Articles in PresS. J Neurophysiol (March 10, 2010). doi:10.1152/jn.00506.2009
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Article type: Research Article
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Dendritic HCN channels shape excitatory postsynaptic potentials at the inner hair
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cell afferent synapse in the mammalian cochlea
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Eunyoung Yi (이은영), Isabelle Roux & Elisabeth Glowatzki
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The Johns Hopkins School of Medicine, Department of Otolaryngology-Head and Neck
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Surgery, 720 Rutland Ave, Ross 824, Baltimore MD 21205, USA
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Running Head: Dendritic Ih at the hair cell synapse
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Elisabeth Glowatzki (corresponding author)
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The Johns Hopkins School of Medicine
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Department of Otolaryngology Head and Neck Surgery
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720 Rutland Avenue, Ross 824
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Baltimore MD 21205, USA
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Tel.: 410-502-7008
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Fax: 410-614-4748
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Email: [email protected]
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Number of figures: 7; Supplemental material: 2 figures
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Number of words: Abstract: 239; Introduction: 432; Methods: 1502; Results: 3531;
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Discussion: 1831
Copyright © 2010 by the American Physiological Society.
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Abstract
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Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the
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auditory pathway, is specialized for rapid and reliable signaling. Here we investigated
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the properties of a hyperpolarization activated current (Ih) expressed in the afferent
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dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity.
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We used whole-cell patch-clamp recordings from afferent dendrites directly where they
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contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials
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(EPSPs) of variable amplitude (1-35 mV) were found with 10-90% rise time of ~ 1 ms
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and time constant of decay of ~ 5 ms at room temperature. Current voltage relations
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recorded in afferent dendrites revealed a hyperpolarization-activated current (Ih). The
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pharmacological profile and reversal potential (-45 mV) indicated that Ih is mediated by
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hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN
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channel subunits HCN1, HCN2 and HCN4 were found to be expressed in afferent
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dendrites using immunolabeling. Raising intracellular cAMP levels sped up the
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activation kinetics and increased the magnitude of Ih and shifted the half activation
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voltage (Vhalf) to more positive values (-104 ± 3 mV to -91 ± 2 mV). Blocking Ih with 50
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μM ZD7288 resulted in hyperpolarization of the resting membrane potential (~ 4 mV)
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and slowing the decay of the EPSP by 47 %, suggesting that Ih is active at rest and
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shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first
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synapse in the auditory pathway.
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Keywords: hair cell, spiral ganglion, dendrite, Ih, auditory nerve
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Introduction
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To perform tasks such as the localization of sound in space, neurons in the auditory
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pathway are specialized to accurately preserve timing information within sound signals
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(Oertel 1999; Trussell 1999). Several pre- and postsynaptic mechanisms enabling rapid
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and reliable transmission at auditory synapses have been described. Presynaptically,
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large calyceal structures release vesicles from many release sites synchronously resulting
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in large EPSPs that reliably activate action potentials (APs) (Schneggenburger and
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Forsythe 2006). Postsynaptically, rapid kinetics of AMPA receptors result in brief
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excitatory postsynaptic currents (EPSCs) (Gardner et al. 2001; 1999; Parks 2000; Raman
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et al. 1994). Voltage-gated ion channels active near the resting membrane potential
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decrease the membrane resistance of the postsynaptic neurons and shorten the membrane
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time constant (Magee and Johnston 2005). This mechanism keeps EPSPs brief and
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prevents temporal summation of synaptic events. A hyperpolarization activated cation
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channel (Ih) has been described in auditory brainstem neurons as one of the ion channels
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serving this role (Banks and Smith 1992; Golding et al. 1995; Rothman and Manis
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2003b). To further shape EPSPs, auditory neurons can receive modulatory inputs that
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either open receptor-coupled ion channels (Funabiki et al. 1998; Smith et al. 2000) or
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regulate Ih via G-protein coupled signaling pathways (Banks et al. 1993; Yamada et al.
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2005).
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The first synapse in the auditory pathway, the synapse between the inner hair cell (IHC)
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and auditory nerve fiber, also employs highly specialized mechanisms to preserve timing
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information (Fuchs 2005; Glowatzki et al. 2008; Moser et al. 2006). The auditory nerve
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fiber receives input from only one IHC via a single dendrite, and large EPSCs are
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activated by multivesicular release at this ribbon-type synapse (Glowatzki and Fuchs
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2002; Goutman and Glowatzki 2007; Grant et al. in press; Keen and Hudspeth 2006; Li et
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al. 2009). Similar to EPSCs recorded from auditory brainstem synapses, AMPA-
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mediated EPSCs at this synapse are brief (Glowatzki and Fuchs 2002; Grant et al. in
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press). However, not much is known regarding the expression pattern of voltage-gated
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ion channels in afferent dendrites and their involvement in shaping postsynaptic activity.
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In a first survey, using voltage clamp recordings from afferent dendrites of the postnatal
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rat cochlea, we identified several voltage-gated conductances in afferent dendrites (Na+-,
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Ca 2+-, K+- conductances) including a hyperpolarization activated conductance.
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Here we focus on the characterization of Ih in IHC afferent dendrites. We find that Ih is
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mediated by HCN channels. Ih is active at rest, and is modulated by intracellular levels of
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cAMP. Ih shortens the EPSP, and therefore is a good candidate for enabling rapid and
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reliable signaling at this first synapse in the auditory pathway.
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Materials and Methods
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Animal protocols were approved by the Johns Hopkins University Animal Care and Use
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Committee. Rats (Sprague Dawley; Charles River, Wilmington, MA) were anaesthetized
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(pentobarbital 0.045mg g -1 i.p. or isoflurane inhalation), decapitated and cochleae were
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quickly removed from temporal bones.
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Electrophysiological recordings. Excised apical cochlear turns of 7-14 day old rats
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were placed into a chamber under an upright microscope (Axioskop2 FS plus, Zeiss,
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Oberkochen, Germany) and superfused with external solution at 1-3 ml/minute (chamber
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volume ~ 2 ml). IHCs and contacting afferent dendrites were visualized on a monitor via
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a 40x water immersion objective, 4x magnification, differential interference contrast
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optics using a green filter, and a NC 70 Newvicon camera (Dage, MTI, Michigan City,
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IN). The pipette solution was (in mM): 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5
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HEPES, 0-2.5 Na2ATP; or 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 HEPES, 4
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Na2ATP, 0.2 Na2GTP; 290 mOsm, pH 7.2 (KOH). In some recordings where the effect
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of cAMP was tested, the pipette solution contained (in mM):131 KCl, 1 MgCl2, 5 EGTA,
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5 HEPES, 5 Na2ATP and 10 Na2phosphocreatine. In addition, a small number of
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recordings (n = 6) was performed using a pipette solution containing (in mM): 110 K-
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methanesulfonate, 20 KCl, 5 EGTA, 5 HEPES, 0.1 CaCl2, 5 Na2phosphocreatine, 4
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MgATP, 0.3 TrisGTP. No significant differences were found in basic membrane
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properties such as input resistance and membrane time constant of the afferent fiber
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between recordings with KCl- or K-methanesulfonate-based pipette solutions. The
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external solution was (in mM): 5.8 KCl, 155 NaCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4,
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5.6 Glucose, 10 HEPES; 300 mOsm, pH 7.4 (NaOH). Drugs were dissolved daily in the
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external solution to their final concentrations from frozen stocks. Application of drug
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solutions was performed using a gravity-driven flow pipette (100 μm diameter) placed
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near the row of IHCs, connected with a VC-6 channel valve controller (Warner
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Instrument, Hamden, CT). ZD7288, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 4-
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aminopyridine (4-AP) were purchased from Tocris Bioscience (Ellisville, MO), and
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tetrodotoxin (TTX) either from Alomone (Jerusalem, Israel) or Sigma (St. Louis, MO).
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All other chemicals were purchased from Sigma.
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Recording pipettes were fabricated from 1 mm borosilicate glass (WPI, Sarasota, FL).
