Original Paper
Audiology
Audiol Neurotol 2011;16:145–157
Neurotology
DOI: 10.1159/000316674
The Actions of Dopamine Receptors in
the Guinea Pig Cochlea
Andrew R. Garrett Donald Robertson Peter M. Sellick
Wilhelmina H.A.M. Mulders
Auditory Laboratory, Discipline of Physiology M 311, School of Biomedical, Biomolecular and Chemical Sciences,
University of Western Australia, Crawley, W.A., Australia
Key Words
Cochlea ⴢ Compound action potential ⴢ Electrophysiology ⴢ
Lateral efferents ⴢ Summating potential
Abstract
Dopamine, a major lateral olivocochlear efferent neurotrans-
mitter, exerts both excitatory and inhibitory effects on the
central nervous system depending on the receptor involved.
We investigated the effects of different dopamine receptors
on the cochlea by perilymphatic perfusion with D1/5, D2 and
D3 receptor agonists and antagonists and recording neural
and hair cell responses (compound action potential – CAP;
summating potential – SP) before, during and after perfu-
sions. The D1/5 agonist resulted in marked suppression of CAP
amplitudes whilst leaving SP amplitudes unchanged, sug-
gesting an inhibitory action of these receptors on afferent
dendrites. The D1/5 antagonist had little or no effect, suggest-
ing that there is no influence of tonic dopamine release on
these receptors. In contrast, perfusing a D2 receptor antago-
nist resulted in marked suppression of CAP suggesting an ex-
citatory action of the receptors and a strong influence of ton-
ic dopamine release on the D2 receptors. The D2 agonist had
little effect, implying that tonic dopamine release is maximal-
ly activating this class of dopamine receptors. D2 antagonists
resulted in reduction of SP, cochlear microphonic and distor-
© 2010 S. Karger AG, Basel
1420–3030/11/0163–0145$38.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/aud
Received: November 12, 2009
Accepted after revision: June 1, 2010
Published online: July 29, 2010
tion product otoacoustic emission amplitudes, suggesting
that D2 receptor action is not confined to afferent dendrites.
Perfusion with D3 agonists and antagonists had no effect.
Copyright © 2010 S. Karger AG, Basel
Introduction
Medial and lateral olivocochlear efferent systems orig-
inate in the brainstem and project to the mammalian
cochlea [Warr and Guinan, 1979]. While a considerable
amount is known regarding the neurochemistry and
physiology of the medial system, no clear picture exists
for the lateral system. Lateral efferents contain a wide
range of neurotransmitters including dopamine [Alt-
schuler et al., 1984, 1985; Eybalin et al., 1993; Fex and
Altschuler, 1984; Fex et al., 1986; Gil-Loyzaga et al., 1997;
Jones et al., 1987; Maison et al., 2003; Safieddine et al.,
1997; Sliwinska-Kowalska et al., 1989]. Excitatory [Le
Prell et al., 2003, 2005] and inhibitory actions [Darrow et
al., 2006] attributed to the lateral innervation have been
inferred from lesion studies in guinea pigs and mice. Al-
though species differences may be involved [Le Prell,
2007], divergent effects have also been found within one
species following putative lateral efferent stimulation
[Groff and Liberman, 2003].
Wilhelmina H.A.M. Mulders
Auditory Laboratory, Discipline of Physiology M 311
School of Biomedical, Biomolecular and Chemical Sciences
University of Western Australia, 35 Stirling Highway, Crawley, W.A. 6009 (Australia)
Tel. +61 8 6488 3321, Fax +61 8 6488 1025, E-Mail hmulders @ cyllene.uwa.edu.au
 In the central nervous system, dopamine can exert Table 1. Affinity binding constants (K i; in nM) for all agonists and
both excitatory and inhibitory effects depending on the antagonists
receptor class activated [Civelli et al., 1993; Missale et al.,
D1/5 D2 D3 D4
1998], and several studies have identified diverse dopa-
mine receptor subtypes in the cochlea [Inoue et al., 2006; Agonists
Karadaghy et al., 1997; Niu and Canlon, 2006]. Real-time SKF 81297 5.9 d7d >1000d >1000d >1000d
polymerase chain reaction (RT-PCR) studies in rat co- SKF 38393 18d >1000d >1000d >1000d
chlear homogenates identified only the mRNA for the (+)PHNO 80h 0.14 /0.24 0.6f
e f
79j
PD 128907 6428l 620b 0.99b 720b
D2L and D3 receptor subtypes [Karadaghy et al., 1997], but
a more recent study identified all dopamine receptors in Antagonists
spiral ganglion cells and in regions beneath inner hair SCH 23390 0.3c 760i 480k 3560j
L 741626 ND 11.2a 163a 1520a
cells (IHCs) consistent with receptors being localized to U 99194A 100000m 1572g 78g *
neural elements [Inoue et al., 2006]. Niu and Canlon
[2006] used immunocytochemical methods and Western ND = No data available; * = no measurable affinity [Waters et
blot analysis to show that dopamine D1 receptor protein al., 1993, cited by LaHoste et al., 2000].
a Grundt et al. [2007]. b This is for the D isoform [Bowery et
is located both on spiral ganglion cells and underneath 2S
al., 1996]. c Sunahara et al. [1991]. d Nielsen et al. [1989]. e Seeman
IHCs. The data of Niu and Canlon [2006] and Inoue et al. et al. [1993]. f Seeman et al. [2005] calculated with spiperone dis-
[2006] suggest that dopamine receptors reside postsynap- placement. g Waters et al. [1993]. h Cited in Nobrega and Seeman
tically on the primary afferent dendrites and spiral gan- [1994]. i Spiperone binding [Andersen et al., 1985]. j Van Tol et al.
glion neurons. It remains unknown whether dopamine [1991]. k Freedman et al. [1994]. l K i calculated using IC50 = 10000
receptors also reside presynaptically (on lateral efferent nM from Pugsley et al. [1995]. m Chen et al. [2006].
membranes) within the cochlea.
A number of previous studies have used pharmaco-
logical agents directed at dopamine receptors. These
studies suffer from several defects that the present study pride, which was reported to decrease nerve activity [Ruel
seeks to address. Niu and Canlon [2006] used the spe- et al., 2001], acts on D2 and D4 receptors [Missale et al.,
cific D1/5 agonist (SKF 38393) and reported an excitatory 1998; Sokoloff et al., 1990].
effect on cochlear neural responses. SKF 38393 is, how- Hence the results of all previous investigations may be
ever, only a partial agonist of D1/5 receptors and it is there- due to co-activation or co-blockade of several different
fore not clear whether the full repertoire of D1/5 effects receptor subtypes. Therefore, in the present study we em-
would have been observed at the concentrations used. No ployed a range of specific agonists and antagonists for the
previous studies have employed a highly selective D2 ago- D1/5, D2 and D3 receptor subtypes in the guinea pig co-
nist or antagonist. For example, the agonist piribedil, re- chlea [Andersen et al., 1985; Bowery et al., 1996; Chen et
ported to reduce cochlear nerve responses and increase al., 2006; Freedman et al., 1994; Grundt et al., 2007; La-
release of dopamine [d’Aldin et al., 1995a; Gaborjan et al., Hoste et al., 2000; Nielsen et al., 1989; Nobrega and See-
1999], has similar affinities for D2, D3 and D4 receptors man, 1994; Pugsley et al., 1995; Seeman et al., 1993, 2005;
[Millan et al., 2002]. The agonist bromocriptine, reported Sunahara et al., 1991; Van Tol et al., 1991; Waters et al.,
to be inhibitory on cochlear neural responses and to en- 1993] and monitored sound-evoked cochlear potentials
hance dopamine release [Halmos et al., 2002; Oestreicher in anaesthetized animals before and after perilymphatic
et al., 1997; Sun and Salvi, 2001], has similar affinity for perfusion of these compounds. The available details on
D2 and D3 though lower for D4 receptors [Missale et al., specificity are summarised in table 1.
1998]. Sun and Salvi [2001] reported that the agonist
quinpirole increases inward currents in spiral ganglion
neurons, an effect that is blocked by the antagonist eticlo- Materials and Methods
pride. Quinpirole binds with greater affinity to D3 and D4
than to D2 receptor subtypes [Burris et al., 1995]. The an- Animals
tagonist sulpiride, which blocks dopamine-mediated ef- Adult pigmented guinea pigs of either sex weighing between
300 and 450 g were used in this study. Animals were pre-treated
fects on single unit activity and increases dopamine re- with atropine (0.1 ml s.c.) and anaesthetized with Nembutal (30
lease in the cochlea [Halmos et al., 2002; Oestreicher et mg/kg, i.p.) and Hypnorm (0.15 ml/animal, i.m.). This anaesthe-
al., 1997], acts on D2, D3 and D4 receptors, while eticlo- sia protocol was maintained by bi-hourly injections of Nembutal

