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1Oregon Hearing Research Center, Department of Otolaryngology/Head and Neck Surgery, Oregon Health & Science University Portland, Oregon 97239; 2Department of Otolaryngology, Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, 200031 People's Republic of China; 3Department of Otolaryngology, Chonbuk National University Medical School, Chonju, Chonbuk, 561-712 Korea; 4Department of Otolaryngology, Albert Szent-Gyorgyi Medical University, H-6724 Szeged, Hungary; and 5Kresge Hearing Research Institute, The University of Michigan, Ann Arbor, Michigan 48109-0506
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ABSTRACT |
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INTRODUCTION |
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;
Szallasi and Blumberg 1999).
Capsaicin exerts its effect on nociceptors by selectively activating vanilloid
receptors (VRs), the ligand-gated nonselective cation channels, leading to
membrane depolarization and action potential generation. In addition, the
activation of VRs in peripheral vanilloid-sensitive nerve endings is
responsible for neurogenic inflammation that is characterized by vasodilation
and vascular permeability increase (Kress
et al. 1997; Szallasi and
Blumberg 1999; Vyklicky et al.
1998). A subtype of VR, called VR1 (now named TRPV1. TRP stands
for transient receptor potential channel)
(Montell et al. 2002), has
been cloned and shown to be expressed in a subset of sensory neurons in the
dorsal root (DRG) and trigeminal ganglia (TG)
(Caterina et al. 1997). The
expression of TRPV1 is not restricted to primary sensory neurons. Instead, it
is reported that TRPV1 and TRPV2 (formerly named VRL-1, VR-like protein)
receptors are widely expressed in the brain and some nonneuronal tissues,
which places VRs in a much broader perspective than pain perception
(Mezey et al. 2000;
Minke and Cook 2002;
Sasamura et al. 1998;
Szallasi and Blumberg 1999).
Moreover, the expression of a VR-related osmotically activated channel has
been demonstrated in mouse inner ear (i.e., the inner and outer hair cells and
stria vascularis) as well as in neurosensory cells in the CNS responsible for
systemic osmotic pressure, TG, and Merkel cells
(Liedtke et al. 2000). In
cochlear physiology studies, it has been reported that capsaicin regulates
guinea pig cochlear blood flow via substance P (SP) and nitric oxide
(NO)-mediated mechanism (Vass et al.
1994,
1995,
1996). However, the existence
and distribution of VRs in the organ of Corti remain unknown as is whether VRs
may play any role in hearing sensitivity if they do exist in the cochlea.
Here, we report that vanilloids can regulate cochlear sensitivity and that
TRPV1 receptors are present in the organ of Corti. Some of the preliminary
data of this study have been presented
(Zheng et al. 2001a). |
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METHODS |
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In mammalian cochlea, auditory signal transduction is accomplished by concerted action of the inner and outer hair cells (IHCs and OHCs) in the organ of Corti. The IHCs detect the acoustic signal and transfer it into action potentials of the auditory nerve. OHCs amplify the sound-evoked motion of the basilar membrane (BM) through an active feed-back mechanism that assures the high sensitivity, sharp tuning, and large dynamic range for acoustic signal processing in the cochlea. Cochlear sensitivity is also determined by other factors including the blood supply, and the electrical and ionic environment of the cochlear fluids. In this study, we investigated the effects of capsaicin and its agonist on the cochlear physiology at various aspects as described in the following text, to provide a comprehensive description of how the vanilloids act in the cochlea.
ANIMAL PREPARATION. Thirty-six pigmented guinea pigs (strain 2NCR, obtained from the Charles River Laboratory) with Preyer's reflex weighing 250–350 g were used in this study for physiological experiments. The animals were housed in American Association for Accreditation of Laboratory Animal Care approved facilities. Experimental protocols were approved by the Committee on the Use and Care of Animals, Oregon Health & Science University. The animals were anesthetized using both ketamine (40 mg/kg im) and xylazine (10 mg/kg im). Supplemental doses of ketamine and xylazine were given on a schedule or as needed, judging by leg withdrawal to a toe pinch.
Rectal temperature of the animals was maintained at 38 ± 1°C with a servo-regulated heating blanket. Cochlear temperature was additionally controlled by supplemental heat to the head from a lamp and a heated head-holder. The electrocardiogram and heart rate were continuously monitored as measures of anesthesia level and the general condition of the animal. All presented data were collected from animals with normal electrocardiograms and with heart rate between 270 and 380 beats/min.
The guinea pig's head was firmly fixed in a heated head-holder, which was mounted on a custom-made manipulator and electrically isolated from the operation table. A tracheotomy was performed, and a ventilation tube was inserted into the trachea to insure free breathing. A ventral and postauricular combined approach was used to expose the left auditory bulla with a large part of the external ear being removed, while the medial 2/3 of the external auditory canal was preserved to facilitate placement of the acoustic speculum. The bulla was widely opened to expose the cochlea. The middle ear muscle tendons were carefully sectioned.