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Pipettes were pulled with a multi-step horizontal puller (Sutter, San Rafael, CA) and fire-
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polished (10-15 MΩ). Pipettes were coated with Sylgard® (Dow Corning, Midland, MI).
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Experiments were done at 22-25° C. Recordings were performed with a Multiclamp
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700A or 700B amplifier (Molecular Devices, Sunnyvale, CA), pClamp version 9.2 and a
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Digidata 1322A board, digitized at 50 kHz, and filtered at 10 kHz.
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In voltage clamp mode, series resistance (Rs) was calculated from capacitative current
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responses to a 10 mV voltage step (-84 to -94 mV). The capacitative current responses
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were fitted with a sum of 2 or more exponential equations. The fastest component of the
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fit was considered to represent the capacitative current for the membrane area near the
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recording electrode (Cfast) and the slower components for current spreading further along
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the afferent nerve fiber (Cslow) (Llano et al. 1991). From the fastest component, the
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voltage clamp time constant (36 ± 12 µs, n = 38), membrane capacitance Cfast (1.32 ±
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0.43 pF) and Rs (30 ± 12 MΩ) were derived. Voltage clamp data were discarded if Rs
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was > 50 MΩ. Most Ih currents were < 300 pA, and with Rs at ~ 30 MΩ, the estimated
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voltage error was < 9 mV.
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Assuming that the dendrite is formed like a cylinder, and with a specific capacitance of 1
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µF/cm2 and a diameter of 1 µm (most likely an overestimation), the first component
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corresponds to the dendrite at a length of about 40 µm. This distance covers the extent of
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the HCN channel expression along the terminal; the HCN specific labeling ceases in the
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region of the first hemi-node of the peripheral dendrite, ~ 50 µm away from the afferent
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contacts with the IHC. Synaptic currents are generated within less than 3 µm of the
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pipette tip, as the tip is directly positioned on the bouton ending. Therefore, voltage
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clamp conditions for recording synaptic currents and HCN channels should be sufficient.
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The input resistance (Rin) of afferent dendrites (394 ± 253 MΩ, n = 173) was determined
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in voltage clamp with voltage steps from -64 mV to -84 mV.
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In current clamp mode, errors due to Rs were compensated using bridge balance and
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pipette capacitance neutralization. Membrane voltage responses to -10 pA current steps
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were fitted with a monoexponential equation and provided a membrane time constant
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(τm) of 3.97 ± 1.81 ms (n = 10). τm measured in current clamp mode is larger than τm
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estimated from voltage clamp data (0.5 ms) when only Cfast is used to estimate cell
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capacitance. This happens most likely because in current clamp mode the effective cell
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capacitance is not limited to Cfast but also includes Cslow.
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Liquid junction potentials (4 mV for KCl-based and 9 mV for K-methanesulfonate based
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pipette solution) were corrected off-line. Data were analyzed off-line using pClamp
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version 9.2 (Axon Instruments, Union City, CA), Minianalysis (Synaptosoft, Decatur,
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GA) and Origin 7.5 (OriginLab, Northampton, MA). For statistical comparisons
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Sigmastat 3.5 (SYSTAT Software Inc, Point Richmond, CA) was used. Statistical
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significances of irreversible drug effects (ZD7288) were tested using a paired-t-test.
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Effects of ZD7288 on the EPSP waveform (measurements were taken at 3 different
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conditions) were tested using one-way repeated measures analysis of variance followed
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by Student-Newman-Keuls test. Effects of reversible drugs (CsCl, and BaCl2) were
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tested using one-way repeated measures analysis of variance followed by Student-
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Newman-Keuls test. Effects of cAMP analogues on Ih amplitude and activation kinetics
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were tested using two-way repeated measures analysis of variance one factor repetition.
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Effects of cAMP on the Vhalf and slope factor of Ih activation curves were tested using
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Student t-test. Values are presented as mean ± standard deviation.
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Immunolabeling. Cochleae from 9-10 and 20-21 day old rats (P9-10, P20-21), were
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perfused through the round and oval windows with cold 4% paraformaldehyde prepared
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in phosphate buffered saline (PBS), pH 7.4, and then post-fixed for 1 h at 4°C under
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agitation. Additionally, for HCN1 immuno-detection, cochleae were rinsed three times in
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PBS and incubated 15 min in methanol at -20°C. Thereafter, preparations were washed 3
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times in PBS and the cochleae were microdissected to facilitate access of the antibodies
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to the tissue. Whole-mount preparations were incubated for 1h at room temperature in a
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blocking and permeabilizing solution (PBS with 20% of either normal goat serum or
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donkey serum and 0.3% Triton X-100), and were then incubated overnight at 4°C with
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the primary antibodies diluted in the same solution. After three 15 min washes in PBS,
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samples were incubated for 1 h at room temperature with fluorescently labeled secondary
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antibodies diluted at 1:800 in PBS with 10% of either normal goat serum or donkey
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serum and 0.15% Triton X-100. Samples were then rinsed once in PBS with 10% of
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either normal goat serum or donkey serum and 0.15% Triton X-100 and twice with PBS
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(15 min each, at room temperature) before the organs of Corti were mounted on slides
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using FluorSave mounting medium (Calbiochem, San Diego, CA). Specific labeling was
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initially examined with an Axio Observer inverted microscope (Zeiss) and further
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detailed images were obtained using a confocal laser scanning microscope (LSM 510
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META, Zeiss) with a 20x air and a 100x oil objectives (optical section steps of 0.25 and
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0.20 microns, respectively). Analysis and reconstruction were carried out using LSM
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Image Examiner (Zeiss) and Volocity 4.2.1 software (Improvision Inc., Waltham, MA).
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No labeling was observed when the primary antibodies were omitted. Again, no labeling
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was observed when the primary antibodies were absorbed onto target peptides, except in
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the stereocilia of the sensory hair cells which displayed staining using HCN1 and HCN4
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antibodies, when the target peptides were used at the recommended concentration or at a
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25 times (HCN1) or 5 times (HCN4) higher concentration. For HCN2, no test for
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preabsorption with a peptide was performed. For HCN3 a shorter fixation (15 min) was
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also tested.
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Antibodies. Rabbit polyclonal antibodies against HCN1 and HCN4 (Alomone) were
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used at dilutions of 1:200 and 1:400, respectively. Rabbit polyclonal antibodies against
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HCN3 (Alpha Diagnostic Intl. Inc., San Antonio, TX) were used at dilutions of 1:200 to
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1:20. Contrary to the antibodies against HCN3 from Chemicon and Alomone, this
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antibody does not show cross reactivity for hHCN1, hHCN2, or hHCN4 (Kouranova et al.
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2008). Monoclonal antibodies against HCN2 and HCN3 from UC Davis/NINDS/NIMH
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NeuroMab Facility (Davis, CA) were used at 1:25. Mouse monoclonal and rabbit
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polyclonal antibodies against recombinant rat calretinin (Chemicon, Temecula, CA) were
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diluted at 1:1000. Guinea-pig serum against VGLUT3 was kindly provided by Dr. Robert
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H. Edwards’ laboratory (Department of Physiology, School of Medicine, University of
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California San Francisco) and used at a 1:1000 dilution. Alexa Fluor 488 F(ab')2 fragment
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of goat anti-rabbit and Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 555 goat
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anti-mouse and Alexa Fluor 594 donkey anti-rabbit IgG, Alexa Fluor 633 goat anti-
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guinea pig IgG (Molecular Probes, Eugene, OR) were used as secondary antibodies.
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Results
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Voltage-gated ion channels in IHC afferent dendrites
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To characterize voltage-gated conductances in IHC afferent dendrites, we used whole-
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cell recordings from afferent dendrites directly where they contact the IHC. Recordings
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were performed in acutely excised apical cochlear turns from 7-14 day old rats at room
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temperature. Resting membrane potentials of afferent dendrites were typically ~ -64 mV.
