J Neurophysiol 103: 2532–2543, 2010.
First published March 10, 2010; doi:10.1152/jn.00506.2009.
Dendritic HCN Channels Shape Excitatory Postsynaptic Potentials at the
Inner Hair Cell Afferent Synapse in the Mammalian Cochlea
Eunyoung Yi, Isabelle Roux, and Elisabeth Glowatzki
Department of Otolaryngology–Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, Maryland
Submitted 10 June 2009; accepted in final form 8 March 2010
INTRODUCTION
To perform tasks such as the localization of sound in space,
neurons in the auditory pathway are specialized to accurately
preserve timing information within sound signals (Oertel 1999;
Trussell 1999). Several pre- and postsynaptic mechanisms
enabling rapid and reliable transmission at auditory synapses
have been described. Presynaptically, large calyceal structures
release vesicles from many release sites synchronously, resulting in large excitatory postsynaptic potentials (EPSPs) that
reliably activate action potentials (APs) (Schneggenburger and
Forsythe 2006). Postsynaptically, rapid kinetics of ␣-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors result in brief excitatory postsynaptic currents (EPSCs)
(Gardner et al. 1999, 2001; Parks 2000; Raman et al. 1994).
Voltage-gated ion channels active near the resting membrane
potential decrease the membrane resistance of the postsynaptic
neurons and shorten the membrane time constant (Magee and
Johnston 2005). This mechanism keeps EPSPs brief and preAddress for reprint requests and other correspondence: E. Glowatzki, The
Johns Hopkins School of Medicine, Department of Otolaryngology–Head and
Neck Surgery, 720 Rutland Avenue, Ross 824, Baltimore, MD 21205 (E-mail:
[email protected]).
2532
vents temporal summation of synaptic events. A hyperpolarization-activated cation channel (Ih) has been described in
auditory brain stem neurons as one of the ion channels serving
this role (Banks and Smith 1992; Golding et al. 1995; Rothman
and Manis 2003b). To further shape EPSPs, auditory neurons
can receive modulatory inputs that either open receptor-coupled ion channels (Funabiki et al. 1998; Smith et al. 2000) or
regulate Ih via G protein coupled signaling pathways (Banks et
al. 1993; Yamada et al. 2005).
The first synapse in the auditory pathway, the synapse
between the inner hair cell (IHC) and auditory nerve fiber, also
uses highly specialized mechanisms to preserve timing information (Fuchs 2005; Glowatzki et al. 2008; Moser et al. 2006).
The auditory nerve fiber receives input from only one IHC via
a single dendrite and large EPSCs are activated by multivesicular release at this ribbon-type synapse (Glowatzki and
Fuchs 2002; Goutman and Glowatzki 2007; Grant et al. 2010;
Keen and Hudspeth 2006; Li et al. 2009). Similar to EPSCs
recorded from auditory brain stem synapses, AMPA-mediated
EPSCs at this synapse are brief (Glowatzki and Fuchs 2002;
Grant et al. 2010). However, not much is known regarding the
expression pattern of voltage-gated ion channels in afferent
dendrites and their involvement in shaping postsynaptic activity. In a first survey, using voltage-clamp recordings from
afferent dendrites of the postnatal rat cochlea, we identified
several voltage-gated conductances in afferent dendrites (Na⫹,
Ca 2⫹, K⫹ conductances) including a hyperpolarization-activated conductance.
Here we focus on the characterization of Ih in IHC afferent
dendrites. We find that Ih is mediated by HCN channels. Ih is
active at rest and is modulated by intracellular levels of cyclic
adenosine monophosphate (cAMP). Ih shortens the EPSP and
is thus a good candidate for enabling rapid and reliable signaling at this first synapse in the auditory pathway.
METHODS
Animal protocols were approved by the Johns Hopkins University
Animal Care and Use Committee. Rats (Sprague–Dawley; Charles
River, Wilmington, MA) were anesthetized (pentobarbital 0.045
mg·g⫺1, administered intraperitoneally or by isoflurane inhalation)
and decapitated and cochleae were quickly removed from temporal
bones.
Electrophysiological recordings
Excised apical cochlear turns of 7- to 14-day-old rats were placed
into a chamber under an upright microscope (Axioskop2 FS plus,
Zeiss, Oberkochen, Germany) and superfused with external solution
at 1–3 ml/min (chamber volume ⬃2 ml). IHCs and contacting afferent
dendrites were visualized on a monitor via a ⫻40 water immersion
0022-3077/10 Copyright © 2010 The American Physiological Society
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Yi E, Roux I, Glowatzki E. Dendritic HCN channels shape excitatory
postsynaptic potentials at the inner hair cell afferent synapse in the
mammalian cochlea. J Neurophysiol 103: 2532–2543, 2010. First
published March 10, 2010; doi:10.1152/jn.00506.2009. Synaptic
transmission at the inner hair cell (IHC) afferent synapse, the first
synapse in the auditory pathway, is specialized for rapid and reliable
signaling. Here we investigated the properties of a hyperpolarizationactivated current (Ih), expressed in the afferent dendrite of auditory
nerve fibers, and its role in shaping postsynaptic activity. We used
whole cell patch-clamp recordings from afferent dendrites directly
where they contact the IHC in excised postnatal rat cochlear turns.
Excitatory postsynaptic potentials (EPSPs) of variable amplitude
(1–35 mV) were found with 10 –90% rise times of about 1 ms and
time constants of decay of about 5 ms at room temperature. Current–
voltage relations recorded in afferent dendrites revealed Ih. The
pharmacological profile and reversal potential (⫺45 mV) indicated
that Ih is mediated by hyperpolarization-activated cyclic nucleotidegated cation (HCN) channels. The HCN channel subunits HCN1,
HCN2, and HCN4 were found to be expressed in afferent dendrites
using immunolabeling. Raising intracellular cAMP levels sped up the
activation kinetics, increased the magnitude of Ih and shifted the half
activation voltage (Vhalf) to more positive values (⫺104 ⫾ 3 to ⫺91
⫾ 2 mV). Blocking Ih with 50 ␮M ZD7288 resulted in hyperpolarization of the resting membrane potential (⬃4 mV) and slowing the
decay of the EPSP by 47%, suggesting that Ih is active at rest and
shortens EPSPs, thereby potentially improving rapid and reliable
signaling at this first synapse in the auditory pathway.
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
J Neurophysiol • VOL
Cfast is used to estimate cell capacitance. This happens most likely
because in current-clamp mode the effective cell capacitance is not
limited to Cfast but also includes Cslow.
Liquid junction potentials (4 mV for KCl-based and 9 mV for
K-methanesulfonate-based pipette solution) were corrected off-line.
Data were analyzed off-line using pClamp version 9.2 (Axon Instruments, Union City, CA), Minianalysis (Synaptosoft, Decatur, GA),
and Origin 7.5 (OriginLab, Northampton, MA). For statistical comparisons Sigmastat 3.5 (Systat Software, San Jose, CA) was used.
Statistical significances of irreversible drug effects (ZD7288) were
tested using a paired t-test. Effects of ZD7288 on the EPSP waveform
(measurements were taken at three different conditions) were tested
using one-way repeated measures ANOVA followed by Student–
Newman–Keuls test. Effects of reversible drugs (CsCl and BaCl2)
were tested using one-way repeated measures ANOVA followed by
Student–Newman–Keuls test. Effects of cAMP analogs on Ih amplitude and activation kinetics were tested using two-way repeated
measures ANOVA one factor repetition. Effects of cAMP on the Vhalf
and slope factor of Ih activation curves were tested using Student’s
t-test. Values are presented as means ⫾ SD.
Immunolabeling
Cochleae from 9- to 10- and 20- to 21-day-old rats (P9 –P10,
P20 –P21) were perfused through the round and oval windows with
cold 4% paraformaldehyde prepared in phosphate buffered saline
(PBS), pH 7.4, and then postfixed for 1 h at 4°C under agitation.
Additionally, for HCN1 immunodetection, cochleae were rinsed three
times in PBS and incubated 15 min in methanol at ⫺20°C. Thereafter,
preparations were washed three times in PBS and the cochleae were
microdissected to facilitate access of the antibodies to the tissue.
Whole-mount preparations were incubated for 1 h at room temperature in a blocking and permeabilizing solution (PBS with 20% of
either normal goat serum or donkey serum and 0.3% Triton X-100)
and were then incubated overnight at 4°C with the primary antibodies
diluted in the same solution. After three 15 min washes in PBS,
samples were incubated for 1 h at room temperature with fluorescently
labeled secondary antibodies diluted at 1:800 in PBS with 10% of
either normal goat serum or donkey serum and 0.15% Triton X-100.
Samples were then rinsed once in PBS with 10% of either normal goat
serum or donkey serum and 0.15% Triton X-100 and twice with PBS
(15 min each, at room temperature) before the organs of Corti were
mounted on slides using FluorSave mounting medium (Calbiochem,
San Diego, CA). Specific labeling was initially examined with an
Axio Observer inverted microscope (Zeiss) and further detailed images were obtained using a confocal laser scanning microscope (LSM
510 META, Zeiss) with ⫻20 air and ⫻100 oil objectives (optical
section steps of 0.25 and 0.20 microns, respectively). Analysis and
reconstruction were carried out using LSM Image Examiner (Zeiss)
and Volocity 4.2.1 software (Improvision, Waltham, MA). No labeling was observed when the primary antibodies were omitted. Again,
no labeling was observed when the primary antibodies were preabsorbed onto target peptides, except in the stereocilia of the sensory
hair cells, which displayed staining using HCN1 and HCN4 antibodies, when the target peptides were used at the recommended concentration or at a 25-fold (HCN1) or 5-fold (HCN4) higher concentration.
For HCN2, no test for preabsorption with a peptide was performed.
For HCN3 a shorter fixation (15 min) was also tested.
