Departments of Neurobiology and Physiology, and Communication Sciences and Disorders, Auditory Physiology Laboratory, The Hugh Knowles Center; and The Institute for Neuroscience, Northwestern University, Evanston, Illinois 60208
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ABSTRACT |
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He, David Z. Z. and Peter Dallos. Development of acetylcholine-induced responses in neonatal gerbil outer hair cells. Cochlear outer hair cells (OHCs) are dominantly innervated by efferents, with acetylcholine (ACh) being their principal neurotransmitter. ACh activation of the cholinergic receptors on isolated OHCs induces calcium influx through the ionotropic receptors, followed by a large outward K+ current through nearby Ca2+-activated K+ channels. The outward K+ current hyperpolarizes the cell, resulting in the fast inhibitory effects of efferent action. Although the ACh receptors (AChRs) in adult OHCs have been identified and the ACh-induced current responses have been characterized, it is unclear when the ACh-induced current responses occur during development. In this study we attempt to address this question by determining the time of onset of the ACh-induced currents in neonatal gerbil OHCs, using whole cell patch-clamp techniques. Developing gerbils ranging in age from 4 to 12 days were used in these experiments, because efferent synaptogenesis and functional maturation of OHCs occur after birth. Results show that the first detectable ACh-induced current occurred at 6 days after birth (DAB) in 12% of the basal turn cells with a small outward current. The fraction of responsive cells and the size of outward currents increased as development progressed. By 11 DAB, the fraction of responsive cells and the current size were comparable with those of adult OHCs. The results indicate that the maturation of the ACh-induced response begins around 6 DAB. It appears that the development of ACh-induced responses occur during the same time period when OHCs develop motility but before the onset of auditory function, which is around 12 DAB when cochlear microphonic potentials can first be evoked with acoustic stimulation in gerbils.
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INTRODUCTION |
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It is a common assumption that mammalian hearing
owes its remarkable sensitivity and frequency selectivity to a local
mechanical feedback process within the cochlea. Termed the "cochlear
amplifier," it is widely assumed that its motor arm is a somatic
length change of outer hair cells (Brownell et al.
1985). In adult mammals, outer hair cells (OHCs) are innervated
predominantly by efferents that originate in the brain stem
(Spoendlin 1972
, 1986
; Warr et al.
1986
). The efferent fibers form chemical synapses at the bases of the OHCs with ACh being their principal neurotransmitter
(Altschuler et al. 1985
; Jasser and Guth
1974
; Schuknecht et al. 1956
,
1959
). Activation of the efferents can alter
micromechanical events within the cochlear partition and thereby
provide a "gain control" of the cochlear amplifier (for review see
Guinan 1996
).
Traditionally, cholinergic receptors have been characterized as
nicotinic or muscarinic. Nicotinic receptors are ionotropic, whereas
muscarinic receptors are linked to second-messenger systems via G
proteins. The two types of receptors can often be distinguished pharmacologically, because they can be selectively activated and blocked by different pharmacological agents. However, ACh receptors on
OHCs have been demonstrated to have unusual pharmacology with some
characteristics of both nicotinic-and muscarinic-receptor types (for
reviews, see Bobbin 1996; Eybalin 1993
;
Guth and Norris 1996
). Recently, a new subunit (
9) of
the nicotinic AChR family has been cloned from a rat genomic library
(Elgoyhen et al. 1994
). When expressed in oocytes, this
subunit produces functional AChRs and demonstrates pharmacological
properties similar to those seen in cochlear hair cells.
It has been demonstrated that activation of the cholinergic receptors
on isolated OHCs by ACh induces rapid calcium influx through the
ionotropic receptors, followed by a large outward K+
current through nearby Ca2+-activated K+
channels (Fuchs and Murrow 1992a,b
; Housley and
Ashmore 1991
). The outward K+ current
hyperpolarizes the cell, resulting in the fast inhibitory effects of
efferent action. It has been suggested that the ACh receptor-mediated
K+ current is carried by small-conductance
Ca2+-activated K+ channels (Nenov et al.
1996
), as opposed to the large-conductance Ca2+-activated K+ channels ("maxi"
K+ channels) that have been described in mammalian OHCs
(Ashmore and Meech 1986
; Housley and Ashmore
1992
; Santos-Sacchi and Dilger 1988
).