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We assume that the peripheral neurites of the recorded auditory nerve fibers were intact
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and connected with their spiral ganglion somata for the following reasons: Firstly, in
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preparations where we separated the spiral ganglion from the cochlear coil, afferent
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dendrites were severely swollen and no recordings could be achieved. Secondly, in 9 of
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9 experiments, where a fluorescent dye, Alexa Fluor 488 hydrazide salt (10 µM), had
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been included in the pipette solution and recordings had lasted longer than 10 min, the
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unbranched afferent fibers could be traced back from the IHC towards the spiral ganglion
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for 200-500 μm (Supplemental Fig. 1) and in 2 cases the fluorescent marker had reached
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the spiral ganglion somata at a distance of 450-500 μm from the IHC.
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Our study focused on the characterization of Ih. However, as voltage-gated conductances
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have not been described for IHC afferent dendrites, in the following paragraph we will
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briefly summarize the different conductances that were observed in response to voltage
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step protocols. From a holding potential of -84 mV, voltage steps between -104 and -4
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mV were applied (Fig. 1A). Voltage-gated sodium currents were found in 114 of 155
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afferent dendrite recordings. Sodium currents often ‘escaped’ the voltage clamp (Fig. 1A
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inset). This is not surprising as the recording site is at the very tip of the unmyelinated
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afferent fiber ending and the action potential initiation site is most likely located further
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away along the myelinated peripheral process (Hossain et al. 2005; Lacas-Gervais et al.
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2004; McLean et al. 2009). For this study sodium channels were not further investigated,
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but rather blocked with 1-2 µM TTX (Fig. 1B). Additionally, small calcium currents that
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could be blocked with 200 µM CdCl2 were detected in some recordings (data not shown).
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As these small currents did not interfere with questions asked in this study, no effort was
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made to block them.
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During depolarizing voltage steps, outward currents were observed in all 161 afferent
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dendrites recorded (Fig. 1). Outward currents reached their maximum within ~5 ms. The
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current voltage relations showed that outward currents activated at potentials as low as -
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64 mV (Fig. 1B-C). We tested the effects of 4-AP (2-4 mM) and TEA (10-30 mM),
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drugs previously shown to inhibit the low voltage activating potassium current (IKL) and
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the high voltage activating potassium current (IKH) respectively, in spiral ganglion
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neurons (Szabo et al. 2002) and auditory brainstem neurons (Bal and Oertel 2001; Brew
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and Forsythe 1995; Cao et al. 2007; Manis and Marx 1991; Rathouz and Trussell 1998;
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Reyes et al. 1994; Rothman and Manis 2003a). 4-AP-sensitive and TEA-sensitive
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currents were observed in afferent dendrites and exhibited similar voltage dependent
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profiles to IKL and IKH, respectively. These conductances are still under investigation.
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During hyperpolarizing voltage steps, a slowly developing inward current was found in
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69 of 79 afferent recordings (Fig. 1B-C). Voltage dependence and activation kinetics of
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this inward current were reminiscent of Ih recorded in dissociated spiral ganglion somata
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(Chen 1997; Mo and Davis 1997b).
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Ih in afferent dendrites is mediated by HCN channels
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To test whether Ih currents are mediated by HCN channels, we monitored Ih during the
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application of different blockers. In response to repeatedly applied negative voltage steps
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from -84 to -124 mV, an instantaneous inward current (Iinst) was followed by a slowly
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developing inward current (I0.5s) (Fig. 2C). Iinst is partially blocked by Ih blockers and
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consists of a mixture of Ih and other conductances (see Fig. 2A-C) (Bal and Oertel 2000;
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Rodrigues and Oertel 2006). We therefore report only on the amplitude of the slowly
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developing component of Ih (I0.5s - Iinst). Ih currents showed some run-down during
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whole-cell recordings and test protocols were designed accordingly. 2 mM CsCl
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reversibly inhibited inward currents by 84 ± 11% (n = 8) whereas 2 mM BaCl2 caused a
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reversible inhibition by 14 ± 8 % (n = 4) (Fig. 2A, B, D, E, F). This combination of a
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strong Cs+ block and a weak Ba2+ block has been described for HCN channels whereas
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inward rectifier potassium channels typically show a substantial Ba2+ block (Kubo et al.
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2005; Robinson and Siegelbaum 2003; van Welie et al. 2005). Additionally, 50 µM
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ZD7288, an antagonist of HCN channels (Shin et al. 2001), irreversibly inhibited Ih by 93
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± 13% (n = 4) (Fig. 2C, E, F).
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The reversal potential of Ih was estimated using the following protocol: different
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conditioning voltages were applied for 3 s (-124 mV, -104 mV and -84 mV), followed by
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10 ms voltage ramps (from -144 mV to -74 mV) (Fig. 3F). The initial current response
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(~ 3 ms) during the voltage ramp was discarded due to contamination with
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uncompensated capacitance currents. The linear portions of the current responses (-120
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to -74 mV) were extrapolated to the region where the responses intersect, corresponding
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to an estimated reversal potential of Ih at -45 ± 2 mV (n = 4, Fig. 3F). This reversal
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potential is consistent with a mixed cation channel permeable for sodium and potassium,
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and is in the range of reversal potentials described for HCN channels (Bal and Oertel
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2000; Banks et al. 1993; Cao et al. 2007; Chen 1997; Cuttle et al. 2001; Mo and Davis
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1997a; Moosmang et al. 2001; Rodrigues and Oertel 2006; Santoro et al. 2000; van Welie
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et al. 2005). In summary, the pharmacological profile and reversal potential suggest that
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Ih in afferent dendrites is mediated by HCN channels.
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HCN channels in afferent dendrites are modulated by cAMP
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The current voltage relation of Ih was recorded while blocking voltage-gated sodium
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channels (1-2 μM TTX), potassium channels (2 mM 4-AP and 10-30 mM TEA) and
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AMPA receptors (10 μM CNQX) (Fig. 3A-E). From a holding potential of -64 mV,
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voltage steps were applied for 3 s from –144 mV to -54 mV in 10 mV increments, and
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were followed by a voltage step to -74 mV, during which tail currents were recorded (Fig.
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3A). Ih was measured as I3s - Iinst. The current voltage relation showed larger activation
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towards more negative voltages, with an Ih amplitude of 174 ± 48 pA at -144 mV (n = 3)
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(Fig. 3C, black trace). The voltage dependence of Ih was measured from tail currents and
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fitted by a Boltzmann equation (Fig. 3D, black traces). Half maximum activation voltage
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(Vhalf) was -104 ± 3 mV and the slope factor was 11 ± 1 (n = 3). The activation curve
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shows that about 3 % of Ih channels would be open at the resting membrane potential of
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the afferent dendrites (-65 mV). The activation range measured here corresponds to those
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measured under similar conditions for HCN channels in heterologous expression systems
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(Moosmang et al. 2001; Santoro et al. 2000) and Ih recorded in other neurons and spiral
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ganglion somata (Bal and Oertel 2000; Banks et al. 1993; Cao et al. 2007; Chen 1997;
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Cuttle et al. 2001; Mo and Davis 1997b; Rodrigues and Oertel 2006; van Welie et al.
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2005).
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Different HCN channel subunits activate on different time scales, with activation time
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constants ranging from milliseconds to seconds (Moosmang et al. 2001; Santoro et al.
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2000). We measured the activation kinetics of Ih for current responses to voltage steps
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between -134 and -104 mV from a holding potential of -64 mV (Fig. 3E, black data
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points). The time course of activation was best fitted with two exponentials. At -134 mV,
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the time constants of the fast (τfast) and the slow (τslow) component were 66 ± 13 ms (n =
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3) and 929 ± 40 ms, respectively (Afast/(Afast+Aslow) = 0.83 ± 0.09). At more positive
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potentials, activation slowed down for both components.