Antibodies
Rabbit polyclonal antibodies against HCN1 and HCN4 (Alomone)
were used at dilutions of 1:200 and 1:400, respectively. Rabbit
polyclonal antibodies against HCN3 (Alpha Diagnostic International,
San Antonio, TX) were used at dilutions of 1:200 to 1:20. Contrary to
the antibodies against HCN3 from Chemicon (Temecula, CA) and
Alomone, this antibody does not show cross reactivity for hHCN1,
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objective, ⫻4 magnification, differential interference contrast optics
using a green filter, and a NC 70 Newvicon camera (Dage, MTI,
Michigan City, IN). The pipette solution consisted of (in mM): 135
KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 HEPES, and 0 –2.5 Na2ATP;
or 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 HEPES, 4 Na2ATP,
and 0.2 Na2GTP; 290 mOsm, pH 7.2 (KOH). In some recordings in
which the effect of cAMP was tested, the pipette solution contained
(in mM):131 KCl, 1 MgCl2, 5 EGTA, 5 HEPES, 5 Na2ATP, and 10
Na2-phosphocreatine. In addition, a small number of recordings (n ⫽
6) was performed using a pipette solution containing (in mM): 110
K-methanesulfonate, 20 KCl, 5 EGTA, 5 HEPES, 0.1 CaCl2, 5
Na2-phosphocreatine, 4 MgATP, and 0.3 Tris-GTP. No significant
differences were found in basic membrane properties such as input
resistance and membrane time constant of the afferent fiber between
recordings with KCl- or K-methanesulfonate-based pipette solutions.
The external solution consisted of (in mM): 5.8 KCl, 155 NaCl, 1.3
CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 glucose, and 10 HEPES; 300
mOsm, pH 7.4 (NaOH). Drugs were dissolved daily in the external
solution to their final concentrations from frozen stocks. Application
of drug solutions was performed using a gravity-driven flow pipette
(100 ␮m diameter) placed near the row of IHCs, connected with a
VC-6 channel valve controller (Warner Instruments, Hamden, CT).
4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and
4-aminopyridine (4-AP) were purchased from Tocris Bioscience (Ellisville, MO) and tetrodotoxin (TTX) either from Alomone (Jerusalem, Israel) or Sigma (St. Louis, MO). All other chemicals were
purchased from Sigma.
Recording pipettes were fabricated from 1 mm borosilicate glass
(WPI, Sarasota, FL). Pipettes were pulled with a multistep horizontal
puller (Sutter Instrument, San Rafael, CA) and fire-polished (10 –15
M⍀). Pipettes were coated with Sylgard (Dow Corning, Midland,
MI). Experiments were done at 22–25°C. Recordings were performed
with a Multiclamp 700A or 700B amplifier (Molecular Devices,
Sunnyvale, CA), pClamp version 9.2, and a Digidata 1322A board,
digitized at 50 kHz, and filtered at 10 kHz.
In voltage-clamp mode, series resistance (Rs) was calculated from
capacitative current responses to a 10 mV voltage step (⫺84 to ⫺94
mV). The capacitative current responses were fitted with a sum of two
or more exponential equations. The fastest component of the fit was
considered to represent the capacitative current for the membrane area
near the recording electrode (Cfast) and the slower components for
current spreading further along the afferent nerve fiber (Cslow) (Llano
et al. 1991). From the fastest component, the voltage-clamp time
constant (36 ⫾ 12 ␮s, n ⫽ 38), membrane capacitance Cfast (1.32 ⫾
0.43 pF), and Rs (30 ⫾ 12 M⍀) were derived. Voltage-clamp data
were discarded if Rs was ⬎50 M⍀. Most Ih currents were ⬍300 pA
and, with Rs at about 30 M⍀, the estimated voltage error was ⬍9 mV.
Assuming that the dendrite is formed like a cylinder and with a
specific capacitance of 1 ␮F/cm2 and a diameter of 1 ␮m (most likely
an overestimation), the first component corresponds to the dendrite at
a length of about 40 ␮m. This distance covers the extent of the HCN
channel expression along the terminal; the HCN specific labeling
ceases in the region of the first heminode of the peripheral dendrite,
about 50 ␮m away from the afferent contacts with the IHC. Synaptic
currents are generated within ⬍3 ␮m of the pipette tip as the tip is
directly positioned on the bouton ending. Therefore voltage-clamp
conditions for recording synaptic currents and HCN channels should
be sufficient. The input resistance (Rin) of afferent dendrites (394 ⫾
253 M⍀, n ⫽ 173) was determined in voltage clamp, with voltage
steps from ⫺64 to ⫺84 mV.
In current-clamp mode, errors due to Rs were compensated using
bridge balance and pipette capacitance neutralization. Membrane
voltage responses to ⫺10 pA current steps were fitted with a monoexponential equation and provided a membrane time constant (␶m) of
3.97 ⫾ 1.81 ms (n ⫽ 10). ␶m measured in current-clamp mode is
larger than ␶m estimated from voltage-clamp data (0.5 ms) when only
2533
2534
E. YI, I. ROUX, AND E. GLOWATZKI
hHCN2, or hHCN4 (Kouranova et al. 2008). Monoclonal antibodies
against HCN2 and HCN3 from UC Davis/National Institute of Neurological Disorders and Stroke (NINDS)/National Institute of Mental
Health (NIMH) NeuroMab Facility (Davis, CA) were used at 1:25.
Mouse monoclonal and rabbit polyclonal antibodies against recombinant rat calretinin (Chemicon) were diluted at 1:1,000. Guinea-pig
serum against VGLUT3 was kindly provided by Dr. Robert H.
Edwards’ laboratory (Department of Physiology, School of Medicine,
University of California, San Francisco) and used at a 1:1,000 dilution. Alexa Fluor 488 F(ab=)2 fragment of goat anti-rabbit and Alexa
Fluor 488 donkey anti-mouse IgG, Alexa Fluor 555 goat anti-mouse
and Alexa Fluor 594 donkey anti-rabbit IgG, and Alexa Fluor 633
goat anti-guinea pig IgG (Molecular Probes, Eugene, OR) were used
as secondary antibodies.
RESULTS
To characterize voltage-gated conductances in IHC afferent
dendrites, we used whole cell recordings from afferent dendrites directly where they contact the IHC. Recordings were
performed in acutely excised apical cochlear turns from 7- to
14-day-old rats at room temperature. Resting membrane potentials of afferent dendrites were typically about ⫺64 mV. We
assume that the peripheral neurites of the recorded auditory
nerve fibers were intact and connected with their spiral ganglion somata for the following reasons: First, in preparations
where we separated the spiral ganglion from the cochlear coil,
afferent dendrites were severely swollen and no recordings
could be achieved. Second, in nine of nine experiments, where
a fluorescent dye, Alexa Fluor 488 hydrazide salt (10 ␮M), had
been included in the pipette solution and recordings had lasted
⬎10 min, the unbranched afferent fibers could be traced back
from the IHC toward the spiral ganglion for 200 –500 ␮m
(Supplemental Fig. S1)1 and, in two cases, the fluorescent
marker had reached the spiral ganglion somata at a distance of
450 –500 ␮m from the IHC.
Our study focused on the characterization of Ih. However,
because voltage-gated conductances have not been described
for IHC afferent dendrites, in the following paragraph we will
briefly summarize the different conductances that were observed in response to voltage step protocols. From a holding
potential of ⫺84 mV, voltage steps between ⫺104 and ⫺4 mV
were applied (Fig. 1A). Voltage-gated sodium currents were
The online version of this article contains supplemental data.
A
B
0.6
I (nA)
I (nA)
0.5
0
-0.5
-4
-1.5
0.4
0.3
0.4
0.2
0.2
0.1
0
-104
3 ms
C
1 µM TTX
-44
-84
-1
To test whether Ih currents are mediated by HCN channels,
we monitored Ih during the application of different blockers. In
response to repeatedly applied negative voltage steps from
⫺84 to ⫺124 mV, an instantaneous inward current (Iinst) was
followed by a slowly developing inward current (I0.5s) (Fig. 2C).
Iinst is partially blocked by Ih blockers and consists of a mixture
of Ih and other conductances (see Fig. 2, A–C) (Bal and Oertel
I (nA)
1
Ih in afferent dendrites is mediated by HCN channels
0
Ih
50 ms
-0.2
50 ms
-0.1
-80
-40
0
V (mV)
FIG. 1. Current–voltage (I–V) relation recorded in an inner hair cell (IHC) afferent dendrite. Current responses to voltage steps in the absence (A) and the
presence (B) of 1 ␮M tetrodotoxin (TTX). Voltage step protocol (inset in A): 200 ms voltage steps from ⫺104 to ⫺4 mV in 10 mV increments, from a holding
potential of ⫺84 mV. A: rapidly activating and inactivating sodium currents that sometimes escaped the voltage clamp (expanded trace shown in inset) were
blocked by 1 ␮M TTX (B). C: current responses at 20 ms (solid circle) and 200 ms (open triangle) into the voltage steps after leak subtraction. A slowly activating
inward current (i.e., a hyperpolarization-activated current [Ih]) was found at voltage steps to ⫺94 mV or more negative potentials. Fast activating outward currents
(within 5 ms) were activated at ⫺64 mV and more positive voltages.
J Neurophysiol • VOL
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Voltage-gated ion channels in IHC afferent dendrites
found in 114 of 155 afferent dendrite recordings. Sodium
currents often “escaped” the voltage clamp (Fig. 1A, inset).
This is not surprising because the recording site is at the very
tip of the unmyelinated afferent fiber ending and the AP
initiation site is most likely located further away along the
myelinated peripheral process (Hossain et al. 2005; LacasGervais et al. 2004; McLean et al. 2009). For this study sodium
channels were not further investigated, but rather blocked with
1–2 ␮M TTX (Fig. 1B). Additionally, small calcium currents
that could be blocked with 200 ␮M CdCl2 were detected in
some recordings (data not shown). Because these small currents did not interfere with questions asked in this study, no
effort was made to block them.
During depolarizing voltage steps, outward currents were
observed in all 161 afferent dendrites recorded (Fig. 1). Outward currents reached their maximum within about 5 ms. The
current–voltage (I–V) relations showed that outward currents
activated at potentials as low as ⫺64 mV (Fig. 1, B and C). We
tested the effects of 4-AP (2– 4 mM) and tetraethylammonium
(TEA, 10 –30 mM), drugs previously shown to inhibit the
low-voltage activating potassium current (IKL) and the highvoltage activating potassium current (IKH), respectively, in
spiral ganglion neurons (Szabo et al. 2002) and auditory brain
stem neurons (Bal and Oertel 2001; Brew and Forsythe 1995;
Cao et al. 2007; Manis and Marx 1991; Rathouz and Trussell
1998; Reyes et al. 1994; Rothman and Manis 2003a). 4-APsensitive and TEA-sensitive currents were observed in afferent
dendrites and exhibited similar voltage dependent profiles to
IKL and IKH, respectively. These conductances are still under
investigation.