146 Audiol Neurotol 2011;16:145–157 Garrett/Robertson/Sellick/Mulders

(15 mg/kg) and hourly injections of Hypnorm (0.15 ml). The elec-
trocardiogram was monitored on an oscilloscope following an-
aesthesia as well as during surgical procedures. All procedures
conformed to the Code of Practice of the National Health and
Medical Research Council of Australia and were approved by the
Animal Ethics Committee of the University of Western Australia.
Surgery and Cochlear Perfusion
Animals were transferred to a sound-attenuating room, tra-
cheotomised and artificially respired with Carbogen (95% O2/5%
CO2). They were then fixed into a head holder with hollow ear
bars. The external pinna was removed and the auditory bulla was
exposed by gentle reflection of the temporalis muscle and shaving
of the temporal bone. Animals were then supinated and the ven-
tral bulla was approached through the neck. Gentle shaving of the
ventral temporal bone revealed the cochlear apex and a small hep-
arinised insect pin was gently pressed into the cochlear apex re-
sulting in the formation of a small hole at the helicotrema (Ø 100
m). A tissue wick was inserted into the bulla to absorb excess
fluid from the cochlear perfusion. The animals were then re-
turned to a prone position and a small hole (100 m) was made in
the basal turn of the cochlea with a fine hand drill (Titex, 0.1 mm).
Single-barrel perfusion pipettes were pulled from borosilicate
glass with a laser glass puller (P2000, Brown Flaming). An etched
silver wire was inserted into the perfusion pipette. This electrode
was in contact with the perilymph within scala tympani and re-
corded sound-evoked cochlear potentials. To prevent insertion of
the pipette too far into scala tympani and to provide a seal, a bead
of silicone (Dow Corning, RTV 734) was made 60–80 m from
the pipette tip. The pipette was attached to a pressure pump (KD
scientific, 100 A) and cochleae were perfused at 2.5 l/min for 15
min. Artificial perilymph contained (in mM) NaCl 137, KCl 5,
MgCl2 1, NaH2PO4 1, NaHCO3 12, CaCl2 2 and glucose 11 [Zhang
et al., 1999]. This solution was made every 2 weeks and stored at
–20 ° C. Prior to use, pH and osmolarity of the working solution
   