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It is well known that intracellular cyclic nucleotides can modulate HCN channel activity
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(Moosmang et al. 2001; Santoro et al. 2000; Wainger et al. 2001). Typically, with
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increased cAMP levels, the activation curve is shifted to more positive values and the
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activation kinetics speeds up. We therefore measured the current voltage relation in the
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presence of cAMP analogues (Fig. 3B). 200 µM cAMP was added to the pipette solution
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and additionally 200 µM of the membrane permeable 8-Br-cAMP was added to the
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external solution. In the presence of cAMP analogues, the current amplitude of Ih
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increased significantly compared to control (from 174 ± 48 pA in control to 332 ± 91 pA
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in cAMP; at -144 mV, n = 4, p < 0.05 for all voltages tested) (Fig. 3B, C, red traces). The
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activation curve shifted to more positive values, by ~ 12 mV (Fig. 3D, red traces) with
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Vhalf at -91 ± 2 mV (n = 4, p < 0.05) and no significant change in the slope factor (11 ± 1,
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n = 4, p = 0.611). Under these conditions, at -65 mV, about 9 % of Ih would be active
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and have an estimated conductance of 1.4 nS (332 pA x 0.085/ 20 mV; reversal potential
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-45 mV). In the presence of cAMP analogues, activation time constants were
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significantly faster with a τfast of 23 ± 6 ms and a τslow of 175 ± 76 ms at a holding
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potential of -134 mV (n = 4, p < 0.05 for all voltages tested; Afast/(Afast+Aslow) = 0.85 ±
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0.05) (Fig. 3E, red data points).
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HCN channel subunits expression in IHC afferent dendrites
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To investigate the expression pattern of HCN channel subunits in IHC afferent dendrites,
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we performed immunolabeling experiments with antibodies raised against the four known
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HCN channel subunits HCN1-4 (Moosmang et al. 2001; Robinson and Siegelbaum 2003).
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We examined HCN distribution in the apical part of the cochlea in pre-hearing animals,
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at P9-10, to match our recordings from afferent dendrites, and at P20-21, to analyze HCN
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subunit expression in hearing animals. No immunolabeling of HCN3 above background
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was found and therefore the result could not be interpreted. Figure 4 shows overviews of
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apical cochlear turns at P9-10 labeled for HCN1 or HCN4. At this age, we did not find
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HCN2 labeling. For better orientation, IHCs were labeled for the vesicular glutamate
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transporter VGLUT3 (Seal et al. 2008) (Fig. 4B). HCN1 and HCN4 specific labeling in
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the organ of Corti was found in the area directly below the IHCs, suggestive of a labeling
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of the unmyelinated peripheral processes of the afferent neurons. Additionally, HCN1
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and HCN4 immunoreactivity was found in most of the spiral ganglion somata (Fig. 4,
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supplemental Fig. 2). The labeling was concentrated at the plasma membrane of the
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somata and in their processes within the ganglion close to the somata (supplemental Fig.
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2). At P20-21, similar to the labeling pattern at P9-10, labeling for HCN1, HCN4 and
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additionally for HCN2 was found directly below the IHCs (Fig. 5) and in the spiral
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ganglion somata (supplemental Fig. 2).
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IHCs are surrounded by different cell types, including afferent fibers, efferent fibers and
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supporting cells. To confirm that the afferent dendrites in the IHC area express the
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different HCN subunits, as indicated by our electrophysiological recordings, we
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performed double immunolabeling experiments for calretinin and HCN channel subunits.
349
Calretinin is a calcium-binding protein involved in calcium buffering and transport. In
350
the rat cochlea, calretinin has been detected both in the cytoplasm of IHCs and in most
351
spiral ganglion neurons including their afferent dendrites (Dechesne et al. 1991;
352
Dechesne et al. 1993). High resolution confocal imaging confirmed the coexpression of
353
the HCN subunits and calretinin in the same afferent dendrites at P9-10 and P20-21 (Fig.
354
5). When assessing the labeling close to the IHCs at P9-10, some afferent dendrites
355
could be identified by their strong labeling with calretinin and were also found positive
356
for HCN1 (Fig. 5A-D, arrowheads) or HCN4 (data not shown). At P20-21, HCN
357
immunolabeling was more sharply defined, most likely reflecting a higher concentration
358
or more intense clustering of the HCN channel subunits in afferent dendrites in the more
359
mature organ of Corti. Most afferent dendrites positive for HCN1, HCN2 and HCN4
360
were also positive for calretinin labeling, indicating that HCN1, HCN2, and HCN4 are
361
indeed localized in the afferent dendrites (Fig. 5E-M, arrowheads). In some dendrites,
362
HCN labeling appeared to surround the calretinin labeling in a ring-like fashion (open
363
arrowheads), consistent with a clustering of the HCN channels in the plasma membrane.
364
Some HCN positive dendrites were not calretinin positive (Fig. 5, asterisks). Similarly,
17
365
not all HCN positive spiral ganglion somata were positive for calretinin labeling (data not
366
shown). We conclude that the HCN subunits 1, 2 and 4 are expressed in afferent
367
dendrites at the IHC afferent synapse.
368
369
EPSP waveforms in IHC afferent dendrites
370
In the CNS, dendritic Ih has been shown to shape EPSPs and modulate excitability in
371
neurons (Magee 2000). To investigate whether Ih affects the EPSP waveform in IHC
372
afferent dendrites, we first characterized the EPSP waveform at rest. To isolate synaptic
373
activity and block the generation of APs, experiments were performed in 1 μM TTX.
374
Resting membrane potentials of afferent dendrites were -64 ± 8 mV (n = 34) when
375
recorded in our standard extracellular solution containing 5.8 mM K+ and 1.3 mM Ca2+.
376
Synaptic events occurred at a rate of 0.7 ± 0.9 /s (n = 47; 13575 synaptic events analyzed).
377
As shown before for EPSCs (Glowatzki and Fuchs 2002), some EPSPs appeared
378
‘multiphasic’, composed of multiple overlapping events. Other EPSPs appeared
379
‘monophasic’, presenting a mono-exponential decay (Fig. 6F). To investigate rise and
380
decay times, only monophasic events were analyzed. In 3 recordings, for direct
381
comparison, both EPSCs and EPSPs were characterized in the same recording (Fig. 6A-
382
K). EPSCs were recorded at a holding potential of -94 mV and EPSPs at the resting
383
membrane potential of the afferent dendrite in 5.8 mM K+. EPSPs were about 3-4 times
384
slower than EPSCs, with a 10-90 % rise time of 1.14 ± 0.17 ms compared to 0.36 ± 0.09
385
ms, and a time constant of decay of 5.13 ± 1.69 ms compared to 1.16 ± 0.13 ms at room
386
temperature (n = 3, 381 EPSPs and n = 8, 1807 EPSCs analyzed) (Fig. 6C, D, G, H). For
387
EPSPs, we found a wide range of amplitudes in every recording, varying from 1 to 35
18
388
mV. Also the shape of the amplitude distributions varied widely. The amplitude
389
distributions of two fibers were highly skewed, with median EPSP amplitudes of 2.3 mV
390
and 2.4 mV, and the majority of EPSPs was smaller than 10 mV (Fig. 6I, J). For a third
391
fiber, EPSP amplitudes spread more evenly within a range between 1 and 35 mV, with a
392
median EPSP amplitude of 13.8 mV (Fig. 6K). However, for both EPSCs and EPSPs,
393
rise and decay times did not change much over this wide range of amplitudes (Fig. 6C, D,
394
G, H).
395
In auditory nerve fibers, the spike initiation zone is believed to be located close to the
396
IHC afferent synapse, on the peripheral process (Hossain et al. 2005; Robertson 1976). A
397
heminode with a high expression level of sodium channels, is less than 50 µm away from
398
the afferent synapse (Hossain et al. 2005; McLean et al. 2009). The expression of sodium
399
channels was also reported on the unmyelinated ending of the afferent fiber, peripheral to
400
the heminode (Hossain et al. 2005). Indeed, when recording in the absence of TTX, at a
401
resting membrane potential of -67 ± 5 mV (n = 7), 18 ± 22 % of the events were spikes
402
rather than EPSPs (range 0.5-50.5 %, n = 4 afferent dendrites with more than 100
403
analyzable events, each, total number of events analyzed: 829) (Fig. 6L-N). Spikes were
404
discriminated from EPSPs by their large and uniform amplitudes (39 ± 7 mV, n = 7, 171
405
spikes) that appeared as a separate group of events in the amplitude histograms (Fig. 6N).