During hyperpolarizing voltage steps, a slowly developing
inward current was found in 69 of 79 afferent recordings (Fig.
1, B and C). Voltage dependence and activation kinetics of this
inward current were reminiscent of Ih recorded in dissociated
spiral ganglion somata (Chen 1997; Mo and Davis 1997b).
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
-200
-200 200 ms
I0.5s
n=4
n=8
CsCl BaCl2 ZD7288
FIG. 2. Ih currents in afferent dendrites are mediated by hyperpolarizationactivated cyclic nucleotide-gated cation (HCN) channels. A–C: in afferent
dendrite recordings, hyperpolarizing voltage steps from ⫺84 to ⫺124 mV
were applied every 10 s for 0.5 s (in 1 ␮M TTX). Current responses consisted
of an instantaneous inward current (Iinst) (most obvious in C) and a slowly
developing inward current (I0.5). A–C: representative traces before, during, and
after application of 2 mM CsCl, 2 mM BaCl2, or 50 ␮M ZD7288 (4ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride). Fast
inward deflections during the recordings in A to B represent excitatory
postsynaptic currents (EPSCs). D and E: diary plots of the Ih amplitude during
application of drugs. Ih amplitude was measured as I0.5 ⫺ Iinst. The effects of
CsCl and BaCl2 were mostly reversible; the effect of ZD7288 was irreversible.
F: percentage reduction in Ih amplitude during CsCl, BaCl2, and ZD7288
application. The Ih amplitudes were determined from mean values of 5
consecutive traces in each condition. To compensate for rundown of Ih, current
amplitudes in 2 mM CsCl or 2 mM BaCl2 were compared with mean value of
respective control and recovery. The Ih amplitude in 50 ␮M ZD7288 was
compared with control.
2000; Rodrigues and Oertel 2006). We therefore report only on
the amplitude of the slowly developing component of Ih (I0.5s ⫺
Iinst). Ih currents showed some run-down during whole cell
recordings and test protocols were designed accordingly. CsCl
(2 mM) reversibly inhibited inward currents by 84 ⫾ 11% (n ⫽
8), whereas 2 mM BaCl2 caused a reversible inhibition by
14 ⫾ 8% (n ⫽ 4) (Fig. 2, A, B, D, E, and F). This combination
of a strong Cs⫹ block and a weak Ba2⫹ block has been
described for HCN channels, whereas inward rectifier potassium channels typically show a substantial Ba2⫹ block (Kubo
et al. 2005; Robinson and Siegelbaum 2003; van Welie et al.
2005). Additionally, 50 ␮M ZD7288, an antagonist of HCN
channels (Shin et al. 2001), irreversibly inhibited Ih by 93 ⫾ 13%
(n ⫽ 4) (Fig. 2, C, E, and F).
The reversal potential of Ih was estimated using the following protocol: different conditioning voltages were applied for 3
s (⫺124, ⫺104, and ⫺84 mV), followed by 10 ms voltage
ramps (from ⫺144 to ⫺74 mV) (Fig. 3F). The initial current
response (⬃3 ms) during the voltage ramp was discarded due
to contamination with uncompensated capacitance currents.
The linear portions of the current responses (⫺120 to ⫺74 mV)
were extrapolated to the region where the responses intersect,
corresponding to an estimated reversal potential of Ih at ⫺45 ⫾
2 mV (n ⫽ 4, Fig. 3F). This reversal potential is consistent
J Neurophysiol • VOL
cAMP+8-Br-cAMP
B
0
0
-200
-200
n=4
0
control
A
50
-54
-400 Iinst -64
-74
mV
-400
I3s
-144
-600
0
C
I (pA)
control
Iinst
% Block
50 µM
ZD7288
-100
600
1
2
3
time (s)
1
2
time (s)
4
3
D
0
-200
-400
-140
E
-600
0
4
-120 -100
V (mV)
-80
1.0
0.5
0
-150
-120
-90
V (mV)
F
1.0
-74
-84
-104
-124
0.5
0.2
-144
0
0.1
0
4
-84
-200 -104
2
0
-140
-60
-124
-120
V (mV)
-100
-400
-120
-80
V (mV)
-40
0
FIG. 3. Properties of Ih in afferent dendrites. A and B: Ih currents in
response to voltage steps (every 10 s for 3 s, from a holding potential of ⫺64
mV to voltages between ⫺144 and ⫺54 mV in 10 mV increments; see inset).
External solution with: TTX (1–2 ␮M), 4-aminopyridine (4-AP, 2 mM),
tetraethylammonium (TEA, 10 –30 mM), and 6-cyano-7-nitroquinoxaline-2,3dione (CNQX, 10 ␮M). A: in control solution. B: with 200 ␮M cyclic
adenosine monophosphate (cAMP) intracellularly and additionally 200 ␮M
8-bromoadenosine-3=,5= cyclic monophosphate (8-Br-cAMP) extracellularly.
C: I–V relations in control (n ⫽ 3, black) and with cAMP analogs (n ⫽ 4, red).
D: voltage dependence of Ih measured from tail currents. Tail current amplitudes were normalized and fit with a Boltzmann equation. Vhalf and slope
factors were ⫺104 ⫾ 3 mV, 11 ⫾ 1 in control (n ⫽ 3, black), and ⫺91 ⫾ 2
mV, 11 ⫾ 1 in cAMP analogs (n ⫽ 4, red). E: activation kinetics of Ih currents.
Current responses to voltage steps from ⫺134 to ⫺104 mV were fit with 2
exponentials, providing 2 time constants (␶fast, ␶slow). Both time constants were
significantly faster for currents recorded with cAMP analogs (n ⫽ 4, red)
compared with control (n ⫽ 3, black). F: reversal potential of Ih. Conditioning
voltages were applied for 3 s (to ⫺124, ⫺104, or ⫺84 mV), followed by 10
ms voltage ramps (from ⫺144 to ⫺74 mV) (top traces: voltage commands;
bottom traces: current responses to the commanding voltage ramps). The
reversal potential was ⫺45.5 mV for this recording.
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100
400
200
time (s)
(I-Imin)/(Imax-Imin)
F
0
0
The I–V relation of Ih was recorded while blocking voltagegated sodium channels (1–2 ␮M TTX), potassium channels (2
mM 4-AP and 10 –30 mM TEA), and AMPA receptors (10 ␮M
CNQX) (Fig. 3, A–E). From a holding potential of ⫺64 mV,
I (pA)
-80
-300 200 ms
I (pA)
HCN channels in afferent dendrites are modulated by cAMP
-40
I (pA)
2 mM BaCl2
recovery
control
ZD7288
0
I3s- Iinst (pA)
-100
CsCl
600
Af/(Af+As)
0
I0.5-Iinst (pA)
I (pA)
E
-200
400
200
time (s)
0
200 ms
C
with a mixed cation channel permeable for sodium and potassium and is in the range of reversal potentials described for
HCN channels (Bal and Oertel 2000; Banks et al. 1993; Cao et
al. 2007; Chen 1997; Cuttle et al. 2001; Mo and Davis 1997a;
Moosmang et al. 2001; Rodrigues and Oertel 2006; Santoro et
al. 2000; van Welie et al. 2005). In summary, the pharmacological profile and reversal potential suggest that Ih in afferent
dendrites is mediated by HCN channels.
BaCl2
-100
2 mM CsCl
recovery
control
-200
-300
B
CsCl
τfast (s)
-100
0
τslow (s)
D
0
I0.5-Iinst (pA)
I (pA)
A
2535
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E. YI, I. ROUX, AND E. GLOWATZKI
significant change in the slope factor (11 ⫾ 1, n ⫽ 4, P ⫽
0.611). Under these conditions, at ⫺65 mV, about 9% of Ih
would be active and have an estimated conductance of 1.4 nS
(332 pA ⫻ 0.085/20 mV; reversal potential ⫺45 mV). In the
presence of cAMP analogs, activation time constants were
significantly faster with a ␶fast of 23 ⫾ 6 ms and a ␶slow of
175 ⫾ 76 ms at a holding potential of ⫺134 mV [n ⫽ 4, P ⬍
0.05 for all voltages tested: Afast/(Afast ⫹ Aslow) ⫽ 0.85 ⫾
0.05] (Fig. 3E, red data points).
HCN channel subunit expression in IHC afferent dendrites
To investigate the expression pattern of HCN channel subunits in IHC afferent dendrites, we performed immunolabeling
experiments with antibodies raised against the four known
HCN channel subunits HCN1–HCN4 (Moosmang et al. 2001;
Robinson and Siegelbaum 2003). We examined HCN distribution in the apical part of the cochlea in prehearing animals, at
P9 –P10, to match our recordings from afferent dendrites, and
at P20 –P21, to analyze HCN subunit expression in hearing
animals. No immunolabeling of HCN3 above background was
found and therefore the result could not be interpreted. Figure 4
shows overviews of apical cochlear turns at P9 –P10 labeled for
HCN1 or HCN4. At this age, we did not find HCN2 labeling.
For better orientation, IHCs were labeled for the vesicular
glutamate transporter VGLUT3 (Seal et al. 2008) (Fig. 4B).
HCN1 and HCN4 specific labeling in the organ of Corti was
found in the area directly below the IHCs, suggestive of a
labeling of the unmyelinated peripheral processes of the afferent neurons. Additionally, HCN1 and HCN4 immunoreactivity
was found in most of the spiral ganglion somata (Fig. 4 and
Supplemental Fig. S2). The labeling was concentrated at the
plasma membrane of the somata and in their processes within
the ganglion close to the somata (Supplemental Fig. S2). At
P20 –P21, similar to the labeling pattern at P9 –P10, labeling
for HCN1, HCN4, and additionally for HCN2 was found
directly below the IHCs (Fig. 5) and in the spiral ganglion
somata (Supplemental Fig. S2).
IHCs are surrounded by different cell types, including
afferent fibers, efferent fibers, and supporting cells. To
confirm that the afferent dendrites in the IHC area express
the different HCN subunits, as indicated by our electrophysiological recordings, we performed double immunolabeling
experiments for calretinin and HCN channel subunits. Calretinin is a calcium-binding protein involved in calcium
buffering and transport. In the rat cochlea, calretinin has
been detected both in the cytoplasm of IHCs and in most
spiral ganglion neurons including their afferent dendrites
FIG. 4. HCN subunit expression pattern in the rat cochlea at
P9. Three-dimensional reconstruction of confocal images from
cochlear whole-mount preparations, apical turns. A: HCN1
labeling (green). B: HCN4 labeling (green). Vesicular glutamate transporter VGLUT3 (red) was used as a marker for IHCs.