MEASUREMENT OF COCHLEAR POTENTIALS. Compound action potential
(CAP) and cochlear microphonic (CM) are sound-evoked cochlear potentials used
for cochlear function assessment. The CAP is the action potential produced by
synchronized auditory nerve firing in response to brief acoustic stimuli and
is used to assess overall cochlear sensitivity at different frequencies. In
contrast, the CM is an AC receptor potential produced primarily by the OHCs of
the organ of Corti during acoustic stimulation. For CAP and CM measurement, a
ball electrode made of Teflon-coated silver wire (75 µm in diameter) was
placed in the round window niche and fixed on the bulla with carboxylate
cement. An Ag/AgCl wire was inserted into neck soft tissue medial to the
exposed bulla to serve as the ground electrode. A plastic three-way coupler
with two speakers (made of 
-in B&K microphones) and an Etymotic 10
B+ microphone were fitted to the ear canal to deliver acoustic
stimuli and record the otoacoustic emissions (OAEs). Tone bursts (10 ms in
duration, 1-ms rise/fall) were generated using a 16-bit D/A converter (Tucker
Davis Technologies) and delivered to the ear canal as acoustic stimuli to
evoke the CAP and CM. The round window signal was amplified 1,000 times by an
AC preamplifier (Grass Instrument, Model P15) and a custom-designed AC
amplifier. After A/D conversion and averaging, the evoked electrical responses
from the round window were digitalized and saved for post processing for the
CAP and CM. The amplified signal was also displayed on an oscilloscope for CAP
threshold assessment to monitor the hearing sensitivity during the experiment.
The N1 detection of 10 µV without averaging was used as the CAP
threshold criterion.
The electrophysiology of the IHCs and OHCs relies on the normal level of
the endocochlear potential (EP), a large positive resting DC potential in the
scala media of the cochlea duct, which is generated and maintained by the
normal function of the stria vascularis, and integrity of the structure of the
scala media (i.e., the perilymph-endolymph barrier). For EP recording, a small
hole was carefully drilled in the cochlear bony wall of the second turn. A
glass micro-electrode (tip diameter: 
0.5 µm) filled with 150 mM KCl
and held on a micromanipulator was inserted into the scala media through the
spiral ligament and stria vascularis. An Ag/AgCl wire was inserted into neck
muscles to serve as the ground electrode. The DC potential was measured with
an amplifier (BMA 200 AC/DC Bioamplifier, CWE) and recorded with a
computerized chart recorder.
EXTRACOCHLEAR CURRENT STIMULATION AND EEOAE MEASUREMENT.
Electrically evoked OAE (EEOAEs) produced by extracochlear current stimulation
have been used as a noninvasive tool to investigate the in vivo
electromotility of OHCs (Nuttall et al.
2001; Ren and Nuttall
1995). The method for EEOAE recording used in this study was the
same as what has been described (Ren and
Nuttall 1995; Zheng et al.
2001b). In brief, a Teflon-insulated platinum-iridium wire (75
µm diam) with a bare end was placed in the round window niche for
electrical stimulation. A sinusoidal signal was generated by a
computer-controlled lock-in amplifier (SR830 DSP, Stanford Research Systems),
and AC (35 µA RMS) was delivered to the stimulation electrode by a
custom-made opto-isolated constant current stimulator. An Ag/AgCl ground
electrode was inserted into the neck muscle next to the ipsilateral bulla. The
EEOAE was recorded from the ear canal with a microphone (Etymotic Research
ER-10B+, Elk Grove Village, IL). The magnitude and phase of the
output signal from the microphone preamplifier was measured at the stimulus
frequency using the lock-in amplifier and was recorded with a computerized
chart recorder at the sampling rate of two samples per second. Each amplitude
spectrum and its corresponding phase spectrum were obtained by linearly
sweeping the current from 400 Hz to 40 kHz at 50 Hz/s with a lock-in time
constant of 1 s.
A recently developed multiple-component analysis method (MCA)
(Ren and Nuttall 2000;
Ren et al. 2000) was used to
measure the multiple delays of the EEOAEs in this study. The real part of the
electrically evoked OAE (X) was calculated from the amplitude
(A) and phase (
) spectra according to: X = A
cos () and presented as a function of the frequency. The
"delay" spectrum of X was obtained using the fast Fourier
transform (FFT).