406
Additionally, about half of the spikes exhibited sudden slope changes during their rise
407
(Fig. 6M, arrow) suggestive of a spike threshold at -50 ± 4 mV (n = 7, 97 spikes). 10-
408
90 % rise time of the spikes (including the rise of the initiating EPSPs) was 2.29 ± 0.75
409
ms and the spike half-width was 2.83 ± 0.86 ms (n = 7, 171 spikes).
410
19
411
HCN channel activity shortens the EPSP waveform in IHC afferent dendrites
412
Next, we tested whether Ih is involved in shaping the EPSP waveform. To block Ih, we
413
first applied CsCl, to allow for recovery of a possible effect. Without any cAMP analog
414
added, 2 mM CsCl significantly increased τdecay of the EPSP by 27 ± 17 % (from 6.37 ±
415
0.64 ms to 8.04 ±1.02 ms, and after washout to 6.98 ± 0.53 ms; n = 4 fibers, 617 EPSPs
416
analyzed, p < 0.05). Neither mean EPSP amplitude (control vs. CsCl: 13 ± 6 mV vs. 14 ±
417
8 mV, p = 0.839) nor 10-90 % rise time (1.31 ± 0.20 ms vs. 1.48 ± 0.20 ms, p = 0.146)
418
changed significantly. In the presence of cAMP analogs (200 µM cAMP intracellularly
419
and additionally 200 µM 8-Br-cAMP extracellularly), application of CsCl increased τdecay
420
by 37 ± 31 % (from 5.25 ± 1.37 ms to 7.15 ± 1.29 ms, n = 7, 878 EPSPs analyzed, p <
421
0.05) (Fig. 7 A-C). Again, EPSP mean amplitude (control vs. CsCl: 10 ± 4 mV vs. 11 ± 6
422
mV, p = 0.337) and 10-90 % rise time (1.23 ± 0.20 ms vs. 1.31 ± 0.30 ms, p = 0.376) did
423
not significantly change. Application of CsCl hyperpolarized the resting membrane
424
potential of the afferent dendrite by ~ 1 mV (from -64 ± 3 to -65 ± 3 mV, n = 11, p =
425
0.002).
426
Immature IHCs express inward rectifier potassium channels that can be inhibited by
427
extracellular Cs+ (Marcotti et al. 2003). Indeed, the frequency of synaptic events was
428
higher in CsCl (control versus CsCl: 1.01 ± 1.66 events/s vs. 2.23 ± 2.44 events/s, n = 16,
429
4357 EPSP or EPSCs analyzed, p=0.025). To exclude the possibility that CsCl by some
430
presynaptic effect changes the EPSC waveform and therefore affects the EPSP waveform,
431
we tested the effect of CsCl on EPSCs and found no significant difference compared to
432
control (control versus CsCl: EPSC mean amplitude: 159 ± 91 pA vs. 168 ± 99 pA, p =
433
0.838; 10-90 % rise time: 0.38 ± 0.14 ms vs. 0.38 ± 0.14 ms, p = 0.796; τdecay: 1.24 ± 0.40
20
434
ms vs. 1.43 ± 0.57 ms, p = 0.383; n = 3, 626 EPSCs analyzed). These data suggest that
435
changes in the EPSP waveform during CsCl application are due to its effect on the
436
afferent dendrite.
437
Application of the irreversible HCN channel blocker, ZD7288, exhibited larger effects on
438
the EPSP waveform than CsCl without changing the EPSP frequency (control versus
439
ZD7288: 0.73 ± 0.76 events/s versus 0.70 ± 0.71 events/s, p = 0.582) (Fig. 7D-F).
440
During application of 50 μM ZD7288 (in the presence of 200 µM cAMP intracellularly
441
and additionally 200 µM 8-Br-cAMP extracellularly), τdecay increased by 47 ± 24 % (from
442
4.22 ± 0.98 ms to 6.19 ± 1.67 ms, n = 6, 593 EPSPs analyzed, p = 0.002) and the resting
443
membrane potential was hyperpolarized by ~ 4 mV (from -63 ± 6 mV to -67 ± 7 mV,
444
p<0.001, n = 11). EPSP amplitude (8 ± 3 mV versus 9 ± 4 mV, p = 0.378) and 10-90%
445
rise time (1.40 ± 0.32 ms versus 1.49 ± 0.31 ms, p = 0.082) did not significantly change.
446
The Ih-induced hyperpolarization of the membrane potential could change the activity of
447
additional ion channels that also might affect the EPSP waveform as it has been shown in
448
a computational model of cochlear nucleus neurons (Rothman and Manis, 2003b) or in
449
recordings from dendrites of both hippocampal neurons (George et al. 2009) and frontal
450
cortex pyramidal neurons (Day et al. 2005). Therefore, to exclude possible effects on the
451
EPSP waveform by hyperpolarization, membrane potentials were manually reset during
452
ZD7288 application to their control values by constant current injection in 3 recordings.
453
A significant increase of 30 ± 11 % in τdecay compared to control was still observed (Fig.
454
7F, τdecay in control: 4.22 ± 0.98 ms; τdecay in ZD7288: 5.25 ± 1.51 ms, p = 0.03). This
455
result indicates that Ih shapes the EPSP waveform directly by contributing to the
456
membrane resting conductance. Both the application of CsCl and ZD7288 increased
21
457
τdecay over the entire range of EPSP amplitudes (Fig 7B, E). We conclude that the activity
458
of Ih in afferent dendrites shortens the EPSP waveform over the whole range of EPSP
459
amplitudes.
460
Application of ZD7288 also caused minor changes in the spike waveform. The spike
461
amplitude increased by 9 ± 4 % (from 32 ± 5 mV to 35 ± 4 mV, p = 0.026; n = 3, 155
462
spikes analyzed) and spike half-width increased by 14 ± 8 % (from 3.05 ± 0.53 ms to
463
3.45 ± 0.39 ms, p = 0.046). 10-90 % rise time did not change significantly (control
464
versus ZD7288: 1.24 ± 0.20 ms versus 1.29 ± 0.19 ms, p = 0.757).
465
466
Discussion
467
Properties of Ih in IHC afferent dendrites
468
Dendritic Ih has been shown to play an important role in setting firing rates in nerve fibers
469
(Magee and Johnston 2005). Here we have used whole-cell recordings to provide an
470
initial characterization of Ih present in postnatal IHC afferent dendrites. The
471
pharmacological profile of Ih, its reversal potential, its sensitivity to cAMP and the
472
expression of the HCN subunits 1, 2 and 4 in afferent dendrites indicate that Ih is
473
mediated by HCN channels. In immunolabeling experiments we found HCN1 and 4 in
474
postnatal afferent fibers and HCN1, 2 and 4 after hearing onset. No immunolabeling of
475
HCN3 above background was found in either the spiral ganglion or the afferent dendrites
476
at both ages, and therefore we cannot make a statement about the expression of HCN3.
477
In adult guinea pig spiral ganglion somata, the expression of all 4 HCN subunits has been
478
reported (Bakondi et al. 2009). The different result regarding HCN3 expression might be
479
due to a difference in the species or to the different antibodies used. No specific HCN1
22
480
or HCN2 labeling has been found in mouse cochlear hair cells and mice lacking HCN1,
481
HCN2 or both exhibited normal transduction currents (Horwitz et al. 2010). Similarly,
482
we found no specific HCN labeling in rat IHCs.