Although the ACh receptors and the ACh-induced current responses in adult OHCs have been identified and characterized, it is unclear when the ACh-induced current responses are expressed during development. The goal of this work is to determine when the ACh-induced current responses develop in neonatal OHCs. ACh-induced outward K+ currents were used as an index in this study. By pinpointing the time of onset of this outward K+ current in neonatal OHCs with whole cell patch-clamp techniques, it was possible to determine when OHCs are functionally ready for mediating efferent action.
Neonatal gerbils (Meriones unguiculatus) ranging in age
between 4 and 12 days after birth (DAB) were chosen for the
experiments. The cochlea of the gerbil (or that of other altricial
rodents such as mouse and hamster) offers a useful model to study the development of the cochlear amplifier, inasmuch as the important period
of hair cell development occurs between birth and the onset of hearing,
which is around 10-12 DAB (Woolf and Ryan 1984).
Furthermore, ultrastructural studies in developing altricial rodents
have shown that efferent synaptogensis of OHCs occurs several days
after birth (Romand 1983
). Therefore it is of interest
to determine whether the development of ACh receptors is related to the
commencement of efferent synapse formation as it is in the case of the
neuromuscular junctions (for review see Jacobson 1992
).
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METHODS |
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Newborn gerbils ranging in age from 4 to 12 DAB were used in this experiment. Births in the breeding colony were monitored at 9 am and 5 pm daily. We designated the day when the litters were born as 0 DAB and the following day as 1 DAB and so on. For each age group, a minimum of four litters were used. Adult gerbils (~30-40 days old) were also used for control.
Hair cell preparation
A detailed description of dissecting the organ of Corti of
newborn gerbils is given by He et al. (1994) and
He (1997)
. Briefly, cochleae from gerbil pups were
dissected out after killing (60 mg/kg pentobarbital sodium), and kept
in cold Ca2+-free Hank's balanced salt solution (GIBCO)
with osmolarity of 310 mOsm and pH of 7.4. The organ of Corti and
associated basilar membrane was carefully unwrapped from the modiolus.
Only the basal segment was used because development of the cochlea is
baso-apical and because the richest cholinergic innervation is found in
the base (Liberman et al. 1990
). The basal segment was
dissected out (Fig. 2A) and transferred to a droplet of
enzymatic digestion medium in the center of a Petri dish. The enzymatic
digestion medium was Ca2+-free Hank's with 2 mg/ml
collagenase type IV (Sigma). After 20 min incubation at room
temperature (22 ± 2°), the tissue was transferred to a small
plastic chamber containing enzyme-free culture medium (Leibovitz's
L-15, 7.35 pH, 310 mOsm). Hair cells were separated after gentle
trituration of the tissue with a 100-µL Hamilton syringe. The chamber
containing the hair cells was then mounted onto the stage of an
inverted microscope (Zeiss) equipped with video cameras. Experiments
were performed under video monitoring, and images of the cells were
captured by a camera and later processed with Adobe Photoshop (version
3.0 for Power Macintosh). The length of the cells was measured from a
video image of the cell on a TV monitor, using a transparency on which
a calibrated grid was printed.
Whole cell patch-clamp recordings
Experiments were performed at room temperature (22 ± 2°)
on an inverted Zeiss microscope with ×10 and ×16 objectives. OHCs were bathed in L-15 medium (components in mM: 136 NaCl, 5.8 NaH2PO4, 5.4 KCl, 1.3 CaCl2, 0.9 MgCl2, and 0.4 MgSO4) buffered with 10 mM
HEPES. Whole cell voltage-clamp tight-seal recordings were established
(Fig. 1) in the same manner as described
by Ashmore and Meech (1986) and Santos-Sacchi and
Dilger (1988)
for guinea pig OHCs. The patch electrodes were
pulled from 1.5-mm glass capillaries (Dagan) using a two-stage puller
(Narashige, Model PB-7). The electrodes were backfilled with a solution
containing (in mM) 120 KF, 20 KCl, 2 MgCl2, 5 EGTA, and 10 HEPES. The solution was buffered to pH 7.4 with Trizma Base (Sigma) and
osmolarity adjusted to 310 mOsm with glucose. The pipettes had initial
bath resistances of 3-5 M
. The access resistance, which is the
actual electrode resistance obtained on establishment of whole cell
configuration, typically ranged from 6 to 10 M
. Approximately 75%
of the series resistance was compensated. To maintain low access
resistance, small transient positive or negative pressure was applied
to electrodes to keep the tip of patch electrodes unblocked.