MEASUREMENT OF BM VELOCITY. To establish whether vanilloid
agonists alter the cochlear mechanical responses, the magnitude and phase
transfer function of BM transverse velocity was measured at the 17-kHz
best-frequency location. To record the responses of the BM, a small opening
was made in the first-turn scala tympani bony wall of the cochlea. Gold coated
glass beads (20 µm diam) were "dropped" onto the BM (at
approximately the 17-kHz place) to serve as reflective objects that track the
motion of the BM. The laser beam of a laser Doppler velocimeter (Polytec, OFV
1102) was focused on a bead with the aid of a compound microscope
(Nuttall et al. 1991). Sounds
were presented to the external ear as described in the preceding text. The
signal output of the velocimeter was digitalized at the sampling rate of 250
kHz and stored in the computer. BM velocities were determined after fast
Fourier transform of the Hanning-windowed responses from the velocimeter.
COCHLEAR BLOOD FLOW MEASUREMENT. It has been reported that round
window membrane application of capsaicin resulted in an increase of the
cochlear blood flow (CBF) (Vass et al.
1995). This study determines the CBF change that occurs when
capsaicin is directly infused into the cochlea. We also determine the
relationship between the alterations of CBF and EP during capsaicin perfusion,
inasmuch as the EP depends on the CBF. The CBF was measured using the laser
Doppler flowmetry (LDF) technique (Miller
and Nuttall 1990). Briefly, the cochlear mucosa was gently removed
with a cotton pledget. The probe of the LDF (Laser Doppler System, Type PF
4001, Perimed) was placed on the lateral wall of the basal turn of the cochlea
to detect the blood flow. Petroleum gel was applied to the probe tip to
provide efficient laser light coupling to the cochlea. All recordings were
done under stable light illumination. The change of CBF is presented as
percentage value relative to preexposure level.
PERILYMPHATIC PERFUSION OF THE SCALA TYMPANI. Perilymphatic
perfusion was performed to deliver chemicals into the scala tympani of the
cochlea. An inlet hole (diameter: 70 µm) was made in the scala tympani
of the basal turn of the cochlea close to the round window niche and the
outlet hole (diameter: 80 µm) on the apex of the cochlea. A three-way
perfusion device that allows solution substitution was used for scala tympani
perfusion. A polyethylene tube was connected to this device and its fine tip
(diameter: 60 µm) was inserted into the inlet hole of the cochlea.
Tissue glue was applied to seal the inlet hole and fasten the tube in
position. Stock solutions were prepared as follows. Capsaicin (20 mM) was
dissolved with a mix solution of ethanol (10%) and Tween-80 (10%).
Resiniferatoxin (RTX; 2 mM) was dissolved in absolute ethanol. Capsazepine (20
mM) was dissolved in absolute methanol. The preceding stock solutions were
diluted with artificial perilymph [(in mM) 125 NaCl, 3.5 KCl, 25
NaHCO3, 1.3 CaCl2, 1.14 MgCl2 ·
6H2O, 0.51 NaH2PO4 · H2O,
5.0 Tris, 3.3 glucose, 2.1 urea] for required concentrations just prior to
use. The pH of all solutions was 7.4 and the osmolality was 300 ± 10
mOsm. Perfusates were infused into the scala tympani at a perfusion rate of 2
µl/min using a syringe pump (Sage Instruments, Model 351). The duration of
perfusion for each chemical was usually 10 min but was prolonged to as long as
30 min in certain cases as needed. Artificial perilymph was infused into the
cochlea in control experiments before chemical perfusion and for drug washout.
The duration for drug washout was usually 30 min. Effluent from the outlet
hole on the apex was absorbed within the bulla using cotton wicks.
Immunolabeling of TRPV1 receptors
Eight adult guinea pigs and 10 rats with Preyer's reflex weighing
300–350 g were used, since the available antibodies for TRPV1
immunolabeling are specific for rats. Animals were anesthetized with ketamine
(100 mg/kg im) and xylazine (2 mg/kg im), and a cardiac perfusion with saline
followed by 4% paraformaldehyde in 0.02 M PBS was performed. The organ of
Corti and DRG (positive control) of either the guinea pig or rats were
dissected and fixed in the solution of 4% paraformaldehyde in 0.02 M PBS for 3
h. The fixed tissues were washed in 0.02 M PBS (pH 7.4), permeabilized with
0.5% Triton X-100 (Sigma) for 1 h and immunoblocked in 10% goat serum in 1%
bovine albumin in 0.02 M PBS for 1 h. They were then incubated with rabbit
anti-TRPV1 polyclonal antibody (gift from Dr. Caterina) diluted 1:1,000 in 1%
BSA-PBS for 48 h. After washing in 1% BSA-PBS, tissues were subsequently
incubated with Alexa-488-conjugated goat anti-rabbit IgG for 3 h (Molecular
Probes; 1:100 in 0.02 M PBS). The labeled tissues were mounted as cochlear
surface preparations or slices of DRG in VectaShield (Vector Labs) and
observed under a combined microscopy system (a Nikon Eclipse TE 300 inverted
microscope fitted with a Bio-Rad MRC 1024 confocal scanning laser system).