was adjusted to 7.3–7.5 and 285–315 mosm, respectively.
With the exception of the D2 agonist (+)PHNO, which was
provided by Merck Sharp and Dome, all chemicals were pur-
chased from Sigma. Test compounds (table 2) had to be dissolved
in either ddH2O or ethanol (D2 antagonist, L 741626 only) before
they could be made up in artificial perilymph. Control perfusions
therefore were performed using artificial perilymph modified to
contain the same amount of either ddH2O or ethanol (0.1%). In
the majority of perfusions, the control perfusion preceded the test
perfusion. In one cochlea in every group, the test perfusate was
presented before the control and the order of perfusion did not
influence the changes seen in compound action potential (CAP)
or summating potential (SP) responses (data not shown). In the
experiments using the D2 antagonist (L 741626) vehicle effects
were found to be substantial. Therefore in order to quantitatively
assess any possible confounding effect of the preceding vehicle
perfusion, an additional 3 animals were used to measure the effect
of the drug on CAP amplitudes without a preceding vehicle (eth-
anol) perfusion. In all experiments, effects were evaluated by sta-
tistical comparison of drug perfusions and their appropriate
matched controls.
Sound Generation and Electrophysiological Recording
Cochlear responses were recorded via either a small silver wire
in contact with the cochlear round window (for initial audio-
Dopamine Receptors in the Cochlea
Table 2. Concentrations of the various dopamine receptor ago-
nists and antagonists used in this study
Dopamine receptor Compound Concentrations, M
Agonist
D1/5 SKF 38393 200, 100, 20
SKF 81297 160, 16, 1.6
D2 (+)PHNO 200, 2
D3 PD 128907 200, 20, 2
Antagonist
D1/5 SCH 23390 20
D2 L 741626 70
D3 U 99194A 20
grams) or the silver wire inserted into the perfusion pipette with-
in scala tympani. The sound-evoked cochlear response was am-
plified (!1000, WPI DAM 50) and filtered (1-Hz high-pass/3-
kHz low-pass filters) and then displayed on a computer monitor
for online analysis. A chlorided silver reference electrode was in-
serted into the temporalis muscle.
A custom-made program (courtesy G. Yates) was used to gen-
erate sinusoidal tone bursts of 10 ms duration (1 ms rise/fall time,
cosine gated) presented every 166 ms and the sound-evoked co-
chlear response was averaged 20 times. Sound stimuli were pre-
sented to the animals via a reverse-driven Brüel and Kjær micro-
phone (type 4134). Initial assessments of cochlear function in-
volved the estimation of N1 audiograms between 4 and 20 kHz.
Animals with normal acoustic thresholds were accepted for fur-
ther study [Johnstone et al., 1979]. CAP and SP input-output (I/O)
functions were generated by a custom software program which
presented a 16-kHz tone burst (10 ms duration, 1 ms rise/fall time)
between 30 and 84 dB SPL (86 dB SPL randomly presented) and
averaged 20 times. I/O functions were recorded before (0 min)
perfusion, immediately after perfusion (18 min) and then every
10 min for 30 min after perfusion (28, 38 and 48 min). The ampli-
tude of the CAP was measured as the potential difference between
the N1 and P1 components of the gross cochlear waveform. Acous-
tic thresholds were defined as the minimum sound pressure level
required to evoke an N1 amplitude of approximately 10 V. In or-
der to evaluate hair cell function, we also monitored the ampli-
tude of the SP. Although traditional measures of SP (see for ex-
ample Durrant et al. [1998]) have used the sustained baseline shift
during the tone presentation, it has been shown more recently that
this measure is contaminated by firing of the auditory nerve fibres
[Sellick et al., 2003]. We therefore used the SP measure described
by Sellick et al. [2003] and McMahon et al. [2004], which involved
measuring the peak of the initial positive deflection of the gross
cochlear responses. Monitoring of the CAP and SP amplitudes
can help to determine if the effects are confined to neural and/or
synaptic elements only (a change in CAP) or if hair cell responses
are also being disrupted (CAP and SP changes).
In some of the D2 antagonist perfusions, the endocochlear
potential (EP) and the maximum (saturation) amplitude of the
cochlear microphonic (CM) generated in response to a 207-Hz
probe tone were monitored at the same time as CAP responses to
a 16-kHz tone burst presented at 82 dB SPL. In a separate series
Audiol Neurotol 2011;16:145–157 147
 84 dB SPL
100 100
80 80
CAP (%)
CAP (%)
60 60
40 40
SKF 38393
20 SKF 81297 20
(+)PHNO
0 0