HCN1 and HCN4 labeling was found in the inner spiral plexus
(isp) under the row of IHCs (ihc, arrowhead) as well as in the
somata of spiral ganglion neurons (sgn). Scale bars: 50 ␮m.
J Neurophysiol • VOL
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voltage steps were applied for 3 s, from ⫺144 to ⫺54 mV in
10 mV increments, and were followed by a voltage step to ⫺74
mV, during which tail currents were recorded (Fig. 3A). Ih was
measured as I3s ⫺ Iinst. The I–V relation showed greater
activation toward more negative voltages, with an Ih amplitude
of 174 ⫾ 48 pA at ⫺144 mV (n ⫽ 3) (Fig. 3C, black trace).
The voltage dependence of Ih was measured from tail currents
and fitted by a Boltzmann equation (Fig. 3D, black traces).
Half-maximum activation voltage (Vhalf) was ⫺104 ⫾ 3 mV
and the slope factor was 11 ⫾ 1 (n ⫽ 3). The activation curve
shows that about 3% of Ih channels would be open at the
resting membrane potential of the afferent dendrites (⫺65
mV). The activation range measured here corresponds to
ranges measured under similar conditions for HCN channels
in heterologous expression systems (Moosmang et al. 2001;
Santoro et al. 2000) and Ih recorded in other neurons and
spiral ganglion somata (Bal and Oertel 2000; Banks et al.
1993; Cao et al. 2007; Chen 1997; Cuttle et al. 2001; Mo
and Davis 1997b; Rodrigues and Oertel 2006; van Welie et
al. 2005).
Different HCN channel subunits activate on different timescales, with activation time constants ranging from milliseconds to seconds (Moosmang et al. 2001; Santoro et al. 2000).
We measured the activation kinetics of Ih for current responses
to voltage steps between ⫺134 and ⫺104 mV from a holding
potential of ⫺64 mV (Fig. 3E, black data points). The time
course of activation was best fitted with two exponentials. At
⫺134 mV, the time constants of the fast (␶fast) and the slow
(␶slow) component were 66 ⫾ 13 ms (n ⫽ 3) and 929 ⫾ 40 ms,
respectively [Afast/(Afast ⫹ Aslow) ⫽ 0.83 ⫾ 0.09]. At more
positive potentials, activation slowed down for both components.
It is well known that intracellular cyclic nucleotides can
modulate HCN channel activity (Moosmang et al. 2001; Santoro et al. 2000; Wainger et al. 2001). Typically, with increased
cAMP levels, the activation curve is shifted to more positive
values and the activation kinetics speeds up. We therefore
measured the I–V relation in the presence of cAMP analogs
(Fig. 3B). cAMP (200 ␮M) was added to the pipette solution
and, additionally, 200 ␮M of the membrane permeable 8-BrcAMP (8-bromoadenosine-3=,5= cyclic monophosphate) was
added to the external solution. In the presence of cAMP
analogs, the current amplitude of Ih increased significantly
compared with control (from 174 ⫾ 48 pA in control to 332 ⫾
91 pA in cAMP; at ⫺144 mV, n ⫽ 4, P ⬍ 0.05 for all voltages
tested) (Fig. 3, B and C, red traces). The activation curve
shifted to more positive values, by about 12 mV (Fig. 3D, red
traces), with Vhalf at ⫺91 ⫾ 2 mV (n ⫽ 4, P ⬍ 0.05) and no
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
2537
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FIG. 5. HCN subunits are localized in afferent dendrites. A: 3-dimensional reconstruction of calretinin and HCN1-labeled whole-mount rat organ of Corti
preparation, apical turn at P9. HCN1 labeling (green) is concentrated in the basolateral region of the IHCs. Calretinin (red) labels IHCs (ihc) and afferent
dendrites. B–D: close-up view showing single confocal laser-scanning micrographs. As seen in the merged view (D), HCN1 (B) and calretinin (C)
immunolabeling overlap in some afferent dendrites (arrowheads). E–M: single confocal laser-scanning micrographs of whole-mount organs of Corti preparations,
apical turns at P21. Preparations were colabeled for HCN1, HCN2, or HCN4 (green) and calretinin (red). Arrowheads indicate examples of double-labeled
afferent dendrites. Note examples of ringlike HCN labeling surrounding calretinin labeling (open arrowheads). Some fibers were labeled for HCN but not for
calretinin (asterisks). Scale bars: 5 ␮m.
(Dechesne et al. 1991, 1993). High resolution confocal
imaging confirmed the coexpression of the HCN subunits
and calretinin in the same afferent dendrites at P9 –P10 and
P20 –P21 (Fig. 5). When assessing the labeling close to the
IHCs at P9 –P10, some afferent dendrites could be identified
by their strong labeling with calretinin and were also found
positive for HCN1 (Fig. 5, A–D, arrowheads) or HCN4 (data
not shown). At P20 –P21, HCN immunolabeling was more
sharply defined, most likely reflecting a higher concentration or more intense clustering of the HCN channel subunits
in afferent dendrites in the more mature organ of Corti. Most
J Neurophysiol • VOL
afferent dendrites positive for HCN1, HCN2, and HCN4
were also positive for calretinin labeling, indicating that
HCN1, HCN2, and HCN4 are indeed localized in the afferent dendrites (Fig. 5, E–M, arrowheads). In some dendrites,
HCN labeling appeared to surround the calretinin labeling in
a ringlike fashion (open arrowheads), consistent with a
clustering of the HCN channels in the plasma membrane.
Some HCN positive dendrites were not calretinin positive
(Fig. 5, asterisks). Similarly, not all HCN positive spiral
ganglion somata were positive for calretinin labeling (data
not shown). We conclude that the HCN subunits 1, 2, and 4
103 • MAY 2010 •
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2538
E. YI, I. ROUX, AND E. GLOWATZKI
EPSP waveforms in IHC afferent dendrites
In the CNS, dendritic Ih has been shown to shape EPSPs and
to modulate excitability in neurons (Magee 2000). To investigate whether Ih affects the EPSP waveform in IHC afferent
dendrites, we first characterized the EPSP waveform at rest. To
isolate synaptic activity and block the generation of APs,
experiments were performed in 1 ␮M TTX. Resting membrane
potentials of afferent dendrites were ⫺64 ⫾ 8 mV (n ⫽ 34)
when recorded in our standard extracellular solution containing
5.8 mM K⫹ and 1.3 mM Ca2⫹. Synaptic events occurred at a
rate of 0.7 ⫾ 0.9/s (n ⫽ 47; 13,575 synaptic events analyzed).
As shown before for EPSCs (Glowatzki and Fuchs 2002),
some EPSPs appeared “multiphasic,” composed of multiple
overlapping events. Other EPSPs appeared “monophasic,” presenting a monoexponential decay (Fig. 6F). To investigate rise
and decay times, only monophasic events were analyzed. In three
recordings, for direct comparison, both EPSCs and EPSPs
were characterized in the same recording (Fig. 6, A–K). EPSCs
were recorded at a holding potential of ⫺94 mV and EPSPs at
the resting membrane potential of the afferent dendrite in 5.8
mM K⫹. EPSPs were about three to four times slower than
C
B
0
EPSC amplitude (pA)
I (pA)
n=57
400
400
-350
100 pA
3 ms
10 s
E -50
-60
5 mV
3 ms
-70
H
30
n=109
20
10
10
0
0
J
K
n=676
100
0
0
L -20
n=307
50
0
10
30
40
20
EPSP amplitude (mV)
number of events
200
number of events
number of events
100
0
N
V (mV)
10 mV
3 ms
-60
0
3 0
3
6
9
τdecay (ms)
20
n=417
10
0
10
20
30
40
EPSP amplitude (mV)
M
-40
1
2
rise (ms)
3
6
9
τdecay (ms)
30
20
10 s
I
0
1
2
3 0
rise (ms)
0
G
F
-80
0
0
10
20
30
40
EPSP amplitude (mV)
30
number of events
-700
V (mV)
D
800
800
EPSP amplitude (mV)
A
n=100
20
10
0
0
30 s
J Neurophysiol • VOL
10 20 30 40 50
EPSP amplitude (mV)
103 • MAY 2010 •
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FIG. 6. EPSCs and excitatory postsynaptic
potentials (EPSPs) recorded at the IHC afferent
synapse. A–K: whole cell recording from an
afferent dendrite in the presence of 1 ␮M TTX
showing EPSCs (A, B) (holding potential ⫺94
mV) and EPSPs (E, F). B and F: overlaid
representative traces of monophasic EPSCs
and EPSPs on an expanded timescale. C, D, G,
and H: 10 –90% rise time (rise) or decay time
constants (␶decay) plotted against the EPSC or
EPSP amplitude. Rise and ␶decay for EPSCs
were 0.33 ⫾ 0.14 and 1.24 ⫾ 0.20 ms (324
EPSCs analyzed). Rise and ␶decay for EPSPs
were 0.96 ⫾ 0.12 and 3.81 ⫾ 0.36 ms (241
EPSPs analyzed). EPSP waveforms remained
relatively invariable over the wide range of
EPSP amplitudes. I–K: EPSP amplitude distributions (bin size 1 mV) from 3 afferent dendrite recordings. Median EPSP amplitudes
were 2.3, 2.4, and 13.8 mV and resting membrane potentials were ⫺75, ⫺56, and ⫺68
mV, respectively. The number of events analyzed is indicated in each panel. L–N: whole
cell current-clamp recording in the absence of
TTX. A mixture of EPSPs and spikes was
observed. M: overlaid representative traces of
spikes on an expanded timescale. Spike threshold (arrow) was ⫺47 mV for this recording.
N: amplitude distribution (bin size 1 mV). A
wide gap in amplitude histogram distinguishes
spikes from EPSPs.