Mounted tissues were imaged using confocal optical slicing techniques, with a
typical step size of 2 µm, and stacks of images were postprocessed for
image analysis. For negative immunocytochemical controls, the primary antibody
was replaced with either 1% BSA-PBS or primary antibodies adsorbed with a
peptide encoding the predicted carboxyl terminus of VR1-EDAEVFKDSMVPGEK
(Tominaga et al. 1998), prior
to imaging at the same laser and confocal settings as experimental
specimens.
Statistical analysis
Animals were divided into two major groups: the cochlear physiology group and the immunohistochemistry group. In the cochlear physiological group 36 guinea pigs were used. This group was further divided into four subgroups according to different measurements of the cochlear physiology and the use of different VR agonists: capsaicin and capsazepine on the CAP, CM, and EEOAEs (n = 15); RTX and capsazepine on the CAP, CM, and EEOAEs (n = 10); capsaicin and capsazepine on the CBF and EP (n = 8); and capsaicin and capsazepine on the BM (n = 3). However, the numbers shown in the results may be different due to different combinations of physiological measurement and chemical administration in each subgroup. Group data of VR agonists or antagonist-induced changes in cochlear physiology indexes are presented in means ± SE. An ANOVA with a repeated-measures design was utilized to determine significant difference across treatment groups. A probability of <0.05 is considered a statistically significant difference.
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RESULTS |
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The CAP, CM, and EEOAEs provide information on various aspects of cochlear
function and therefore were used in this study as indexes of cochlear
performance. It was found that perfusion of 20 µM capsaicin into the scala
tympani resulted in reduction of cochlear sensitivity as shown by the
alterations of the CAP and CM (Fig.
1). The CAP thresholds were tested with tones from 2 to 36 kHz
that cover most of the hearing frequency range of guinea pigs. Elevation of
CAP threshold by 10 dB was observed after 20 µM capsaicin perfusion
(Fig. 1A). To further
investigate the effect of capsaicin on the auditory nerve activity, CAP
response versus sound level (input-output) functions at 8 and 18 kHz were
analyzed. The magnitude of the CAP was reduced by capsaicin by a small but
statistically significant degree and with a parallel shift of the input-output
function curves (Fig. 1, B and
C). Furthermore, the reduction of the CM magnitude (at 8
and 18 kHz) by capsaicin was similar to that of CAP input-output functions in
both reduction degree and pattern (Fig. 1,
D and E). Both the CAP and CM recovered to
normal after washout with artificial perilymph, indicating the effect of
capsaicin administered in this experiment to be reversible (data not shown).
In vitro experiments have shown capsaicin's effective concentrations for
specific effects to be below 1 µM (EC50 = 0.52–0.9 µM)
(Caterina et al. 1997;
Welch et al. 2000). However,
in our pilot experiment, we did not observe any effects of capsaicin until the
concentration reached 10 µM, and the effects at this concentration were
very subtle. At 20 µM, consistent and obvious effects on cochlear
potentials were observed. Due to the concern of possible nonspecific effects
at high concentration, we limited this study to 20 µM capsaicin. To verify
the specificity of capsaicin's effects on the evoked cochlear potentials,
capsazepine, a competitive antagonist of VRs which alone has no effect on
cochlear potentials, was perfused into the cochlea in advance of capsaicin
application. Capsaicin perfusion immediately after 20 µM capsazepine
application did not affect cochlear potentials
(Fig. 1). After capsazepine was
washed out from the cochlea by artificial perilymph perfusion for ≥30 min,
suppression of cochlear potentials by capsaicin application could be observed
again. This indicates that capsazepine causes a complete and specific block of
capsaicin's effects.
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Capsaicin perfusion at 20 µM reduced overall EEOAE magnitude by 5
dB without apparent effect on the fine structure (the peaks and notches in the
magnitude spectra of EEOAEs; Fig.
2A). In certain cases (data not shown here), the notches
in the fine structure became deeper, making the fine structure more profound.
The effect of capsaicin on EEOAEs was reversible after washout with artificial
perilymph (Fig. 2B).
Also, perfusion with 20 µM capsazepine in advance blocked capsaicin's
effect on EEOAEs (Fig. C). MCA showed that capsaicin reduced both the
short and long delay components of the EEOAEs
(Fig. 2D).
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Effects of RTX on cochlear potentials and EEOAEs
To further verify that the observed capsaicin effects were mediated by VRs,
effects on cochlear sensitivity by a potent agonist of VRs, RTX, was
investigated. RTX has been shown to be at least ten times more potent than
capsaicin (Caterina et al.
1997). Intracochlear perfusion of 2 µM RTX also reduced
cochlear potentials and EEOAEs in a same manner as that induced by 20 µM
capsaicin (see Figs. 3 and
4), suggesting the similar
effects of capsaicin and RTX on cochlear sensitivity with ≥10 times
difference in potency. However, recovery after washout of RTX was difficult to
achieve. The effects of RTX could be completely blocked by capsazepine (20
µM) applied in advance.