1 2 5 10 20 50 100 200
a Agonist (μM) b Agonist (μM)
Fig. 1. CAP dose-response function for the D1/5 and D2 agonists
perfused through the cochlea. The reduction in CAP amplitudes
evoked by a 16-kHz tone burst presented at 60 dB SPL were calcu-
lated immediately after perfusion. IC50 values were calculated to
of experiments involving D2 antagonist perfusions, we also mea-
sured distortion product otoacoustic emissions (DPOAEs) to-
gether with CAP thresholds at a range of frequencies (1–20 kHz)
and 207 Hz CM amplitude. In all these experiments, sound stim-
uli were presented by a Beyer DT 48 headphone connected to the
ear bar via a short length of plastic tubing. The CM and CAP
waveforms were recorded via the electrode within the perfusion
pipette or from an insulated silver wire placed on the round win-
dow membrane. EP was recorded using a borosilicate glass mi-
croelectrode inserted through the round window and basilar
membrane into the scala media. The result was amplified (Cyto
721, WPI) and filtered (DC coupled, 5.0 kHz low pass, Stanford
Research Systems) and sent to a LabVIEW data acquisition card.
A LabVIEW program (courtesy Dr. G. O’Beirne) was used to in-
terleave the CM and CAP stimulus presentations as well as to
record the EP during each perfusion trial. For DPOAE measure-
ments in the external ear canal, primary tones (f1 and f2) were
delivered using two separate Beyer DT48 headphones coupled to
the hollow ear bar with two 1-cm-long probe tubes. The 2f1-f2
distortion product sound pressures were measured using a tech-
nique similar to that of Marcon and Patuzzi [2008]. Sound pres-
sure in the external ear canal was measured using a Brüel and
Kjær 0.5-inch condenser microphone coupled tightly to the ear
bar using a 4-mm probe tube ending approximately 3 mm from
the tympanic membrane. The output of the 0.5-inch microphone
was amplified 20 dB by a Brüel and Kjær 2660 low-noise pre-
amplifier and then passed through a Brüel and Kjær type 2609
measuring amplifier. DPOAE and primary tone levels were mea-
sured by averaging the spectrum of the 0.5-inch microphone out-
put for 32 presentations of each level of the primary tones (50 ms
duration, 5 ms rise/fall time, 5 ms interval). I/O functions for
DPOAEs were constructed using f1 = 15 kHz and f2 = 18 kHz. The
relative intensities of the primary tones were chosen in each ani-
mal to maximize DPOAE amplitudes, and ranged from a ratio of
1.04–1.21. Noise floor was estimated using the same averaging
procedure in the absence of primary tones and was approximate-
ly 2 dB SPL.
148 Audiol Neurotol 2011;16:145–157
60 dB SPL 36 dB SPL
100
80
CAP (%)
60
40
20
0
1 2 5 10 20 50 100 200 1 2 5 10 20 50 100 200
c Agonist (μM)
be 170  M for SKF 81297 and 118  M for SKF 38393. IC50 was not
calculated for (8)PHNO. Data points represent the average
(n = 5) 8 SEM compared to 0 min. Maximum dose for each com-
pound was determined by solubility.
Statistical Analysis
Statistical comparisons were made between vehicle and test
perfusates using paired t test analysis. Comparisons were made
immediately after perfusion and at 10-min intervals for 30 min
after the cessation of perfusion for one intermediate intensity only
(60 dB SPL). ANOVA with Tukey post hoc analysis was used to
compare the changes for low (36 dB SPL)-, medium (60 dB SPL)-
and high-intensity (84 dB SPL) sound stimuli. Normalised CAP
responses (expressed as a percentage of pre-perfusion values) were
used to generate IC50 values for the D1/5 receptor agonists in
GraphPad prism v5.0. Dose-response plots were modelled with
the following equation. %CAP = 100/(1 + 10[agonist] – logIC 50) and the
IC50 values were calculated as the concentration of agonist which
resulted in a 50% reduction in percent CAP responses.
Results
Where agonists caused a significant change in cochle-
ar potentials, there was a clear dose dependence of the
magnitude of effects observed (fig. 1). There was, how-
ever, no qualitative difference in the effects at different
concentrations and for simplicity all subsequent data
shown are for the highest drug concentrations employed
(table 2).
Effects of D1/5 Receptor Compounds on CAP and SP
Amplitudes
Both of the D1/5 receptor agonists resulted in the sup-
pression of CAP amplitudes immediately following per-
fusion (fig. 2a). Figure 2a shows the effect of the full D1/5
receptor agonist SKF 81297 (160 M) at three sound in-
tensities. CAP was significantly suppressed at low (t test,
p = 0.0015), medium (p = 0.0024) and high sound pres-
Garrett/Robertson/Sellick/Mulders
 SKF 81297 SCH 23390
0 0