483
We found each HCN subunit in a high percentage of afferent dendrites. It is therefore
484
highly likely that heteromeric channels are formed. However, it is difficult to
485
unequivocally deduce the composition of HCN channels in afferent dendrites from the
486
properties of the recorded Ih. When measured in the same experimental conditions,
487
homomeric HCN1 channels activate at more positive voltages and show faster kinetics
488
than homomeric HCN2 and HCN4 channels. On the other hand, HCN1 shows the least
489
sensitivity to cyclic nucleotides, followed by HCN2 to 4 (Moosmang et al. 2001; Santoro
490
et al. 2000). Heteromeric HCN channels have been shown to exhibit intermediate
491
properties compared to homomeric channels (Chen et al. 2001; Ulens and Tytgat 2001).
492
Additionally, HCN channels with the same subunit composition exhibit variable
493
characteristics when measured in different expression systems or tissues (Moosmang et al.
494
2001; Santoro et al. 2000; Wainger et al. 2001). Knock-out animals for HCN1, 2, and 4
495
do exist, however, reports on their behavior do not mention any test of auditory function
496
(Harzheim et al. 2008; Herrmann et al. 2007; Ludwig et al. 2003; Nolan et al. 2004;
497
Nolan et al. 2003; Stieber et al. 2003).
498
A range of activation voltages and cyclic nucleotide sensitivities has been described for Ih
499
in studies on auditory neurons. For example, in bushy cells, a Vhalf of -94 to -84 mV and
500
positive shift of Vhalf with cAMP was found (Cuttle et al. 2001; Leao et al. 2006),
501
whereas in octopus cells, a Vhalf of -65 mV and no shift with cAMP was reported (Bal
502
and Oertel 2000). The properties we found for Ih in afferent dendrites are comparable to
23
503
those found for Ih in spiral ganglion somata. In afferent dendrites, Vhalf was -104 mV,
504
similar to Vhalf reported for spiral ganglion somata (-101 mV (Chen 1997); -78 to -122
505
mV (Mo and Davis 1997b). Both in spiral ganglion somata and afferent dendrites, Vhalf
506
shifted positively in the presence of cAMP analogues.
507
In control conditions, we found 3 %, and in the presence of cAMP analogues, 9 % of Ih
508
open at rest. The experimental conditions might underestimate the size of Ih in vivo: We
509
recorded at room temperature, and the amplitude of Ih has been found to be temperature
510
sensitive in auditory neurons (Cao and Oertel 2005; Cao et al. 2007; Rodrigues and
511
Oertel 2006). We recorded in immature cochleae, however, our immunolabeling
512
experiments suggest a maturation of HCN channel subunit expression in hearing animals.
513
For example, HCN2 labeling of afferent dendrites was found in 3 week old cochleae but
514
not before hearing onset. HCN2 has a higher sensitivity to cAMP compared to HCN1
515
and therefore properties of Ih may change with maturation. In our whole-cell recordings,
516
second messengers may be subject to wash-out, and therefore the Vhalf measured may be
517
more negative compared to in vivo conditions. Additionally, in in vivo conditions, input
518
from lateral efferent fibers may upregulate intradendritic cAMP levels (see last
519
paragraph), and in vitro transmitter release from these fibers may be absent or abnormal.
520
521
Role of Ih in auditory neurons
522
Multiple studies have shown that auditory neurons involved in processing timing
523
information express Ih. Examples include bushy cells (Cao et al. 2007; Leao et al. 2006;
524
Leao et al. 2005; Rothman and Manis 2003a), octopus cells (Bal and Oertel 2000; Cao
525
and Oertel 2005; Golding et al. 1999; Golding et al. 1995), and stellate cells (Rodrigues
24
526
and Oertel 2006; Rothman and Manis 2003b) of the cochlear nucleus, MSO neurons
527
(Scott et al. 2005), nucleus laminaris neurons (Kuba et al. 2005; Yamada et al. 2005),
528
lateral superior olive neurons (Barnes-Davies et al. 2004; Leao et al. 2006), MNTB
529
neurons (Banks et al. 1993; Cuttle et al. 2001; Leao et al. 2006), the calyx of Held (Cuttle
530
et al. 2001), and inferior colliculus neurons (Koch and Grothe 2003). Additionally, Ih
531
was found in the somata of spiral ganglion neurons (Chen 1997; Mo and Davis 1997b).
532
Ih can be partially active at rest, contributes to a lower membrane input resistance and
533
thereby shortens EPSPs. Shorter EPSPs reduce the time window of temporal summation
534
and therefore improve coincidence detection (Golding et al. 1995; Koch and Grothe
535
2003; Yamada et al. 2005). Similarly, Ih expressed in dendrites of non-auditory CNS
536
neurons shortened EPSPs and thereby reduced summation of synaptic activity (Day et al.
537
2005; Magee 2000; Magee and Johnston 2005). The non-uniform distribution of HCN
538
channels along dendrites compartmentalized distal dendrites from the somata (Berger et
539
al. 2003), limiting AP back-propagation, and thereby decreasing hyperexcitability (Tsay
540
et al. 2007; Ying et al. 2007). In motion-sensitive neurons in superior colliculus (Endo et
541
al. 2008), dendritic Ih was shown to keep AP timing at a minimum jitter and with short
542
latencies. It is not surprising that similar mechanisms might be employed at the IHC
543
afferent synapse, as discussed below.
544
545
Properties of EPSPs in afferent fibers and effects of Ih on EPSPs
546
Here we describe EPSP waveforms at the IHC afferent synapse. EPSPs have 10-90 %
547
rise times of ~1 ms and time constants of decay of ~5 ms. In comparison, the duration of
548
EPSPs recorded in vivo in guinea pig (Palmer and Russell 1986; Siegel 1992; Siegel and
25
549
Dallos 1986), was ~1-2 ms. The slower kinetics of EPSPs reported here could be due to
550
the immature age of the recorded dendrites. AMPA mediated EPSCs at IHC afferent
551
synapses speed up by a factor of two, when comparing the immature age used in this
552
study with 3 week old animals (Grant et al. in press). Also, our recordings were
553
performed at room temperature and AMPA receptor kinetics are temperature sensitive,
554
with a Q10 of > 2 (Postlethwaite et al. 2007). The amplitudes and gating kinetics of
555
voltage-gated ion channels such as Ih and IKL that are known to shorten EPSPs and APs in
556
auditory neurons are generally larger and faster in post-hearing animals (Scott et al. 2005)
557
and at body temperature (Cao and Oertel 2005; Cao et al. 2007; Rodrigues and Oertel
558
2006).
559
As shown for EPSCs (Glowatzki and Fuchs 2002; Grant et al. in press), EPSP amplitudes
560
covered an impressively wide range, between 1 and 35 mV. The median EPSP
561
amplitudes varied widely between fibers, between 2 and 14 mV. Spikes recorded in
562
afferent dendrites had a threshold at ~ -50 mV, and ~ 20% of EPSPs activated spikes.
563
Therefore, for small EPSPs, postsynaptic summation of EPSPs might be necessary to
564
reach the threshold for AP generation. In simultaneous recordings from hair cells and
565
afferent dendrites, summation of EPSCs was observed during the early response to step
566
depolarizations of the IHC membrane potential (Goutman and Glowatzki 2007). Our
567
results showed that EPSPs slowed down by 47 % when Ih was blocked by ZD7288.
568
These data suggest that depending on the level of Ih active, the time window of EPSP
569
summation may vary, allowing for regulation of firing rate in auditory nerve fibers.
570
Block of Ih with ZD7288 also induced a hyperpolarization of the membrane potential by
571
~ 4 mV. When the membrane potential was reset to control values during block of Ih,
26
572
EPSPs still slowed down by ~ 30 %. This result indicates that Ih shapes the EPSP
573
waveform directly by contributing to the resting membrane conductance. Ih may also act
574
indirectly on the EPSP waveform, by depolarizing the membrane potential and thereby
575
activating other ion channels like IKL that might additionally change the resting
576
membrane conductance (Rothman and Manis 2003b). However, our dataset here does
577
not prove or reject this scenario, as the changes in EPSP waveform during block of Ih
578
with and without hyperpolarization were not significantly different.