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Time dependence of CM alteration during capsaicin and RTX perfusion
To investigate the time course of hearing suppression effects by capsaicin
and RTX, we monitored the alteration of the CM evoked by 8-kHz tone burst at
40 dB SPL during the VR agonist perfusion. Reduction of CM magnitude was
observed within 1–2 min after capsaicin perfusion
(Fig. 5, A and
B). This effect was absent when capsazepine was applied
in advance (Fig. 5C).
In most animals (n = 10), the CM decreased gradually during perfusion
(Fig. 5A). In some
animals (n = 2), the CM decreased with a fast early reduction phase
followed by a slower reduction phase (Fig.
5B). After the termination of capsaicin perfusion the CM
showed a gradual recovery (Fig.
5A). The time course for the onset of RTX's effects was
similar to that of capsaicin; however, we only observed the slow CM reduction
pattern by RTX perfusion (n = 5). In the case of prolonged perfusion
(≤20 min), a partial recovery in CM magnitude occurred 15 min after
the onset of perfusion with either capsaicin or RTX (n = 7 and
n = 5, respectively; Fig. 5,
D and E). The recovery of CM in this case is
evidence of the desensitization phenomenon of VRs that has been observed in
primary sensory neurons (Szallasi and
Blumberg 1999).
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Effect of capsaicin and RTX on cochlear blood flow and EP
Round window membrane application of capsaicin has been shown to increase
cochlear blood flow (CBF) (Vass et at.
1995). In the current study, we found that intracochlear perfusion
of capsaicin (20 µM) could increase CBF in a pattern similar to that with
round window application (Fig.
6A). During a 10-min capsaicin perfusion, the CBF
increased very quickly and reached the peak (about 140% of the initial level)
in 2 min. It fell to a level 10% above the preexposure level in
4 min and then had a slower return to the preexposure level. This CBF
increase effect could be blocked by capsazepine.
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Although an increase of CBF was observed, there is a possibility that the alteration of cochlear sensitivity might be the consequence of EP reduction. To test this possibility, the effect of capsaicin and RTX on EP was investigated in 8 animals. The EP was not altered with either capsaicin or RTX perfusion. Figure 6B presents the EP data during 20 µM capsaicin perfusion.
Effect of capsaicin on BM motion
BM velocity was measured at the 17-kHz best-frequency location in three animals. Data from these animals showed consistent alteration in BM motion by capsaicin. Figure 7 illustrates an example of such alteration in which the velocity growth functions near the best frequency (BF) were altered (Fig. 7A) and relatively unchanged at frequencies well below BF (Fig. 7B). These changes are typical for an insensitive cochlea, indicating a suppressed cochlear amplifier. The mechanical amplification of proposed cochlear amplifier is frequency dependent and most efficient at low sound levels. Thus it is the low sound-level portions of the growth functions near BF that are most affected. Figure 7C shows the iso-velocity tuning curve derived by determining the sound level required to produce a 10-µm/s criterion velocity response at various frequencies. The reduction of the cochlear sensitivity displays as a less sharply tuned curve. This is another hallmark of a reduction of the gain of the cochlear amplifier. Of particular note is that no change in the mechanical response of the BM occurred at low frequencies (e.g., 8 kHz). Therefore the lack of BM motion reduction by capsaicin at low frequencies cannot account for the reduction of the CM (e.g., Figs. 1 and 3) at these low frequencies. Because EP is also not reduced, a depolarization of the OHCs by capsaicin is the likely mechanism.
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Immunolabeling and distribution of TRPV1 receptors in the rodent cochlea
Immunofluorescence microscopy was used to determine the distribution of
TRPV1 in the organ of Corti of rat and guinea pigs using an anti-rat TRPV1
antibody. Confocal microscopy of the organ of Corti was conducted
perpendicular to the lumenal surface of the sensory epithelium, resulting in
horizontal optical sections of the cochlear surface preparation. In the rat
organ of Corti, TRPV1 labeling was evident in the OHCs
(Fig. 8A) with
comparatively weaker labeling in the IHCs
(Fig. 8C). TRPV1
expression could not be detected in the stereocilia of hair cells. Positive
labeling for TRPV1 was also found in some supporting cells and in particular
in the inner and outer pillar cells, and in Hensen's cells
(Fig. 8, A–C,
respectively). The labeling in outer pillar cells was somewhat weaker than in
OHCs (average intensity = 70.7 ± 14.1; and 85.7 ± 11.3,
respectively; n = 10; P < 0.01), and both cell types were
significantly more intensely labeled than IHCs (45.5 ± 4.0; P
< 0.001). TRPV1 expression was also found in the spiral ganglion neurons of
the rat cochlea (Fig.
8D), confirming the previous report of TRPV1 in cochlear
afferent neurons (Balaban et al.
2003).