–20 –20
*

 CAP (%)

 CAP (%)
–40 –40
**
–60 –60
** Vehicle Vehicle
** D1/5 agonist D1/5 antagonist
–80 –80
36 60 84 36 60 84
a Stimulus intensity (dB SPL) d Stimulus intensity (dB SPL)

SKF 81297 SCH 23390

50 30

30 10
 SP (%)

 SP (%)
10 –10

****
–10 –30 ***

–30 –50
36 60 84 36 60 84
b Stimulus intensity (dB SPL) e Stimulus intensity (dB SPL)

120 140
***
100 120

100
80
CAP (%)

80
SP (%)

60
60
40
40 Vehicle
20
*** D1/5 agonist
20 Vehicle
D1/5 antagonist
0 0
0 12 24 36 48 0 12 24 36 48
c Time (min) f Time (min)

Fig. 2. Percentage changes (re 0 min) in CAP and SP amplitudes
after cochlear perfusion with the D1/5 receptor agonist SKF
81297 (a, b) or the antagonist SCH 23390 (d, e) compared with
the effects of perfusion of the matched vehicle. a, b, d, e Data
were measured immediately after perfusion. c, f Time course of
effects on the CAP and SP amplitude (60 dB SPL) compared to
0 min. Shaded areas indicate length of perfusion. Statistical an-
alysis was performed at all time points shown, and effects of
agonist and antagonist against the matched control were as-
sessed using paired t test (**** p ! 0.0001, *** p ! 0.001, ** p !
0.01, *  p ! 0.05). Data points represent the average (n = 5) 8
SEM.

sure levels (p = 0.001) compared to the matched vehicle
perfusates. There were no significant differences in the
magnitude of CAP suppression across the three intensi-
ties (ANOVA, p = 0.151). Although there was partial re-
covery, CAP was still significantly suppressed 30 min af-
ter perfusion (fig. 2c). SP amplitudes were not significant-
ly altered at any intensity or time for either the full or
partial agonist (fig. 2b, f).
Perfusion with the D1/5 receptor antagonist (SCH
23390, 20 M) caused a small reduction in CAP ampli-

Dopamine Receptors in the Cochlea Audiol Neurotol 2011;16:145–157 149

 Vehicle
D2 antagonist
(+)PHNO L 741626
0 0

–20 –20

–40 –40
 CAP (%)

 CAP (%)
**
–60 –60

–80 –80

–100 Vehicle –100

D2 agonist *** **** ****
–120 –120
36 60 84 36 60 84
a Stimulus intensity (dB SPL) d Stimulus intensity (dB SPL)

60 10

–10

40 –30
 SP (%)

 SP (%)
–50 ***
20 –70

–90 ***
–110
**
0
36 60 84 36 60 84
b Stimulus intensity (dB SPL) e Stimulus intensity (dB SPL)

120 140
Vehicle Vehicle
100 120 D2 agonist D2 antagonist

100
80
CAP (%)

80
SP (%)

60
60
40
40
20
***
20 *
0 0
*** **
0 12 24 36 48 0 12 24 36 48
c Time (min) f Time (min)

Fig. 3. Percentage change (re 0 min) in CAP and SP amplitudes
after cochlear perfusion with the D2 receptor agonist (+)PHNO (a,
b) or D2 receptor antagonist L 741626 (d, e). a, b, d, e Data were
measured immediately after perfusion. c, f Time course of effects
on the CAP and SP amplitude (60 dB SPL). Of particular interest
is the marked suppression of both CAP and SP amplitudes follow-
ing perfusion with the D2 receptor antagonist. Shaded areas indi-
cate length of perfusion. Statistical analysis was performed at all
time points shown, and effects of agonist and antagonist against
the matched control were assessed using paired t test (**** p !
0.0001, *** p ! 0.001, ** p ! 0.01, * p ! 0.05). Data points represent
the average (n = 5) 8 SEM.

tudes. Compared to vehicle perfusions (fig. 2d), this effect
was significant only at the highest sound intensity (t test,
p = 0.015) and only immediately after perfusion (fig. 2f).
SP amplitudes following D1/5 receptor blockade were sig-
nificantly enhanced (15–25%) relative to changes caused
by the vehicle perfusion at medium (p = 0.004) and high
sound intensities (p = 0.007). However, similar to the ef-
fects of the antagonist on the CAP amplitudes, this effect
was only significant immediately after perfusion (fig. 2f)
and was not present at later time points.

150 Audiol Neurotol 2011;16:145–157 Garrett/Robertson/Sellick/Mulders

 EP CM change
120 120

100 100

80 80

CM (%)
EP (%)
60 60

40 40
Vehicle 20
20
L 741626
0 0
–600 0 600 1200 1800 2400 3000 3600 0 1000 2000 3000 4000
a Time (s) d Time (s)

CM amplitude N1 threshold change

120 0

100 20

CAP threshold (dB SPL)

80 40
CM (%)

60 60

40 80

20 100

0 120
–600 0 600 1200 1800 2400 3000 3600 0 1000 2000 3000 4000
b Time (s) e Time (s)

CAP Threshold vs. CM change

120 0
Experimental data
100
Theoretical OHC
20 kHz HL (dB SPL)
CAP amplitude (%)

80 20 contribution

60

40 40

20
16 kHz 82 dB SPL
0 60
–600 0 600 1200 1800 2400 3000 3600 0 20 40 60 80 100
c Time (s) f CM (%)

Fig. 4. a–c Simultaneous recording of the EP, CM and CAP reveals
that the D2 receptor antagonist suppresses both the CM and CAP
without altering the EP. The antagonist (n = 3, grey line) or etha-
nol vehicle (n = 2, black line) were perfused for 15 min (vertical
lines). d–f Comparison between the CM amplitude (d) and 20-
kHz CAP threshold (e) shows that the loss of cochlear sensitivity
cannot be explained solely by altered OHC mechanics (f). f Theo-
retical data were derived from Patuzzi et al. [1989]. b, d Compared
to values at 0 min.

