine (4AP) was
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focally applied to the recording sites. To adjust the osmolarity of the solution,
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the concentration of NaCl in the ACSF was lowered by 20 mM. Leakage and
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capacitive currents were subtracted online using P/4 procedures. Series
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resistance (57.2 ± 3.3 MΩ, n = 7) was compensated by 50-70%. In some
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recordings, unclamped tail currents with a very slow time course appeared in
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an all-or-none manner, possibly reflecting spiking of the neighboring axons
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or boutons. These recordings with a time constant longer than 400 μs were
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excluded from analysis. Signals were filtered at 10 kHz with a 4-pole Bessel
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filter, and were digitized at 20 kHz with a DIGIDATA 1322A interface and
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16-bit resolution (Molecular Devices, Sunnyvale, CA). All data were acquired
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and analyzed offline with pClamp 10.7 software (Molecular Devices,
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Sunnyvale, CA) and Origin 8J or 2015 (OriginLab, Northampton, MA).
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Simulation The simulated membrane potential (Vm) at the hippocampal mossy fibers
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was calculated according to the model suggested by Engel and Jonas (2005)
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based on the data recorded from mossy fiber boutons. The model basically
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assumed a Hodgkin-Huxley-type model (Hodgkin and Huxley, 1952) adapted
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to channels in mossy fiber terminals, and K+ channels inactivation was
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implemented multiplicatively with parameters of recombinant KV1.4
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channels (Wissmann et al., 2003). Simulations were performed using
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NEURON 7.5 for Windows (Hines and Carnevale, 1997). The passive
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electrical properties of the axon were assumed to be uniform, with a specific
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membrane capacitance Cm of 1 μF cm−2, a specific membrane resistance Rm
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of 10,000 Ω cm2, and an intracellular resistivity Ri of 110 Ω cm. The
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structure of the mossy fiber was approximated by a soma (diameter, 10 μm),
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10 axonal cylinders (diameter, 0.2 μm; length, 100 μm), and 10 en passant
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boutons (diameter, 4 μm). The number of segments was 1 μm−1, and the time
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step was 5 μs in all simulations. The resting potential was assumed to be −80
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mV. The reversal potential of the leak conductance was set to −81 mV to
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maintain stability. Voltage-gated Na+ channels, K+ channels, and leakage
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channels were inserted into the soma, axon, and boutons, respectively. The
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Na+ conductance density was set to 50 mS cm−2 for the axon and boutons,
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and 10 mS cm−2 for the soma. The K+ conductance density was set to 36 mS
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cm−2 throughout all parts of the neurons. Action potentials were evoked by
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injection of depolarizing current into the 9th bouton (1 ms, 0.15 nA) or the
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soma (2 ms, 0.2 nA) in the simulation shown in Fig. 4G and 4H, respectively.
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We carried out stimulation on the last (10th) bouton in a “pearl chain
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structure” to avoid sealed end effects. The equilibrium potentials for Na+ and
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K+ ions were assumed to be +50 mV and −85 mV, respectively.
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Statistics All data are expressed as the mean ± SEM. Statistical analysis was
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performed by non-parametric two-sided tests (Wilcoxon signed-rank test for
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paired data and Mann-Whitney U test for unpaired data), and a P-value of