579
During block of Ih, and with a change in the resting membrane conductance, we expected
580
to see not only slowing of the EPSP waveform but also an increase in the EPSP
581
amplitude (Magee 1998). The EPSP amplitude increased slightly, however, not
582
significantly. This is most likely due to the wide range of EPSP amplitudes in individual
583
recordings. We suspect that if a larger dataset was available, the difference in amplitude
584
might have reached statistical significance. The fact that there was a small but significant
585
increase in the spike amplitude (representing a less variable waveform compared to the
586
EPSPs) further supports this idea.
587
588
HCN channels in afferent dendrites as a possible target for lateral efferent
589
transmission
590
IHC afferent dendrites receive efferent innervation from the lateral superior olivary
591
complex. Multiple transmitters such as acetylcholine, dopamine, gamma-amino butyric
592
acid, and opiate peptides, have been found in lateral efferent terminals (Eybalin, 1993).
593
Lesion of lateral efferent nerves disrupted temporal coding and increased susceptibility to
594
acute acoustic trauma (Darrow et al. 2006; 2007). Intracochlear perfusion of putative
27
595
lateral efferent neurotransmitters like dopamine affected firing rates in the auditory nerve
596
(Ruel et al. 2001). However, the cellular mechanisms underlying these modulatory
597
effects are unclear. One possibility is that lateral efferent transmitters modulate dendritic
598
ion channels like Ih, for example via second messenger cascades, thereby subsequently
599
affecting auditory nerve firing. Indeed, such modulation of Ih by a neurotransmitter has
600
been demonstrated in auditory neurons. In rat MNTB neurons, noradrenaline and cAMP
601
analogues increased the amplitude and shifted the activation curve of Ih (Banks et al.
602
1993). In chick nucleus laminaris, noradrenaline enhanced temporal precision of EPSPs
603
by regulating Ih activity in the postsynapse (Yamada et al. 2005). In addition to cyclic
604
nucleotides, phosphorylation by protein kinases, pH, and lipid second messengers have
605
been shown to modulate HCN channel activity (Fogle et al. 2007; Pian et al. 2007;
606
Robinson and Siegelbaum 2003; Zolles et al. 2006). Our finding that a cAMP-sensitive
607
Ih in afferent dendrites modulates the EPSP waveform, supports the idea that modulation
608
of temporal coding could occur directly at the first synapse of the auditory pathway,
609
possibly via lateral efferent inputs.
610
611
Acknowledgements
612
Monoclonal antibodies against HCN2 and HCN3 were obtained from UC
613
Davis/NINDS/NIMH NeuroMab Facility, supported by NIH grant U24NS050606 and
614
maintained by the Department of Pharmacolgy, School of Medicine, University of
615
California, Davis, CA 95616. We thank Robert H. Edwards’ laboratory for kindly
616
providing the antibodies against VGLUT3, John Gibas for his excellent technical
617
assistance with confocal microscopy and Lisa Grant for comments on the manuscript.
28
618
This work was supported by NIDCD DC006476 to EG, DC008860 to Dwight Bergles, a
619
research grant from the Deafness Research Foundation to EY, a EMBO fellowship ALTF
620
952-2006 to IR, NIH R24DK064388 to the Ross Confocal Facility, NIDCD P30
621
DC005211 to the Center for Hearing and Balance Histology Core.
622
29
623
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859
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861
40
862
Figure Legends
863
Figure 1. Current voltage relation recorded in an IHC afferent dendrite. Current
864
responses to voltage steps in the absence (A) and presence (B) of 1 µM TTX. Voltage
865
step protocol (inset in A): 200 ms voltage steps from -104 to -4 mV in 10 mV increments,
866
from a holding potential of -84 mV. A, Rapidly activating and inactivating sodium
867
currents that sometimes escaped the voltage clamp (expanded trace shown in inset) were
868
blocked by 1 μM TTX (B). C, Current responses at 20 ms (solid circle) and 200 ms
869
(open triangle) into the voltage steps after leak subtraction. A slowly activating inward
870
current (Ih) was found at voltage steps to -94 mV or more negative potentials. Fast
871
activating outward currents (within 5 ms) were activated at -64 mV and more positive
872
voltages.
873
874
Figure 2. Ih currents in afferent dendrites are mediated by HCN channels. A –C, In
875
afferent dendrite recordings, hyperpolarizing voltage steps from -84 to -124 mV were
876
applied every 10 s for 0.5 s (in 1 µM TTX). Current responses consisted of an
877
instantaneous inward current (Iinst) (most obvious in C) and a slowly developing inward
878
current (I0.5). A-C show representative traces before, during and after application of 2
879
mM CsCl, 2 mM BaCl2 or 50 µM ZD7288. Fast inward deflections during the recordings
880
in A to B represent EPSCs. D, E, Diary plots of the Ih amplitude during application of
881
drugs. Ih amplitude was measured as I0.5.-Iinst. The effects of CsCl and BaCl2 were mostly
882
reversible; the effect of ZD7288 was irreversible. F, Percent reduction in Ih amplitude
883
during CsCl, BaCl2 and ZD7288 application. The Ih amplitudes were determined from
884
mean values of 5 consecutive traces in each condition. To compensate for rundown of Ih,
41
885
current amplitudes in 2 mM CsCl or 2 mM BaCl2 were compared to mean value of
886
respective control and recovery. The Ih amplitude in 50 µM ZD7288 was compared to
887
control.
888
889
Figure 3. Properties of Ih in afferent dendrites. A, B, Ih currents in response to voltage
890
steps (every 10 s for 3 s, from a holding potential of -64 mV to voltages between -144
891
mV and -54 mV in 10 mV increments; see inset). External solution with: TTX (1-2 µM),
892
4-AP (2 mM), TEA (10-30 mM), and CNQX (10 µM). A, In control solution. B, with
893
200 µM cAMP intracellularly and additionally 200 µM 8-Br-cAMP extracellularly. C,
894
Current voltage relations in control (n = 3, black) and with cAMP analogues (n = 4, red).
895
D, Voltage dependence of Ih measured from tail currents. Tail current amplitudes were
896
normalized and fit with a Boltzmann equation. Vhalf and slope factors were -104 ± 3 mV,
897
11 ± 1 in control (n = 3, black) and -91 ± 2 mV, 11 ± 1 in cAMP analogues (n = 4, red).
898
E, Activation kinetics of Ih currents. Current responses to voltage steps from -134 mV to
899
-104 mV were fit with 2 exponentials, providing two time constants (τfast, τslow). Both
900
time constants were significantly faster for currents recorded with cAMP analogues (n =
901
4, red) compared to control (n = 3, black). F, Reversal potential of Ih. Conditioning
902
voltages were applied for 3 s (to -124 mV, -104 mV or -84 mV), followed by 10 ms
903
voltage ramps (from -144 mV to -74 mV) (upper traces: voltage commands, lower traces:
904
current responses to the commanding voltage ramps). The reversal potential was -45.5
905
mV for this recording.
906
42
907
Figure 4. HCN subunit expression pattern in the rat cochlea at P9. Three-dimensional
908
reconstruction of confocal images from cochlear whole-mount preparations, apical turns.
909
A, HCN1 labeling (green). B, HCN4 labeling (green). Vesicular glutamate transporter
910
VGLUT3 (red) was used as a marker for IHCs. HCN1 and HCN4 labeling was found in
911
the inner spiral plexus (isp) under the row of IHCs (ihc, arrowhead) as well as in the
912
somata of spiral ganglion neurons (sgn). Scale bars: 50 μm.
913
914
Figure 5. HCN subunits are localized in afferent dendrites. A, Three-dimensional
915
reconstruction of calretinin and HCN1-labeled whole-mount rat organ of Corti
916
preparation, apical turn at P9. HCN1 labeling (green) is concentrated in the baso-lateral
917
region of the IHCs. Calretinin (red) labels IHCs (ihc) and afferent dendrites. B-D, Close
918
up view showing single confocal laser-scanning micrographs. As seen in the merged
919
view (D), HCN1 (B) and calretinin (C) immunolabeling overlap in some afferent
920
dendrites (arrowheads). E-M, Single confocal laser-scanning micrographs of whole-
921
mount organs of Corti preparations, apical turns at P21. Preparations were colabeled for
922
HCN1, HCN2 or HCN4 (green) and calretinin (red). Arrowheads indicate examples of
923
double-labeled afferent dendrites. Note examples of ring-like HCN labeling surrounding
924
calretinin labeling (open arrowheads). Some fibers were labeled for HCN but not for
925
calretinin (asterisks). Scale bars: 5 µm.