Effects of a D2 Receptor Agonist/Antagonist on CAP
and SP Amplitude
Perfusion of the cochlea with a selective D2 agonist
(+) PHNO (200 M) had no effect on CAP amplitudes
except for a small and short-lasting significant reduction
immediately after perfusion at the highest sound inten-
sity (84 dB SPL, p = 0.005; fig. 3a, c). SP amplitudes re-
mained unchanged following perfusion with the D2 ago-
nist (fig. 3b, c). In contrast, perfusion with the D2 antago-
nist L 741626 (70 M) resulted in a marked and persistent

Dopamine Receptors in the Cochlea Audiol Neurotol 2011;16:145–157 151

 25
120 140

2f1-f2 DPOAE level (dB SPL)

CAP threshold (dB SPL)
100 120 20
100
80 15

CM (%)
80
60
60 10
40 Average
40 noise floor
5
20 20
0 0 0
–200 0 200 400 600 800 1000 1200 30 40 50 60 70 80
a Time (s) b f1 primary level (dB SPL)

50 18
Reduction in CM amplitude (%)

Before
45 16 After
Before
40

amplitude (dB SPL)

14
35

Mean DPOAE
12 After
30 10 ***
25
8
20
15 6
10 4
5 2
0 0
Vehicle perfusion D2 antagonist Vehicle perfusion D2 antagonist
c perfusion d perfusion

Fig. 5. Effects of the D2 antagonist L 741626 (70  M) on measures
of outer hair cell and neural function. a Example in 1 animal of
continuous recording of low-frequency CM (S) and CAP thresh-
old to tones at 4 (!), 15 () ) and 20 kHz (U). CM amplitude ex-
pressed as change relative to the start of perfusion at 0 min in-
dicated by a solid bar. b I/O function of 2f1-f2 DPOAE in the same
animal as in a before (U) and after (S) antagonist perfusion.
c Average reduction (%) in low-frequency CM amplitude (n = 7)
caused by perfusion of vehicle alone and vehicle with D2 antago-
nist. d Average amplitude of 2f1-f2 DPOAE before and after perfu-
sion with vehicle (n = 6) and vehicle with D2 antagonist (n = 7).
Difference between DPOAE amplitude before and after drug per-
fusion was highly significant (*** p ! 0.001, paired t test).

reduction in CAP across all intensities (fig.  3c, d). Al- In addition to the changes in CAP, SP amplitudes were
though the average CAP changes following the ethanol also suppressed following D2 blockade (fig. 3e). SP ampli-
vehicle trials were found to be significant on their own tudes were suppressed by between 80% at 36 dB SPL and
compared to pre-perfusion values (paired t test, p ! 35% at the higher sound intensities. The differences be-
0.0001), there was a dramatic and sustained additional tween vehicle and antagonist perfusions were significant
reduction during the antagonist trials (fig.  3c, d). This (36 dB SPL, p = 0.0009; 60 dB SPL, p = 0.002; 84 dB SPL,
CAP reduction was significant compared to the ethanol p = 0.0009) up to 20 min after perfusion had ceased
vehicle effects at low (p = 0.0007), medium (p ! 0.0001) (fig. 3f).
and high sound pressure levels (p ! 0.0001). There were The marked suppression of SP following D2 receptor
no significant differences in the magnitude of CAP sup- blockade suggests that dopamine D2 receptors may be
pression across the three intensities (ANOVA, p = 0.06). influencing cochlear structures other than the primary
It is possible that an interaction could occur between afferents or the synapse between IHCs and afferents. We
preceding ethanol vehicle effects and that of the subse- therefore made simultaneous measurements of the CAP
quent drug perfusions. For this reason, in 4 animals ef- and CM amplitudes as well as the EP (fig. 4a–c). There
fects of drug perfusion were measured without a preced- were no changes in EP amplitude during or following
ing vehicle perfusion. The average CAP amplitude de- perfusion with vehicle or D2 antagonist (fig. 4a). In con-
crease was 76.6 8 5.3% without and 86.9 8 4.1% with a trast, the CM amplitude was appreciably suppressed to-
preceding vehicle perfusion. This difference was not sig- gether with the CAP amplitude during and following
nificant (unpaired t test, p = 0.17). perfusion of the D2 antagonist (fig. 4b, c). During perfu-

152 Audiol Neurotol 2011;16:145–157 Garrett/Robertson/Sellick/Mulders

 PD 128907 U 99194A
0 0

–10 –10

 CAP (%)

 CAP (%)
–20 –20

–30 –30
Vehicle Vehicle
D3 agonist D3 antagonist
–40 –40
36 60 84 36 60 84
a Stimulus intensity (dB SPL) d Stimulus intensity (dB SPL)

40 60

40 *

 SP (%)
 SP (%)

20 20

0 –20
36 60 84 36 60 84
b Sound intensity (dB SPL) e Sound intensity (dB SPL)

120 140 *
100 120
100
80
CAP (%)