Downloaded from http://jn.physiology.org/ by 10.220.33.1 on October 26, 2016
EPSCs, with a 10 –90% rise time of 1.14 ⫾ 0.17 ms compared
with 0.36 ⫾ 0.09 ms and a time constant of decay of 5.13 ⫾
1.69 ms compared with 1.16 ⫾ 0.13 ms at room temperature
(n ⫽ 3, 381 EPSPs; n ⫽ 8, 1,807 EPSCs analyzed) (Fig. 6, C,
D, G, and H). For EPSPs, we found a wide range of amplitudes
in every recording, varying from 1 to 35 mV; the shape of the
amplitude distributions also varied widely. The amplitude distributions of two fibers were highly skewed, with median EPSP
amplitudes of 2.3 and 2.4 mV, and most of the EPSPs were
⬍10 mV (Fig. 6, I and J). For a third fiber, EPSP amplitudes
spread more evenly within a range between 1 and 35 mV, with
a median EPSP amplitude of 13.8 mV (Fig. 6K). However, for
both EPSCs and EPSPs, rise and decay times did not change
much over this wide range of amplitudes (Fig. 6, C, D, G, and H).
In auditory nerve fibers, the spike initiation zone is believed
to be located close to the IHC afferent synapse, on the peripheral process (Hossain et al. 2005; Robertson 1976). A heminode with a high expression level of sodium channels, is ⬍50
␮m away from the afferent synapse (Hossain et al. 2005;
McLean et al. 2009). The expression of sodium channels was
also reported on the unmyelinated ending of the afferent fiber,
peripheral to the heminode (Hossain et al. 2005). Indeed, when
recording in the absence of TTX, at a resting membrane
potential of ⫺67 ⫾ 5 mV (n ⫽ 7), 18 ⫾ 22% of the events
are expressed in afferent dendrites at the IHC afferent
synapse.
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
A
5 ms
5 ms
E
amplitude (mV)
20
10
10
5
6
decay
(ms)
(% of control)
*
150
8
n=7
F
decay
12
(ms)
18
n=6
*
150
100
n=3
*
n=6
100
decay
50
0
6
control
CsCl
recovery
50
0
control ZD7288 ZD7288
+ Iinj
FIG. 7. Ih shortens EPSPs in afferent dendrites. A–H: whole cell currentclamp recording from afferent dendrites. Recordings were done in the presence
of cAMP analogs (200 ␮M 8-Br-cAMP extracellularly and additionally 200
␮M cAMP intracellularly). A–C: EPSP waveform before and while blocking Ih
with 2 mM CsCl. A: average EPSP waveform before (black) and during
application of 2 mM CsCl (red). B: EPSP decay time constants (␶decay) plotted
against EPSP amplitudes before and while blocking Ih with 2 mM CsCl;
control: ␶decay ⫽ 4.97 ms (black, 34 EPSPs); in 2 mM CsCl: ␶decay ⫽ 7.18 ms
(red, 41 EPSPs). C: summarized results from 7 recordings. D–F: EPSP
waveform before and during application of 50 ␮M ZD7288. D: average EPSP
waveform before (black) and during application of 50 ␮M ZD7288 (magenta).
E: EPSP decay time constants (␶decay) plotted against EPSP amplitudes;
control: ␶decay ⫽ 5.38 ms (black, 22 EPSPs), in 50 ␮M ZD7288: ␶decay ⫽ 8.30
ms (magenta, 16 EPSPs). F: summarized results from 6 recordings.
time (1.40 ⫾ 0.32 vs. 1.49 ⫾ 0.31 ms, P ⫽ 0.082) did not
significantly change. The Ih-induced hyperpolarization of the
membrane potential could change the activity of additional ion
channels that also might affect the EPSP waveform as it has been
shown in a computational model of cochlear nucleus neurons
(Rothman and Manis 2003b) or in recordings from dendrites of
both hippocampal neurons (George et al. 2009) and frontal cortex
pyramidal neurons (Day et al. 2005). Therefore to exclude possible effects on the EPSP waveform by hyperpolarization, membrane potentials were manually reset during ZD7288 application
to their control values by constant current injection in three
recordings. A significant increase of 30 ⫾ 11% in ␶decay compared
with control was still observed (Fig. 7F, ␶decay in control: 4.22 ⫾
0.98 ms; ␶decay in ZD7288: 5.25 ⫾ 1.51 ms, P ⫽ 0.03). This result
indicates that Ih shapes the EPSP waveform directly by contributing to the membrane resting conductance. The application not
only of CsCl but also of ZD7288 increased ␶decay over the entire
range of EPSP amplitudes (Fig. 7, B and E). We conclude that the
activity of Ih in afferent dendrites shortens the EPSP waveform
over the whole range of EPSP amplitudes.
Application of ZD7288 also caused minor changes in the
spike waveform. The spike amplitude increased by 9 ⫾ 4%
103 • MAY 2010 •
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0
4
C
decay
15
(% of control)
B
0
Next, we tested whether Ih is involved in shaping the EPSP
waveform. To block Ih, we first applied CsCl, to allow for
recovery of a possible effect. Without any cAMP analog added,
2 mM CsCl significantly increased ␶decay of the EPSP by 27 ⫾
17% (from 6.37 ⫾ 0.64 to 8.04 ⫾ 1.02 ms and, after washout,
to 6.98 ⫾ 0.53 ms; n ⫽ 4 fibers, 617 EPSPs analyzed, P ⬍
0.05). Neither mean EPSP amplitude (control vs. CsCl: 13 ⫾ 6
vs. 14 ⫾ 8 mV, P ⫽ 0.839) nor 10 –90% rise time (1.31 ⫾ 0.20
vs. 1.48 ⫾ 0.20 ms, P ⫽ 0.146) changed significantly. In the
presence of cAMP analogs (200 ␮M cAMP intracellularly and,
additionally, 200 ␮M 8-Br-cAMP extracellularly), application
of CsCl increased ␶decay by 37 ⫾ 31% (from 5.25 ⫾ 1.37 to
7.15 ⫾ 1.29 ms, n ⫽ 7, 878 EPSPs analyzed, P ⬍ 0.05)
(Fig. 7, A–C). Again, EPSP mean amplitude (control vs. CsCl:
10 ⫾ 4 vs. 11 ⫾ 6 mV, P ⫽ 0.337) and 10 –90% rise time
(1.23 ⫾ 0.20 vs. 1.31 ⫾ 0.30 ms, P ⫽ 0.376) did not
significantly change. Application of CsCl hyperpolarized the
resting membrane potential of the afferent dendrite by about 1
mV (from ⫺64 ⫾ 3 to ⫺65 ⫾ 3 mV, n ⫽ 11, P ⫽ 0.002).
Immature IHCs express inward rectifier potassium channels
that can be inhibited by extracellular Cs⫹ (Marcotti et al.
2003). Indeed, the frequency of synaptic events was higher in
CsCl (control vs. CsCl: 1.01 ⫾ 1.66 vs. 2.23 ⫾ 2.44 events/s,
n ⫽ 16, 4,357 EPSP or EPSCs analyzed, P ⫽ 0.025). To
exclude the possibility that CsCl by some presynaptic effect
changes the EPSC waveform and therefore affects the EPSP
waveform, we tested the effect of CsCl on EPSCs and found no
significant difference compared with control (control vs. CsCl:
EPSC mean amplitude: 159 ⫾ 91 vs. 168 ⫾ 99 pA, P ⫽ 0.838;
10 –90% rise time: 0.38 ⫾ 0.14 vs. 0.38 ⫾ 0.14 ms, P ⫽ 0.796;
␶decay: 1.24 ⫾ 0.40 vs. 1.43 ⫾ 0.57 ms, P ⫽ 0.383; n ⫽ 3, 626
EPSCs analyzed). These data suggest that changes in the EPSP
waveform during CsCl application are due to its effect on the
afferent dendrite.
Application of the irreversible HCN channel blocker ZD7288
exhibited greater effects than those of CsCl on the EPSP waveform, without changing the EPSP frequency (control vs. ZD7288:
0.73 ⫾ 0.76 vs. 0.70 ⫾ 0.71 events/s, P ⫽ 0.582) (Fig. 7, D–F).
During application of 50 ␮M ZD7288 (in the presence of 200 ␮M
cAMP intracellularly and, additionally, 200 ␮M 8-Br-cAMP extracellularly), ␶decay increased by 47 ⫾ 24% (from 4.22 ⫾ 0.98 to
6.19 ⫾ 1.67 ms, n ⫽ 6, 593 EPSPs analyzed, P ⫽ 0.002) and the
resting membrane potential was hyperpolarized by about 4 mV
(from ⫺63 ⫾ 6 to ⫺67 ⫾ 7 mV, P ⬍ 0.001, n ⫽ 11). EPSP
amplitude (8 ⫾ 3 vs. 9 ⫾ 4 mV, P ⫽ 0.378) and 10 –90% rise
ZD7288
CsCl
HCN channel activity shortens the EPSP waveform in IHC
afferent dendrites
J Neurophysiol • VOL
D
amplitude (mV)
were spikes rather than EPSPs (range 0.5–50.5%, n ⫽ 4
afferent dendrites with ⬎100 analyzable events each; total
number of events analyzed: 829) (Fig. 6, L–N). Spikes were
discriminated from EPSPs by their large and uniform amplitudes (39 ⫾ 7 mV, n ⫽ 7, 171 spikes) that appeared as a
separate group of events in the amplitude histograms (Fig. 6N).
Additionally, about half of the spikes exhibited sudden slope
changes during their rise (Fig. 6M, arrow), suggestive of a
spike threshold at ⫺50 ⫾ 4 mV (n ⫽ 7, 97 spikes). The
10 –90% rise time of the spikes (including the rise of the
initiating EPSPs) was 2.29 ⫾ 0.75 ms and the spike half-width
was 2.83 ⫾ 0.86 ms (n ⫽ 7, 171 spikes).
2539
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E. YI, I. ROUX, AND E. GLOWATZKI
(from 32 ⫾ 5 to 35 ⫾ 4 mV, P ⫽ 0.026; n ⫽ 3, 155 spikes
analyzed) and spike half-width increased by 14 ⫾ 8% (from
3.05 ⫾ 0.53 to 3.45 ⫾ 0.39 ms, P ⫽ 0.046). The 10 –90% rise
time did not change significantly (control vs. ZD7288: 1.24 ⫾
0.20 vs. 1.29 ⫾ 0.19 ms, P ⫽ 0.757).