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In control experiments, when the primary TRPV1 antibody was replaced with
PBS-BSA, negligible nonspecific labeling could be observed
(Fig. 8, E and
F). When the primary antibody was replaced with
antibodies preadsorbed with a synthetic blocking peptide sequence
(Tominaga et al. 1998), prior
to incubation with organ of Corti material, negligible nonspecific labeling
was observed in the organ of Corti (Fig. 8,
G and H). In positive control experiments,
strong immunolabeling for TRPV1 was found in rat DRG neurons
(Fig. 8I) that are
known to express TRPV1 in previous studies
(Caterina et al. 1997). When
the primary TRPV1 antibody was replaced with PBS-BSA or adsorbed with a
synthetic blocking peptide sequence prior to incubation with rat DRG tissues,
negligible nonspecific labeling could be observed
(Fig. 8, J and
K).
In the guinea pig organ of Corti, TRPV1 labeling was also present in the
OHCs and IHCs but not in the stereocilia. Positive labeling for TRPV1 was also
present in the outer and inner pillar cells, in Hensen's cells, and in the
spiral ganglion neurons (data not shown). In guinea pig control experiments,
when the primary TRPV1 antibody was replaced with PBS-BSA or antibodies
preadsorbed with a synthetic blocking peptide sequence
(Tominaga et al. 1998),
negligible nonspecific labeling was observed in the organ of Corti. In
positive control experiments, strong immunolabeling for TRPV1 was found in
guinea pig DRG neurons and absent when the primary TRPV1 antibody was replaced
with PBS-BSA or adsorbed with a synthetic blocking peptide sequence (data not
shown). Thus the anti-rat TRPV1 antibody we used in guinea pigs showed the
same pattern of positive labeling (albeit weaker) as in the rat.
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DISCUSSION |
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Capsaicin and its ultrapotent analog RTX specifically activate VRs when
used in proper concentration and hence have long been used to characterize the
VR-mediated physiology of sensory neurons (e.g.,
Helliwell et al. 1998;
Szallasi and Blumberg 1999;
Szallasi et al. 1999) and
nonneruronal cells (e.g., Biro et al.
1998a,b).
Also, it is known that capsaicin has nonspecific effects (non-VR-mediated
capsaicin actions) on other ion channels as well as various enzymes and cell
membrane properties when applied with high concentration
(Bevan et al. 1992;
Szallasi and Blumberg 1999).
Nonspecific effects can be ruled out for three reasons in the current study.
1) Although the capsaicin concentration we used for perilymphatic
perfusion was ≥20 times higher than for in vitro experiments
(EC50 = 0.52–0.9 µM)
(Caterina et al. 1997;
Welch et al. 2000), the
effective concentration in the organ of Corti will be much less than that in
the perilymph because of diffusion from the perilymph of the scala tympani
into the organ. 2) The action of 20 µM capsaicin on the cochlear
sensitivity was completely blocked by capsazepine, a competitive VR antagonist
that is effective against both capsaicin and RTX
(Bevan et al. 1992;
Maggi et al. 1993;
Perkins and Campbell 1992;
Szallasi and Blumberg 1999;
Urban and Dray 1991;
Wardle et al. 1997).
3) RTX was used at a concentration within its effective concentration
range as determined by in vitro experiment
(Docherty et al. 1997).
Identical effects on cochlear sensitivity were seen for RTX, which has a
unique spectrum of biological activities that is devoid of most undesirable,
capsaicin-like side effects (Szallasi and
Blumberg 1999). Therefore the findings in this study imply
functional VRs in the cochlea. This possibility is reinforced by
immunolabeling experiment showing the presence of TRPV1 receptor in the organ
of Corti.
VRs are members of OSM-9 family in the TRP (transient receptor potential)
channel superfamily of Ca2+-permeable ion channels
(Caterina et al. 1997;
Harteneck et al. 2000;
Minke and Cook 2002). Binding
of capsaicin and other vanilloids to VRs initiates a complex and, as yet,
poorly understood cascade of intracellular events, which not only lead to
excitation of nerves but also desensitization and neurotoxicity
(Caterina et al. 1997;
Szallasi and Blumberg 1999).
Electrophysiological studies have shown that the VR channel pore opening leads
to cation (predominantly Ca2+) influx and may in turn
cause depolarization, resulting in neuron excitation
(Bevan and Docherty 1993;
Marsh et al. 1987;
Szallasi and Blumberg 1999;
Wood et al. 1988). In the
current experiment, reduction of CM and EEOAEs suggests a primary site of
capsaicin and RTX's action in the cochlea to be on the OHCs by the rationale
given in the following text and to result in a suppressive effect. It is
possible that the alteration of OHC function by vanilloids occurred through a
VR-mediated downward-regulation of OHC motility. Arguments of this hypothesis
will be further discussed in the following text.