SP (%)

Vehicle 80
60
D3 agonist 60
40 Vehicle
D3 antagonist 40
20 20
0 0
0 12 24 36 48 0 12 24 36 48
c Time (min) f Time (min)

Fig. 6. Percentage changes (re 0 min) in CAP and SP amplitudes
after cochlear perfusion with the D3 receptor agonist PD 128907
(a, b) or D3 receptor antagonist U 99194A (d, e). a, b, d, e Data
were measured immediately after perfusion. c, f Time course of
effects on the CAP and SP amplitude (60 dB SPL). Shaded areas
indicate length of perfusion. Statistical analysis was performed at
all time points shown, and the effects of agonist and antagonist
against the matched control was performed using paired t test
(* p ! 0.05). Data points represent the average (n = 5) 8 SEM.

sion of the control vehicle, the CM amplitude was found
to increase and this may reflect the action of the ethanol
on the outer hair cell (OHC) transduction process, or a
change in cochlear sensitivity following displacement of
the cochlear partition [Patuzzi et al., 1984] which can
happen during pressure perfusions. The CAP amplitude
in response to a fixed-intensity 16-kHz tone burst showed
the expected marked and sustained suppression during
and following perfusion with the D2 antagonist (fig. 4c).
Although there was an appreciable change in the CAP
amplitude during perfusion with the ethanol vehicle,
this effect was short-lived and the CAP amplitude rap-
idly returned to pre-perfusion values when perfusion
ceased.