DISCUSSION
Properties of Ih in IHC afferent dendrites
J Neurophysiol • VOL
Role of Ih in auditory neurons
Multiple studies have shown that auditory neurons involved
in processing timing information express Ih. Examples include
bushy cells (Cao et al. 2007; Leao K et al. 2006; Leao R et al.
2005; Rothman and Manis 2003a), octopus cells (Bal and
Oertel 2000; Cao and Oertel 2005; Golding et al. 1995, 1999),
and stellate cells (Rodrigues and Oertel 2006; Rothman and
Manis 2003b) of the cochlear nucleus, medial superior olive
neurons (Scott et al. 2005), nucleus laminaris neurons (Kuba et
al. 2005; Yamada et al. 2005), lateral superior olive neurons
(Barnes-Davies et al. 2004; Leao K et al. 2006), medial nucleus
of the trapezoid body (MNTB) neurons (Banks et al. 1993;
Cuttle et al. 2001; Leao K et al. 2006), the calyx of Held
(Cuttle et al. 2001), and inferior colliculus neurons (Koch and
Grothe 2003). Additionally, Ih was found in the somata of
spiral ganglion neurons (Chen 1997; Mo and Davis 1997b). Ih
can be partially active at rest, contributing to a lower membrane input resistance and thus shortening EPSPs. Shorter
EPSPs reduce the time window of temporal summation and
therefore improve coincidence detection (Golding et al. 1995;
Koch and Grothe 2003; Yamada et al. 2005). Similarly, Ih
expressed in dendrites of nonauditory CNS neurons shortened
EPSPs and thereby reduced summation of synaptic activity
(Day et al. 2005; Magee 2000; Magee and Johnston 2005). The
nonuniform distribution of HCN channels along dendrites
compartmentalized distal dendrites from the somata (Berger et
al. 2003), limiting action potential (AP) back-propagation, and
thereby decreasing hyperexcitability (Tsay et al. 2007; Ying et
al. 2007). In motion-sensitive neurons in the superior colliculus
(Endo et al. 2008), dendritic Ih was shown to keep AP timing
at a minimum jitter and with short latencies. It is not surprising
that similar mechanisms might be used at the IHC afferent
synapse, as discussed in the following text.
103 • MAY 2010 •
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Dendritic Ih has been shown to play an important role in
setting firing rates in nerve fibers (Magee and Johnston 2005).
Here we have used whole cell recordings to provide an initial
characterization of Ih present in postnatal IHC afferent dendrites. The pharmacological profile of Ih, its reversal potential,
its sensitivity to cAMP, and the expression of the HCN subunits 1, 2, and 4 in afferent dendrites indicate that Ih is
mediated by HCN channels. In immunolabeling experiments
we found HCN1 and HCN4 in postnatal afferent fibers and
HCN1, HCN2, and HCN4 after hearing onset. No immunolabeling of HCN3 above background was found in either the
spiral ganglion or the afferent dendrites at both ages and thus
we cannot make a statement about the expression of HCN3.
In adult guinea pig spiral ganglion somata, the expression of
all four HCN subunits has been reported (Bakondi et al.
2009). The different result regarding HCN3 expression
might be due to a difference in the species or to the different
antibodies used. No specific HCN1 or HCN2 labeling has
been found in mouse cochlear hair cells and mice lacking
HCN1, HCN2, or both exhibited normal transduction currents (Horwitz et al. 2010).
We found each HCN subunit in a high percentage of afferent
dendrites. It is therefore highly likely that heteromeric channels
are formed. However, it is difficult to unequivocally deduce the
composition of HCN channels in afferent dendrites from the
properties of the recorded Ih. When measured in the same experimental conditions, homomeric HCN1 channels activate at more
positive voltages and show faster kinetics than homomeric HCN2
and HCN4 channels. On the other hand, HCN1 shows the least
sensitivity to cyclic nucleotides, followed by HCN2 to HCN4
(Moosmang et al. 2001; Santoro et al. 2000). Heteromeric
HCN channels have been shown to exhibit intermediate properties compared with those of homomeric channels (Chen et al.
2001; Ulens and Tytgat 2001). Additionally, HCN channels
with the same subunit composition exhibit variable characteristics when measured in different expression systems or tissues
(Moosmang et al. 2001; Santoro et al. 2000; Wainger et al.
2001). Knock-out animals for HCN1, HCN2, and HCN4 do
exist, although reports on their behavior do not mention any
test of auditory function (Harzheim et al. 2008; Herrmann et al.
2007; Ludwig et al. 2003; Nolan et al. 2003, 2004; Stieber et al.
2003).
A range of activation voltages and cyclic nucleotide sensitivities has been described for Ih in studies on auditory neurons.
For example, in bushy cells, a Vhalf of ⫺94 to ⫺84 mV and
positive shift of Vhalf with cAMP were found (Cuttle et al.
2001; Leao K et al. 2006), whereas in octopus cells, a Vhalf of
⫺65 mV and no shift with cAMP were reported (Bal and
Oertel 2000). The properties we found for Ih in afferent
dendrites are comparable to those found for Ih in spiral ganglion somata. In afferent dendrites, Vhalf was ⫺104 mV, similar
to Vhalf reported for spiral ganglion somata (⫺101 mV; Chen
1997; ⫺78 to ⫺122 mV; Mo and Davis 1997b). Both in spiral
ganglion somata and afferent dendrites, Vhalf shifted positively
in the presence of cAMP analogs.
In control conditions, we found 3% and, in the presence of
cAMP analogs, 9% of Ih open at rest. The experimental
conditions might underestimate the size of Ih in vivo; we
recorded at room temperature and the amplitude of Ih has been
found to be temperature sensitive in auditory neurons (Cao and
Oertel 2005; Cao et al. 2007; Rodrigues and Oertel 2006). We
recorded in immature cochleae, however, our immunolabeling
experiments suggest a maturation of HCN channel subunit
expression in hearing animals. For example, HCN2 labeling of
afferent dendrites was found in 3-wk-old cochleae but not
before hearing onset. HCN2 has a higher sensitivity to cAMP
compared with that of HCN1 and thus properties of Ih may
change with maturation. In our whole cell recordings, second
messengers may be subject to wash-out and therefore the Vhalf
measured may be more negative compared with that under
in vivo conditions. Additionally, under in vivo conditions,
input from lateral efferent fibers may up-regulate intradendritic
cAMP levels (see last paragraph) and in vitro transmitter
release from these fibers may be absent or abnormal.
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
2541
HCN channels in afferent dendrites as possible targets for
lateral efferent transmission
Here we describe EPSP waveforms at the IHC afferent
synapse. EPSPs have 10 –90% rise times of about 1 ms and
time constants of decay of about 5 ms. In comparison, the
duration of EPSPs recorded in vivo in guinea pig (Palmer and
Russell 1986; Siegel 1992; Siegel and Dallos 1986) was about
1–2 ms. The slower kinetics of EPSPs reported here could be
due to the immature age of the recorded dendrites. AMPA
mediated EPSCs at IHC afferent synapses speed up by a factor
of 2, in a comparison of the immature age used in this study
with 3-wk-old animals (Grant et al. 2010). Also, our recordings
were performed at room temperature and AMPA receptor
kinetics are temperature sensitive, with a Q10 of ⬎2 (Postlethwaite et al. 2007). The amplitudes and gating kinetics of
voltage-gated ion channels, such as Ih and IKL that are known
to shorten EPSPs and APs in auditory neurons, are generally
larger and faster in posthearing animals (Scott et al. 2005) and
at body temperature (Cao and Oertel 2005; Cao et al. 2007;
Rodrigues and Oertel 2006).
As shown for EPSCs (Glowatzki and Fuchs 2002; Grant et
al. 2010), EPSP amplitudes covered an impressively wide
range, between 1 and 35 mV. The median EPSP amplitudes
varied widely between fibers, between 2 and 14 mV. Spikes
recorded in afferent dendrites had a threshold at about ⫺50 mV
and roughly 20% of EPSPs activated spikes. Therefore for
small EPSPs, postsynaptic summation of EPSPs might be
necessary to reach the threshold for AP generation. In simultaneous recordings from hair cells and afferent dendrites,
summation of EPSCs was observed during the early response
to step depolarizations of the IHC membrane potential (Goutman and Glowatzki 2007). Our results showed that EPSPs
slowed down by 47% when Ih was blocked by ZD7288. These
data suggest that depending on the level of Ih active, the time
window of EPSP summation may vary, allowing for regulation
of firing rate in auditory nerve fibers.
Block of Ih with ZD7288 also induced a hyperpolarization of
the membrane potential by about 4 mV. When the membrane
potential was reset to control values during block of Ih, EPSPs
still slowed down by roughly 30%. This result indicates that Ih
shapes the EPSP waveform directly by contributing to the
resting membrane conductance. Ih may also act indirectly on
the EPSP waveform, by depolarizing the membrane potential
and thereby activating other ion channels such as IKL that
might additionally change the resting membrane conductance
(Rothman and Manis 2003b). However, our data set here does
not prove or reject this scenario because the changes in EPSP
waveform during block of Ih with and without hyperpolarization were not significantly different.
During block of Ih, and with a change in the resting membrane conductance, we expected to see not only slowing of the
EPSP waveform but also an increase in the EPSP amplitude
(Magee 1998). The EPSP amplitude increased slightly, although not significantly. This is most likely due to the wide
range of EPSP amplitudes in individual recordings. We suspect
that if a larger data set was available, the difference in amplitude might have reached statistical significance. The fact that
there was a small but significant increase in the spike amplitude
(representing a less variable waveform compared with the
EPSPs) further supports this idea.
IHC afferent dendrites receive efferent innervation from the
lateral superior olivary complex. Multiple transmitters such as
acetylcholine, dopamine, ␥-aminobutyric acid, and opiate peptides have been found in lateral efferent terminals (Eybalin
1993). Lesion of lateral efferent nerves disrupted temporal
coding and increased susceptibility to acute acoustic trauma
(Darrow et al. 2006, 2007). Intracochlear perfusion of putative
lateral efferent neurotransmitters like dopamine affected firing
rates in the auditory nerve (Ruel et al. 2001). However, the
cellular mechanisms underlying these modulatory effects are
unclear. One possibility is that lateral efferent transmitters
modulate dendritic ion channels such as Ih, for example via
second messenger cascades, thereby subsequently affecting
auditory nerve firing. Indeed, such modulation of Ih by a
neurotransmitter has been demonstrated in auditory neurons. In
rat MNTB neurons, noradrenaline and cAMP analogs increased the amplitude and shifted the activation curve of Ih
(Banks et al. 1993). In chick nucleus laminaris, noradrenaline
enhanced temporal precision of EPSPs by regulating Ih activity
in the postsynapse (Yamada et al. 2005). In addition to cyclic
nucleotides, phosphorylation by protein kinases, pH, and lipid
second messengers have been shown to modulate HCN channel activity (Fogle et al. 2007; Pian et al. 2007; Robinson and
Siegelbaum 2003; Zolles et al. 2006). Our finding that a
cAMP-sensitive Ih in afferent dendrites modulates the EPSP
waveform supports the idea that modulation of temporal coding could occur directly at the first synapse of the auditory
pathway, possibly via lateral efferent inputs.