Continuous monitoring of CM revealed the time course of capsaicin and RTX's
effects on cochlear sensitivity being 2 min after the onset of perfusion
(Fig. 5). This may represent
the time for these vanilloids to diffuse and access the OHCs in the organ of
Corti and exert their effects. The CM magnitude gradually decreased during
perfusion, reaching the maximum effect in 15 min, suggesting the pattern
of vanilloid concentration increase during the perfusion. It may also
represent the time course for maximum activation of vanilloids at a given
concentration. Longer continuous application of the vanilloids failed to
sustain the effect at its maximum level
(Fig. 5, D and
E), indicating a possible desensitization phenomenon of
vanilloids' action on the organ of Corti. It was speculated that
desensitization is a consequence of agonist-induced conformational change in
VR protein that decreases the responsiveness of the ion channel.
(Liu and Simon 1996). The
brief CBF increase effect by capsaicin may also be attributable to the
desensitization. However, the time course and shape of this effect are
different from that on the CM.
Characteristics of cochlear function modulation by vanilloids
Physiological results of this study suggest actions of vanilloids on at
least two major sites in the cochlea. 1) Primary sensory nerves on
cochlear vessels. The capsaicin-induced increase of the CBF is identical to
that reported by Vass et al.
(1995). The targeted vessels
may include vessels of the spiral modiolar artery (SMA) and its arterioles.
The basis for a CBF change could be a capsaicin-stimulated release of SP from
possible primary sensory fibers innervating the cochlea (Vass et al.
1995,
2001). Despite the alteration
in CBF, the EP was not affected by either capsaicin or RTX, indicating the
normal functioning of the stria vascularis that maintains the electrical and
ionic environment of the endolymphatic compartment of the cochlea. The
reduction of cochlear sensitivity by vanilloids therefore cannot be attributed
to EP reduction. The most parsimonious explanation is as a consequence of the
action of VR agonists on the organ of Corti, especially, on the OHCs.
2) OHCs. Reduction of the CM by capsaicin and RTX suggests a primary
effect on OHCs, as these cells are the primary site for CM generation and
their responses are known to dominate the CM signal measured at the round
window (e.g., Patuzzi et al.
1989). Decrease of CM magnitude may represent the reduction in
mechanical drive to the cell (i.e., less stereocilia motion) or reduction in
transduction current passing into the cell from an altered electro-chemical
potential (i.e., reduced EP or cell depolarization) or from an altered cell
electrical conductance. The lack of change of BM velocity for low-frequency
stimuli and the lack of EP reduction with capsaicin application indicate that
the observed CM reduction is likely due to an OHC conductance increase from
the TRPV1 channel that also depolarizes the OHC. However, we also observe
TRPV1 immunolabeling in supporting cells of the organ of Corti, with pillar
cells having the strongest signal. It is possible that altered mechanical
properties of supporting cells could reduce the OHC stereocilia motion without
apparent BM velocity change. Our data cannot rule out this possibility.
Further evidence that OHCs are affected by vanilliods is from the change in
electro-mechanical transduction (as evidenced by EEOAE mean value change) and
cochlear mechanics (BM motion). In our previous studies, we observed that fine
structure of the EEOAEs was a feature of sensitive cochlea and was related to
the long delay component of the EEOAEs, whereas the overall mean magnitude of
EEOAEs was mainly related to the short delay component and was relatively less
sensitive to OHC damage (Ren and Nuttall
2000; Zheng et al.
2001b). In addition, fine structure results from
cancellation/enhancement effects of multiple sound waves in the cochlea
(Ren and Nuttall 2000).
Preservation or enhancement of fine structure suggests that the relative
strength of the multiple waves is maintained even in the face of decreased
overall power of the OHC electromotile response. Multiple-component analysis
of the EEOAE data in this study showed that both the long and short delay
components were reduced. This observation provides evidence in favor of the
above speculation.
Based on the physiological data, we propose a hypothesis for the
vanilloid-induced suppression of OHC function. Activation of VRs on the OHCs
(probably on the basolateral wall) by vanilloids may result in
Ca2+ influx through the VR channels and may in turn
cause OHC depolarization and intracellular Ca2+ release.
Because the OHC membrane potential and the EP together provide electrical
force for transduction current through the OHCs, reduction of the OHC membrane
potential (i.e., OHC depolarization) in the face of unchanged EP will result
in a decrease of the transduction current, and hence, a reduction of the AC
receptor potential. Increase in conductance at the base of the OHCs (due to VR
ionic channel opening) could also reduce the transduction current (by a
"shunt") and reduce the AC receptor potential produced by the
stereocillia receptor current (Guinan
1996). Reduction in OHC receptor potential would consequently
reduce the voltage-dependent OHC motion that is involved in the amplification
of BM motion. In addition, Ca2+ influx-induced
intracellular Ca2+ release and the subsequent
intracellular events (probably through phosphorylation in proteins responsible
for OHC motility) may play a role as well because elevated intracellular
Ca2+ causes OHC contractions and stiffness increase in
an ATP-dependent manner that reduces the OHC motility
(Dallos et al. 1997;
Holley 1996;
Sziklai et al. 1997). The
observed decrease in CM, EEOAE and BM responses from capsaicin and RTX thus
are consistent with OHC depolarization and reduction in cochlear
amplification.