Dopamine Receptors in the Cochlea Audiol Neurotol 2011;16:145–157 153

 In order to investigate the quantitative relationship be-
tween CM amplitude and cochlear neural sensitivity, the
16-kHz CAP threshold was also monitored during some
trials with the D2 antagonist at the same time as the CM
(fig.  4c–f). CM amplitude fell by approximately 35%,
whereas CAP thresholds were elevated by up to 55 dB. In
figure 4f the percentage change in CM is plotted against
CAP threshold and is compared to the theoretical curve
that describes the relationship between OHC current
(drive to OHC active process) and sensorineural hearing
loss [Patuzzi et al., 1989a].
Further evidence for an effect of the D2 antagonist on
OHC was obtained by measuring DPOAEs. Figure 5a, b
show the change in CM and CAP thresholds at three dif-
ferent frequencies (4, 15 and 20 kHz) as well as the DPOAE
I/O function in 1 animal. As in figure 4, there was a
marked reduction in the low-frequency CM amplitude
and CAP thresholds at all frequencies tested. In addition,
there was a significant reduction in DPOAE amplitudes.
In figure 5c, d, mean data for CM and DPOAE ampli-
tudes are presented, comparing the results of vehicle and
antagonist perfusions. The effect of the D2 antagonist on
CM amplitude (fig. 5c) was similar to that shown in the
previous set of experiments (fig. 4). The average reduction
in DPOAE amplitude (fig. 5d) caused by the antagonist
perfusion was approximately 6 dB (p ! 0.001; paired t
test). Vehicle perfusions caused no significant change.
Effects of a D3 Receptor Agonist/Antagonist on CAP
and SP Amplitudes
Perfusing scala tympani with the D3 agonist PD
128907 (200 M) did not alter the amplitude of the CAP
or SP at any sound intensity compared to vehicle perfus-
ates (fig. 6a–c). Similarly, the D3 antagonist U 99194A (20
M) did not change the amplitudes of the CAP and SP
compared to vehicle (fig. 6d–f), with the sole exception of
a small relative suppression of the SP amplitude at a single
intensity (60 dB SPL, p ! 0.05) immediately after perfu-
sion.
Discussion
In this study, we observed marked suppression of the
sound-evoked cochlear response following perilymphat-
ic perfusion of specific D1/5 and D2 receptor agonists and
antagonists but not D3 receptor compounds. Further-
more, we observed sustained suppression of the SP, CM
and DPOAE amplitudes following D2 receptor blockade
which suggests that dopamine receptors may influence
154 Audiol Neurotol 2011;16:145–157
cochlear structures other than the primary afferent den-
drites. In contrast to earlier studies, we have employed a
full D1/5 receptor agonist [Arnt et al., 1992; Nielsen et al.,
1989] and other agonists and antagonists for either the D2
or D3 receptors (table 1). This approach enabled for the
first time a distinction between the roles of the latter two
receptor subtypes in the cochlea, since all previous com-
pounds employed activated both these receptors [d’Aldin
et al., 1995b; Gaborjan et al., 1999; Halmos et al., 2002;
Oestreicher et al., 1997; Ruel et al., 2001; Sun and Salvi,
2001].
Although D3 receptor mRNA and protein have been
shown to be present in the mouse and rat cochlea [Inoue
et al., 2006; Karadaghy et al., 1997], in the present study we
found no change in the sound-evoked responses following
perfusion of the D3 receptor compounds. This finding,
however, does not exclude the possibility of more subtle
interactions between the different receptor subtypes [Zeng
et al., 2006]. Future experiments are needed to confirm the
presence of the D3 receptor in the guinea pig cochlea and
to investigate the effects of perfusions combining agents
acting on the different receptor subtypes.
The D1/5 receptor agonist reduced CAP amplitudes
which suggests that these receptors are inhibitory on the
sound-evoked response. This finding is consistent with
previous work [d’Aldin et al., 1995b; Ruel et al., 2001] that
also showed CAP suppression following perfusion with
dopamine (which acts at all receptor subtypes), quin-
pirole (D2/D3 agonist) and piribedil (D2/D3 agonist). We
were unable to replicate the more recent findings by Niu
and Canlon [2006], who reported enhancement of the
CAP amplitudes using the specific D1/5 receptor partial
agonist SKF 38393, suggesting an excitatory role for do-
pamine D1/5 receptors in the cochlea. It may be that a re-
duction in CAP is actually caused by an increased resting
firing rate at the single-neuron level, since this will lead
to adaptation and a reduced synchronous response. How-
ever, it is worth noting that previous studies revealed that
elevated dopamine concentration in the cochlea sup-
presses CAP as well as single-unit firing rates both at rest
and during sound stimulation [Ruel et al., 2001, 2006].
Our dopamine D1/5 receptor antagonist data showed
no significant change in CAP amplitudes at intensities
where the CAP was markedly affected by the D1/5 agonist.
The simplest interpretation of this finding is that there is
little or no tonic dopamine release onto the D1/5 receptors.
There was a small suppression of CAP amplitudes caused
by the antagonist at the highest sound intensity, but the
transient nature of this effect suggests it may be related to
non-specific perturbations caused by the perfusion.
Garrett/Robertson/Sellick/Mulders
 Our D2 receptor antagonist data indicate a strong ex-
citatory influence of tonic dopamine release on these re-
ceptors. The lack of an enhancement of CAP after perfu-
sion of the D2 agonist could be explained if the levels of
endogenous dopamine release are sufficient to keep the
D2 receptors at, or close to, their maximum activation
state. In view of the large and sustained changes caused
by the antagonist, the small suppression of CAP ampli-
tude after perfusion of the D2 receptor agonist is most
likely to be a small artifact of perfusion, since it dissipates
rapidly.
In addition to the suppression on the CAP, perfusion
with the D2 antagonist also caused changes in SP, CM and
DPOAEs, suggesting a separate action of tonic dopamine
release on hair cells. The data plotted in figure 4f show
that these hair cell effects contribute to some, although
by no means all, of the changes in neural thresholds
[Patuzzi et al., 1989].
The mechanism by which the D2 antagonist causes
changes in hair cell responses is unclear. Our measure-
ments of EP show that there is no change in this compo-
nent of the electrical drive on ions through the transduc-
tion channels of the IHCs or OHCs. The drop in the sat-
uration value of the low-frequency CM therefore suggests
either a fall in OHC membrane potential or a disruption
to the OHC mechano-electrical transduction process.
This could result in an SP change in two possible ways.
The SP is thought to emanate largely from the IHCs, es-
pecially for low and moderate sound intensities [Russell
and Sellick, 1983], but it is the OHC active electrome-
chanical feedback process that determines the drive to
the IHCs. Hence actions of a D2 antagonist on the OHC
could alter SP by affecting this active process. This inter-
pretation is supported by the DPOAE data. The situation
is complicated however by the fact that the OHCs them-
selves can generate a significant contribution to the SP
particularly at higher sound pressure levels [Durrant et
al., 1998; Sellick et al., 2003] and this component of the
SP could also be directly affected by the drug or by pres-
sure effects during and after perfusion (see for example
Bobbin and Salt [2005]). Finally, the drug could act di-
rectly on the IHC transduction process causing an SP
change independently of action on the OHCs.
Although dopamine receptors have never been report-
ed on cochlear hair cells, efferent terminals have been
reported on IHCs in the cat [Liberman et al., 1990]. While
no such connection has been shown to exist in the guinea
pig, it is conceivable that dopamine may act directly on
the IHC either via a direct connection or, alternatively,
via diffusion from the lateral efferent terminals to the
Dopamine Receptors in the Cochlea
IHC region. In addition, a dopamine transporter has
been shown to be present in the vicinity of Deiter’s cells
underneath the OHC [Ruel et al., 2006]. Furthermore,
one cannot rule out the possibility that dopamine re-
leased by lateral efferents could diffuse to the OHCs, act-
ing in a paracrine fashion, as has been suggested for uro-
cortin [Vetter et al., 2002]. In a previous study, inhibiting
dopamine reuptake and thus increasing circulating do-
pamine concentrations did not significantly alter hair cell
potentials such as the SP and CM [Ruel et al., 2006]. How-
ever, increasing dopamine levels in the cochlea will acti-
vate all dopamine receptors present, the end result being
possibly different from manipulating a single receptor
subtype, especially as the current data suggest competing
effects of the different receptor subtypes.
In summary, our data are consistent with dopamine
being tonically released into the cochlea, presumably
from the lateral efferent terminals. Our data also suggest
divergent actions of dopamine dependent on the subclass
of receptor involved. In addition, the effects on hair cell
measures suggest that these receptors are not solely con-
fined to the primary afferent dendrites.
Acknowledgements
A.R.G. was a recipient of a UPA scholarship provided by the
University of Western Australia. This work was also supported by
grants from the National Health and Medical Research Council,
the Medical Health Research Infrastructure Fund (W.A., Austra-
lia) and the University of Western Australia.
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