J Neurophysiol • VOL
ACKNOWLEDGMENTS
We thank R. H. Edwards’ laboratory for kindly providing the antibodies
against VGLUT3, J. Gibas for excellent technical assistance with confocal
microscopy, and L. Grant for comments on the manuscript.
Monoclonal antibodies against HCN2 and HCN3 were obtained from UC
Davis/National Institute of Neurological Disorders and Stroke (NINDS)/
National Institute of Mental Health (NIMH) NeuroMab Facility, supported by
NINDS Grant U24-NS-050606 and maintained by the Department of Pharmacology, School of Medicine, University of California, Davis. This work was
supported by National Institute on Deafness and Other Communication Disorders (NIDCD) Grants DC-006476 to E. Glowatzki and DC-008860 to D.
Bergles, a research grant from the Deafness Research Foundation to E. Yi, a
European Molecular Biology Organization Fellowship ALTF 952-2006 to I.
Roux, National Institute of Diabetes and Digestive and Kidney Diseases Grant
R24-DK-064388 to the Ross Confocal Facility, and NIDCD Grant P30
DC-005211 to the Center for Hearing and Balance Histology Core.
DISCLOSURES
No conflicts of interest are declared by the authors.
REFERENCES
Bakondi G, Por A, Kovacs I, Szucs G, Rusznak Z. Hyperpolarizationactivated, cyclic nucleotide-gated, cation non-selective channel subunit
expression pattern of guinea-pig spiral ganglion cells. Neuroscience 158:
1469 –1477, 2009.
Bal R, Oertel D. Hyperpolarization-activated, mixed-cation current (Ih) in
octopus cells of the mammalian cochlear nucleus. J Neurophysiol 84:
806 – 817, 2000.
Bal R, Oertel D. Potassium currents in octopus cells of the mammalian
cochlear nucleus. J Neurophysiol 86: 2299 –2311, 2001.
Banks MI, Pearce RA, Smith PH. Hyperpolarization-activated cation current
(Ih) in neurons of the medial nucleus of the trapezoid body: voltage-clamp
103 • MAY 2010 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.1 on October 26, 2016
Properties of EPSPs in afferent fibers and effects of Ih on
EPSPs
2542
E. YI, I. ROUX, AND E. GLOWATZKI
J Neurophysiol • VOL
Golding NL, Robertson D, Oertel D. Recordings from slices indicate that
octopus cells of the cochlear nucleus detect coincident firing of auditory
nerve fibers with temporal precision. J Neurosci 15: 3138 –3153, 1995.
Goutman JD, Glowatzki E. Time course and calcium dependence of transmitter release at a single ribbon synapse. Proc Natl Acad Sci USA 104:
16341–16346, 2007.
Grant L, Yi E, Glowatzki E. Two modes of release shape the postsynaptic
response at the inner hair cell ribbon synapse. J Neurosci 30: 4210 – 4220,
2010.
Harzheim D, Pfeiffer KH, Fabritz L, Kremmer E, Buch T, Waisman A,
Kirchhof P, Kaupp UB, Seifert R. Cardiac pacemaker function of HCN4
channels in mice is confined to embryonic development and requires cyclic
AMP. EMBO J 27: 692–703, 2008.
Herrmann S, Stieber J, Ludwig A. Pathophysiology of HCN channels.
Pflügers Arch 454: 517–522, 2007.
Horwitz GC, Lelli A, Geleoc GS, Holt JR. HCN channels are not required for
mechanotransduction in sensory hair cells of the mouse inner ear. PloS One
5: e8627, 2010.
Hossain WA, Antic SD, Yang Y, Rasband MN, Morest DK. Where is the
spike generator of the cochlear nerve? Voltage-gated sodium channels in the
mouse cochlea. J Neurosci 25: 6857– 6868, 2005.
Keen EC, Hudspeth AJ. Transfer characteristics of the hair cell’s afferent
synapse. Proc Natl Acad Sci USA 103: 5537–5542, 2006.
Koch U, Grothe B. Hyperpolarization-activated current (Ih) in the inferior
colliculus: distribution and contribution to temporal processing. J Neurophysiol 90: 3679 –3687, 2003.
Kouranova EV, Strassle BW, Ring RH, Bowlby MR, Vasilyev DV. Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein
expression in large versus small diameter dorsal root ganglion neurons:
correlation with hyperpolarization-activated current gating. Neuroscience
153: 1008 –1019, 2008.
Kuba H, Yamada R, Fukui I, Ohmori H. Tonotopic specialization of
auditory coincidence detection in nucleus laminaris of the chick. J Neurosci
25: 1924 –1934, 2005.
Kubo Y, Adelman JP, Clapham DE, Jan LY, Karschin A, Kurachi Y,
Lazdunski M, Nichols CG, Seino S, Vandenberg CA. International Union
of Pharmacology. LIV. Nomenclature and molecular relationships of inwardly rectifying potassium channels. Pharmacol Rev 57: 509 –526, 2005.
Lacas-Gervais S, Guo J, Strenzke N, Scarfone E, Kolpe M, Jahkel M, De
Camilli P, Moser T, Rasband MN, Solimena M. BetaIVSigma1 spectrin
stabilizes the nodes of Ranvier and axon initial segments. J Cell Biol 166:
983–990, 2004.
Leao KE, Leao RN, Sun H, Fyffe RE, Walmsley B. Hyperpolarizationactivated currents are differentially expressed in mice brainstem auditory
nuclei. J Physiol 576: 849 – 864, 2006.
Leao RN, Svahn K, Berntson A, Walmsley B. Hyperpolarization-activated
(I) currents in auditory brainstem neurons of normal and congenitally deaf
mice. Eur J Neurosci 22: 147–157, 2005.
Li GL, Keen E, Andor-Ardo D, Hudspeth AJ, von Gersdorff H. The
unitary event underlying multiquantal EPSCs at a hair cell’s ribbon synapse.
J Neurosci 29: 7558 –7568, 2009.
Llano I, Marty A, Armstrong CM, Konnerth A. Synaptic- and agonistinduced excitatory currents of Purkinje cells in rat cerebellar slices. J
Physiol 434: 183–213, 1991.
Ludwig A, Budde T, Stieber J, Moosmang S, Wahl C, Holthoff K,
Langebartels A, Wotjak C, Munsch T, Zong X, Feil S, Feil R, Lancel M,
Chien KR, Konnerth A, Pape HC, Biel M, Hofmann F. Absence epilepsy
and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.
EMBO J 22: 216 –224, 2003.
Magee JC. Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. J Neurosci 18:
7613–7624, 1998.
Magee JC. Dendritic integration of excitatory synaptic input. Nat Rev 1:
181–190, 2000.
Magee JC, Johnston D. Plasticity of dendritic function. Curr Opin Neurobiol
15: 334 –342, 2005.
Manis PB, Marx SO. Outward currents in isolated ventral cochlear nucleus
neurons. J Neurosci 11: 2865–2880, 1991.
Marcotti W, Johnson SL, Holley MC, Kros CJ. Developmental changes in
the expression of potassium currents of embryonic, neonatal and mature
mouse inner hair cells. J Physiol 548: 383– 400, 2003.
McLean WJ, Smith KA, Glowatzki E, Pyott SJ. Distribution of the Na,KATPase alpha subunit in the rat spiral ganglion and organ of Corti. J Assoc
Res Otolaryngol 10: 37– 49, 2009.
103 • MAY 2010 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.1 on October 26, 2016
analysis and enhancement by norepinephrine and cAMP suggest a modulatory mechanism in the auditory brain stem. J Neurophysiol 70: 1420 –1432,
1993.
Banks MI, Smith PH. Intracellular recordings from neurobiotin-labeled cells
in brain slices of the rat medial nucleus of the trapezoid body. J Neurosci 12:
2819 –2837, 1992.
Barnes-Davies M, Barker MC, Osmani F, Forsythe ID. Kv1 currents
mediate a gradient of principal neuron excitability across the tonotopic axis
in the rat lateral superior olive. Eur J Neurosci 19: 325–333, 2004.
Berger T, Senn W, Luscher HR. Hyperpolarization-activated current Ih
disconnects somatic and dendritic spike initiation zones in layer V pyramidal neurons. J Neurophysiol 90: 2428 –2437, 2003.
Brew HM, Forsythe ID. Two voltage-dependent K⫹ conductances with
complementary functions in postsynaptic integration at a central auditory
synapse. J Neurosci 15: 8011– 8022, 1995.
Cao XJ, Oertel D. Temperature affects voltage-sensitive conductances differentially in octopus cells of the mammalian cochlear nucleus. J Neurophysiol
94: 821– 832, 2005.
Cao XJ, Shatadal S, Oertel D. Voltage-sensitive conductances of bushy cells
of the mammalian ventral cochlear nucleus. J Neurophysiol 97: 3961–3975,
2007.
Chen C. Hyperpolarization-activated current (Ih) in primary auditory neurons.
Hear Res 110: 179 –190, 1997.
Chen S, Wang J, Siegelbaum SA. Properties of hyperpolarization-activated
pacemaker current defined by coassembly of HCN1 and HCN2 subunits and
basal modulation by cyclic nucleotide. J Gen Physiol 117: 491–504, 2001.
Cuttle MF, Rusznak Z, Wong AY, Owens S, Forsythe ID. Modulation of a
presynaptic hyperpolarization-activated cationic current (Ih) at an excitatory
synaptic terminal in the rat auditory brainstem. J Physiol 534: 733–744,
2001.
Darrow KN, Maison SF, Liberman MC. Cochlear efferent feedback balances interaural sensitivity. Nat Neurosci 9: 1474 –1476, 2006.