Positive expression of TRPV1 receptors in the IHCs and in the spiral
ganglion neurons of both rats and guinea pigs (also reported by
Balaban et al. 2003) implies an
action of vanilloids on these sites. Our electrophysiological and BM motion
data are not sufficient to distinguish whether this is the case. However, it
is interesting that a parallel shift of the CAP growth function is observed
after capsaicin application (Fig. 1,
B and C). Because BM velocity returns to control
values at high sound levels (Fig.
7A), the residual effect on CAP could be from IHC or
afferent neuron effects.
Potential roles of VRs in the cochlea
In primary sensory neurons VRs are activated by heat, protons, and
mechanical force, serving to transmit nociceptive information and being
responsible for neurogenic inflammation. The expression and functional roles
of VRs in other tissues remains unclear, although Birder et al.
(2001,
2002) find that bladder
epithelial cells express TRPV1 and that TRPV1 knockout mice have altered
bladder function, and Denda et
al.(2001) and Inoue et al.
(2002) find expression in
epidermal keratinocytes. In the auditory system, we propose that the VRs may
have roles in cochlear homeostasis and participate in pathophysiological
process in the cochlea; however, the endogeonous agonists for VRs in the
cochlea are not known.
First, there is a possible role of the VRs for cochlear nociception.
Because the VRs are sensitive to pH and heat, alterations in pH and
temperature could activate the VRs on the sensory fibers innervating the
cochlear blood vessels. This would result in a brief increase of CBF and
vascular permeability changes (Vass et al.
1995). The sensation that might be perceived from activation of
such sensory afferents is not known. Second, there is a possible role of the
VRs in regulating the activity of the organ of Corti. However, our data
indicate that the effects of TRPV1 activation on the hearing sensitivity will
be small. The sensory, supporting cells and spiral ganglion cells of the
cochlea may be able to respond to environmental alterations in pH,
temperature, and osmotic pressure. Protons (or reduction of pH) are able to
activate TRPV1 in normal physiological temperature (i.e., 37°C)
(Tominaga et al. 1998).
Although cochlear temperature would be expected to be regulated tightly and
the TRPV1 receptor is gated on at 43°C; however, it is also known
that protons can sensitize the channel to a lower temperature activation
threshold (Tominaga et al.
1998). Recently, VR-related osmotically activated channels TRPV4
(also known as VR-OAC or OTRPC4) were reported to be present in the organ of
Corti (Dai et al. 2002;
Liedtke et al. 2000). Specific
ion channel activation-induced Ca2+ influx (through
these channels) and subsequent Ca2+ release from
internal stores triggered by such Ca2+ influx has
already been observed in VR and VR-OAC channels in other tissues
(Caterina et al. 1997;
Liedtke et al. 2000). TRPV4 is
also gated by temperature in the normal physiological range
(Guler et al. 2002). The
molecular relationship between VRs and their TRP family members makes it
likely that this group of ion channels subserves diverse but related
physiological functions. In addition, suppression in EEOAEs and BM response
suggest that VRs may play a role in modulating the mechanics of the organ of
Corti, presumably by changing the cell turgor or stiffness. Third is the
possible role of VRs in cochlear pathophysiology. In the case of inflammation,
a combination of endogenous vanilloids and inflammatory mediators may act on
VRs, which in turn initiates the inflammatory cascade
(Szallasi and Blumberg 1999).
It is possible that the VRs may also play a similar role for hearing loss in
inflammatory processes in the inner ear or in pathological situations that may
affect inner ear lymph chemistry. Meniere's disease and autoimmune
sensorineural hearing loss are examples of such conditions.
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ACKNOWLEDGMENTS |
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This work was supported by the following grants: National Institute on Deafness and Other Communication Disorders Grants R01 DC-00105, DC-00141, and P01 DC-00078 (A. L. Nutttall), DC-04555 (P. S. Steyger), DC-04554 (T. Ren) and Veterans Affairs Research Resource and Development Center Grant RCTR-597-0160, Portland, Veterans Affairs Medical Center.
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FOOTNOTES |
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Address for reprint requests: A. L. Nuttall, Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., NRC04, Portland, OR 97239 (E-mail: nuttall{at}ohsu.edu).
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ABSTRACT
INTRODUCTION
-). B: EP was not
altered by CPS perfusion. Vertical bars on the curves represent SE. Horizontal
bars at the bottom of each figure indicate the duration of CPS perfusion.
*P < 0.05; **P < 0.01.