926
927
Figure 6. EPSCs and EPSPs recorded at the IHC afferent synapse. A-K, Whole-cell
928
recording from an afferent dendrite in the presence of 1 μM TTX showing EPSCs (A, B)
929
(holding potential -94 mV) and EPSPs (E, F). B, F, overlaid representative traces of
43
930
monophasic EPSCs and EPSPs on an expanded time scale. C-D, G-H, 10-90 % rise time
931
(rise) or decay time constants (τdecay) plotted against the EPSC or EPSP amplitude. Rise
932
and τdecay for EPSCs were 0.33 ± 0.14 ms and 1.24 ± 0.20 ms (324 EPSCs analyzed).
933
Rise and τdecay for EPSPs were 0.96 ± 0.12 ms and 3.81 ± 0.36 ms (241 EPSPs analyzed).
934
EPSP waveforms remained relatively invariable over the wide range of EPSP amplitudes.
935
I-K, EPSP amplitude distributions (bin size 1 mV) from 3 afferent dendrite
936
recordings. Median EPSP amplitudes were 2.3 mV, 2.4 mV, and 13.8 mV, and resting
937
membrane potentials were -75 mV, -56 mV and -68 mV, respectively. The number of
938
events analyzed is indicated in each panel. L-N, Whole-cell current clamp recording in
939
the absence of TTX. A mixture of EPSPs and spikes was observed. M, overlaid
940
representative traces of spikes on an expanded time scale. Spike threshold (arrow) was -
941
47 mV for this recording. N, Amplitude distribution (bin size 1mV). A wide gap in
942
amplitude histogram distinguishes spikes from EPSPs.
943
944
Figure 7. Ih shortens EPSPs in afferent dendrites. A-H, Whole-cell current clamp
945
recording from afferent dendrites. Recordings were done in the presence of cAMP
946
analogues (200 µM 8-Br-cAMP extracellularly and additionally 200 µM cAMP
947
intracellularly). A-C, EPSP waveform before and while blocking Ih with 2 mM CsCl. A,
948
Average EPSP waveform before (black) and during application of 2 mM CsCl (red). B,
949
EPSP decay time constants (τdecay) plotted against EPSP amplitudes before and while
950
blocking Ih with 2 mM CsCl; control: τdecay = 4.97 ms (black, 34 EPSPs), in 2mM CsCl:
951
τdecay= 7.18 ms (red, 41 EPSPs). C, Summarized results from 7 recordings. D-F, EPSP
952
waveform before and during application of 50 µM ZD7288. D, Average EPSP waveform
44
953
before (black) and during application of 50 µM ZD7288 (magenta). E, EPSP decay time
954
constants (τdecay) plotted against EPSP amplitudes; control: τdecay = 5.38 ms (black, 22
955
EPSPs), in 50 µM ZD7288: τdecay= 8.30 ms (magenta, 16 EPSPs). F, Summarized results
956
from 6 recordings.
957
958
Supplemental Figure 1. Fluorescent dye-filled auditory nerve fiber. Alexa Fluor 488
959
hydrazide (10 μM) was included in the pipette solution during a whole-cell patch-clamp
960
recording. The recording pipette was located at the terminal of the afferent fiber, close to
961
its contact with the IHC. A, The fiber (in green) could be traced from the IHC region for
962
a distance of ~ 400 μm, until close to its entrance point into the spiral ganglion (SGN).
963
Rows of inner (IHCs) and outer hair cells (OHCs) near the recording site are marked by
964
arrows. B, the same tissue as in (A) viewed in transmitted light. The dotted line marks
965
the attachment of Reissners membrane for orientation. Scale bar: 100 μm.
966
967
Supplemental Figure 2. HCN subunit expression in spiral ganglion neurons. Single
968
confocal laser-scanning micrographs of whole-mount rat cochlea preparations. At P9-10,
969
HCN1 and HCN4, but not HCN2 immunolabeling was detected in spiral ganglion
970
neurons. At P20-21, spiral ganglion neurons were positive for HCN1, HCN2 and HCN4
971
labeling. At both ages, a strong labeling was found in the plasma membrane of the
972
somata and of the most proximal part of the neurites. Scale bars: 10 µm.
B
I (nA)
I (nA)
0.5
0
-0.5
-4
-1.5
0.4
0.3
0.4
0.2
0
-104
3 ms
C
1 μM TTX
-44
-84
-1
0.6
I (nA)
A
50 ms
-0.2
Ih
50 ms
0.2
0.1
0
-0.1
-80
-40
V (mV)
0
Figure 1
$
0
-100
2 mM CsCl
recovery
control
-200
-300
0
I0.5-Iinst (pA)
I (pA)
A
-200
CsCl
I0.5-Iinst (pA)
I (pA)
E
-100
2 mM BaCl2
recovery
control
-200
50 μM
ZD7288
control
Iinst
-200 200 ms
I0.5s
% Block
I (pA)
100
-100
ZD7288
-40
F
0
600
0
-80
-300 200 ms
C
400
200
time (s)
0
0
BaCl2
-100
200 ms
B
CsCl
0
400
200
time (s)
600
n=4
n=8
50
n=4
0
CsCl BaCl2 ZD7288
Figure 2
control
cAMP+8-Br-cAMP
B
0
-200
-200
-400 Iinst -64
Af/(Af+As)
Tfast (s)
Tslow (s)
-400
I3s
2
3
time (s)
-600
0
4
1
2
time (s)
3
4
D
0
1.0
(I-Imin)/(Imax-Imin)
I3s- Iinst (pA)
1
-200
-400
-140
E
-74
mV
-144
-600
0
C
-54
I (pA)
0
-120 -100
V (mV)
-80
0.5
0
-150
-120
-90
V (mV)
-74
-84
-104
-124
0.5
0.2
-144
0
0.1
0
4
-84
-200 -104
2
0
-140
-60
F
1.0
I (pA)
I (pA)
A
-124
-120
V (mV)
-100
-400
-120
-80
V (mV)
-40
0
Figure 3
I (pA)
-350
100 pA
3 ms
10 s
F
-60
5 mV
3 ms
-70
-80
n=57
0
200
n=676
100
0
G
H
30
30
n=109
20
20
10
10
0
0
100
n=307
50
0
N
10 mV
-40
3 ms
-60
0
3 0
3
6
9
Tdecay (ms)
20
n=417
10
0
10
20
30
40
EPSP amplitude (mV)
M
-20
1
2
rise (ms)
3
6
9
Tdecay (ms)
K
0
10
30
40
20
EPSP amplitude (mV)
0
1
2
3 0
rise (ms)
0
J
0
V (mV)
400
400
number of events
number of events
I
L
800
10 s
number of events
V (mV)
E -50
D
800
0
10
20
30
40
EPSP amplitude (mV)
30
number of events
-700
C
EPSC amplitude (pA)
B
0
EPSP amplitude (mV)
A
n=100
20
10
0
30 s
0
10 20 30 40 50
EPSP amplitude (mV)
Figure 6
A
D
ZD7288
CsCl
5 ms
B
5 ms
E 15
amplitude (mV)
amplitude (mV)
20
10
5
0
0
6
Tdecay (ms)
C
*
8
n=7
150
6
F
*
150
100
12
Tdecay (ms)
18
n=6
Tdecay (% of control)
4
Tdecay (% of control)
10
n=3
*
n=6
100
50
0
control
CsCl
recovery
50
0
control ZD7288 ZD7288
+ Iinj
Figure 7
A
B
OHCs
IHCs
SGN
OHCs
IHCs
SGN
Supplimental figure 1