Darrow KN, Maison SF, Liberman MC. Selective removal of lateral
olivocochlear efferents increases vulnerability to acute acoustic injury. J
Neurophysiol 97: 1775–1785, 2007.
Day M, Carr DB, Ulrich S, Ilijic E, Tkatch T, Surmeier DJ. Dendritic
excitability of mouse frontal cortex pyramidal neurons is shaped by the
interaction among HCN, Kir2, and Kleak channels. J Neurosci 25: 8776 –
8787, 2005.
Dechesne CJ, Winsky L, Kim HN, Goping G, Vu TD, Wenthold RJ,
Jacobowitz DM. Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner
ear. Brain Res 560: 139 –148, 1991.
Dechesne CJ, Winsky L, Moniot B, Raymond J. Localization of calretinin
mRNA in rat and guinea pig inner ear by in situ hybridization using
radioactive and non-radioactive probes. Hear Res 69: 91–97, 1993.
Endo T, Tarusawa E, Notomi T, Kaneda K, Hirabayashi M, Shigemoto R,
Isa T. Dendritic Ih ensures high-fidelity dendritic spike responses of motionsensitive neurons in rat superior colliculus. J Neurophysiol 99: 2066 –2076,
2008.
Fogle KJ, Lyashchenko AK, Turbendian HK, Tibbs GR. HCN pacemaker
channel activation is controlled by acidic lipids downstream of diacylglycerol kinase and phospholipase A2. J Neurosci 27: 2802–2814, 2007.
Fuchs PA. Time and intensity coding at the hair cell’s ribbon synapse. J
Physiol 566: 7–12, 2005.
Funabiki K, Koyano K, Ohmori H. The role of GABAergic inputs for
coincidence detection in the neurones of nucleus laminaris of the chick. J
Physiol 508: 851– 869, 1998.
Gardner SM, Trussell LO, Oertel D. Time course and permeation of
synaptic AMPA receptors in cochlear nuclear neurons correlate with input.
J Neurosci 19: 8721– 8729, 1999.
Gardner SM, Trussell LO, Oertel D. Correlation of AMPA receptor subunit
composition with synaptic input in the mammalian cochlear nuclei. J
Neurosci 21: 7428 –7437, 2001.
George MS, Abbott LF, Siegelbaum SA. HCN hyperpolarization-activated
cation channels inhibit EPSPs by interactions with M-type K(⫹) channels.
Nat Neurosci 12: 577–584, 2009.
Glowatzki E, Fuchs PA. Transmitter release at the hair cell ribbon synapse.
Nat Neurosci 5: 147–154, 2002.
Glowatzki E, Grant L, Fuchs P. Hair cell afferent synapses. Curr Opin
Neurobiol 18: 389 –395, 2008.
Golding NL, Ferragamo MJ, Oertel D. Role of intrinsic conductances
underlying responses to transients in octopus cells of the cochlear nucleus.
J Neurosci 19: 2897–2905, 1999.
DENDRITIC Ih AT THE HAIR CELL SYNAPSE
J Neurophysiol • VOL
Ruel J, Nouvian R, Gervais d’Aldin C, Pujol R, Eybalin M, Puel JL.
Dopamine inhibition of auditory nerve activity in the adult mammalian
cochlea. Eur J Neurosci 14: 977–986, 2001.
Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR,
Siegelbaum SA. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J Neurosci 20:
5264 –5275, 2000.
Schneggenburger R, Forsythe ID. The calyx of Held. Cell Tissue Res 326:
311–337, 2006.
Scott LL, Mathews PJ, Golding NL. Posthearing developmental refinement
of temporal processing in principal neurons of the medial superior olive. J
Neurosci 25: 7887–7895, 2005.
Seal RP, Akil O, Yi E, Weber CM, Grant L, Yoo J, Clause A, Kandler K,
Noebels JL, Glowatzki E, Lustig LR, Edwards RH. Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron
57: 263–275, 2008.
Shin KS, Rothberg BS, Yellen G. Blocker state dependence and trapping in
hyperpolarization-activated cation channels: evidence for an intracellular
activation gate. J Gen Physiol 117: 91–101, 2001.
Siegel JH. Spontaneous synaptic potentials from afferent terminals in the
guinea pig cochlea. Hear Res 59: 85–92, 1992.
Siegel JH, Dallos P. Spike activity recorded from the organ of Corti. Hear Res
22: 245–248, 1986.
Smith AJ, Owens S, Forsythe ID. Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive. J Physiol 529:
681– 698, 2000.
Stieber J, Herrmann S, Feil S, Loster J, Feil R, Biel M, Hofmann F,
Ludwig A. The hyperpolarization-activated channel HCN4 is required for
the generation of pacemaker action potentials in the embryonic heart. Proc
Natl Acad Sci USA 100: 15235–15240, 2003.
Szabo ZS, Harasztosi CS, Sziklai I, Szucs G, Rusznak Z. Ionic currents
determining the membrane characteristics of type I spiral ganglion neurons
of the guinea pig. Eur J Neurosci 16: 1887–1895, 2002.
Trussell LO. Synaptic mechanisms for coding timing in auditory neurons. Ann
Rev Physiol 61: 477– 496, 1999.
Tsay D, Dudman JT, Siegelbaum SA. HCN1 channels constrain synaptically
evoked Ca2⫹ spikes in distal dendrites of CA1 pyramidal neurons. Neuron
56: 1076 –1089, 2007.
Ulens C, Tytgat J. Functional heteromerization of HCN1 and HCN2 pacemaker channels. J Biol Chem 276: 6069 – 6072, 2001.
van Welie I, Wadman WJ, van Hooft JA. Low affinity block of native and
cloned hyperpolarization-activated Ih channels by Ba2⫹ ions. Eur J Pharmacol 507: 15–20, 2005.
Wainger BJ, DeGennaro M, Santoro B, Siegelbaum SA, Tibbs GR.
Molecular mechanism of cAMP modulation of HCN pacemaker channels.
Nature 411: 805– 810, 2001.
Yamada R, Kuba H, Ishii TM, Ohmori H. Hyperpolarization-activated
cyclic nucleotide-gated cation channels regulate auditory coincidence detection in nucleus laminaris of the chick. J Neurosci 25: 8867– 8877, 2005.
Ying SW, Jia F, Abbas SY, Hofmann F, Ludwig A, Goldstein PA.
Dendritic HCN2 channels constrain glutamate-driven excitability in reticular thalamic neurons. J Neurosci 27: 8719 – 8732, 2007.
Zolles G, Klocker N, Wenzel D, Weisser-Thomas J, Fleischmann BK,
Roeper J, Fakler B. Pacemaking by HCN channels requires interaction
with phosphoinositides. Neuron 52: 1027–1036, 2006.
103 • MAY 2010 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.1 on October 26, 2016
Mo Z, Davis RL. Endogenous firing patterns of murine spiral ganglion
neurons. J Neurophysiol 77: 1294 –1305, 1997a.
Mo ZL, Davis RL. Heterogeneous voltage dependence of inward rectifier
currents in spiral ganglion neurons. J Neurophysiol 78: 3019 –3027, 1997b.
Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A. Cellular
expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem
268: 1646 –1652, 2001.
Moser T, Neef A, Khimich D. Mechanisms underlying the temporal precision
of sound coding at the inner hair cell ribbon synapse. J Physiol 576: 55– 62,
2006.
Nolan MF, Malleret G, Dudman JT, Buhl DL, Santoro B, Gibbs E,
Vronskaya S, Buzsáki G, Siegelbaum SA, Kandel ER, Morozov A. A
behavioral role for dendritic integration: HCN1 channels constrain spatial
memory and plasticity at inputs to distal dendrites of CA1 pyramidal
neurons. Cell 119: 719 –732, 2004.
Nolan MF, Malleret G, Lee KH, Gibbs E, Dudman JT, Santoro B, Yin D,
Thompson RF, Siegelbaum SA, Kandel ER, Morozov A. The hyperpolarization-activated HCN1 channel is important for motor learning and
neuronal integration by cerebellar Purkinje cells. Cell 115: 551–564, 2003.
Oertel D. The role of timing in the brain stem auditory nuclei of vertebrates.
Ann Rev Physiol 61: 497–519, 1999.
Palmer AR, Russell IJ. Phase-locking in the cochlear nerve of the guinea-pig
and its relation to the receptor potential of inner hair-cells. Hear Res 24:
1–15, 1986.
Parks TN. The AMPA receptors of auditory neurons. Hear Res 147: 77–91,
2000.
Pian P, Bucchi A, Decostanzo A, Robinson RB, Siegelbaum SA. Modulation of cyclic nucleotide-regulated HCN channels by PIP(2) and receptors
coupled to phospholipase C. Pflügers Arch 455: 125–145, 2007.
Postlethwaite M, Hennig MH, Steinert JR, Graham BP, Forsythe ID.
Acceleration of AMPA receptor kinetics underlies temperature-dependent
changes in synaptic strength at the rat calyx of Held. J Physiol 579: 69 – 84,
2007.
Raman IM, Zhang S, Trussell LO. Pathway-specific variants of AMPA
receptors and their contribution to neuronal signaling. J Neurosci 14:
4998 –5010, 1994.
Rathouz M, Trussell L. Characterization of outward currents in neurons of
the avian nucleus magnocellularis. J Neurophysiol 80: 2824 –2835, 1998.
Reyes AD, Rubel EW, Spain WJ. Membrane properties underlying the firing
of neurons in the avian cochlear nucleus. J Neurosci 14: 5352–5364, 1994.
Robertson D. Possible relation between structure and spike shapes of neurones
in guinea pig cochlear ganglion. Brain Res 109: 487– 496, 1976.
Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents:
from molecules to physiological function. Ann Rev Physiol 65: 453– 480,
2003.
Rodrigues AR, Oertel D. Hyperpolarization-activated currents regulate excitability in stellate cells of the mammalian ventral cochlear nucleus. J
Neurophysiol 95: 76 – 87, 2006.
Rothman JS, Manis PB. Differential expression of three distinct potassium
currents in the ventral cochlear nucleus. J Neurophysiol 89: 3070 –3082,
2003a.
Rothman JS, Manis PB. The roles potassium currents play in regulating the
electrical activity of ventral cochlear nucleus neurons. J Neurophysiol 89:
3097–3113, 2003b.
2543