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Under computer control, hyperpolarizing and depolarizing voltage steps,
usually 200 ms long and ranging from 100 to 140 mV in 20-mV steps,
were used to elicit whole cell currents. The low-pass filtered currents
(corner frequency at 5 kHz) were amplified using an Axopatch 200 A
amplifier (Axon Instruments) with headstage (Model CV 201AU).
ACh-evoked current responses were recorded in voltage-clamp mode. The
holding potential of the cells varied, depending on the nature of the
experiments. To obtain large ACh-evoked outward currents, the cells
were usually held between
30 to
10 mV, where the outward currents
were found to be largest (Blanchet et al. 1996
;
Evans 1996
). When we sought to detect early inward current, the cells were held at potentials more negative than
50 mV
to deemphasize the late outward K+ current. Whole cell
currents and ACh-evoked current responses were acquired by software
pClamp (Clampex and Fetchex, Axon Instruments) running on an
IBM-compatible computer equipped with a 12-bit A/D converter (TL-1,
Scientific Solutions). The sampling frequency was 2 kHz for recording
whole cell currents and 2.5 kHz for the ACh-evoked current responses.
For every cell tested, the whole cell currents were first obtained
before recording any ACh-evoked current responses. Data were analyzed
using clampfit and fetchan in the pClamp software package (pClamp 5.01, Axon Instruments).
ACh application
ACh was delivered by pressure ejection from a micropipette with
tip diameter of 3-4 µm positioned 15-20 µm from the synaptic pole
(Fig. 1). In some cases, multibarrel micropipettes were used to deliver
different doses of ACh and antagonists. The tip of the micropipette was
silanized to prevent capillary action. The duration and strength of the
pressure pulse were controlled by a pneumatic micropump (WPI, model
PV820), which, in turn, was controlled by the computer. The
half-activating concentration for ACh receptors in OHCs is 20-25 µM,
and the response is maximal at 100 µM (Dallos et al.
1997; Elgoyhen et al. 1994
;
Eróstegui et al. 1994
; McNiven et al.
1996
). To detect any small ACh-induced currents in neonatal
cells, the ACh concentration used in the experiments was, unless
otherwise stated, 100 µM. The ACh solution was freshly prepared
before each experiment.
Use and care of animals were approved by the National Institutes of Health and the Animal Care Committee of Northwestern University.
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RESULTS |
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One striking feature of the organ of Corti of the gerbil before 6 DAB is that hair cells and supporting cells are closely packed and no
extracellular space is found (Edge et al. 1998; Souter et al. 1997
). The lack of extracellular space
made hair cell isolation difficult and resulted in a low yield of
isolated OHCs. Many OHCs obtained in our preparations before 6 DAB were seen in clusters. Our recordings were made from OHCs regardless of
whether they were in isolation or in clusters. As the tunnel of Corti
and Nuel's space began to appear after 7 DAB (Edge et al.
1998
; Souter et al. 1997
), obtaining isolated
OHCs was no longer difficult.
Because of their cylindrical shape, OHCs were easily recognizable in
mature gerbil hair cell preparations. However, the identification of
isolated OHCs was not so easy in preparations derived from immature cochleae. Before 6 DAB, both types of hair cell looked similar
at the light microscope level, although their stereocilia bundle
configuration appears to be different. Even though it was not easy to
differentiate isolated inner hair cells (IHCs) from isolated OHCs
before 6 DAB, there were a number of reasons why it is believed that
our recordings were made from OHCs. First, most of our recordings
before 7 DAB were made from clusters of hair cells (Fig.
1A), where OHCs could easily be identified based on their
anatomic locations. Second, OHCs are somewhat easier to separate from
the organ of Corti and the associated basilar membrane than IHCs in our
preparations after enzymatic digestion and mechanical trituration.
Among all cells that we measured at all age groups, we encountered only
five isolated IHCs, which were later identified from the
characteristics in their whole cell current responses. Third, and most
important, the whole cell current response was different in OHCs and
IHCs. The differences in current response were used to ascertain that
recordings were from OHCs. These differences are addressed later. As
OHCs gradually acquire motile behavior after 7 DAB (He et al.
1994), identification of these OHCs is quite straightforward.
OHCs were selected for experiments if they showed no obvious signs of
damage and/or deterioration such as swelling, translocation of the
nucleus, and/or granulation. Figure 2b
shows an example, with Hoffmann optics, of an OHC isolated from
7-day-old basal turn cochlea. Whole cell voltage-clamp recordings were
made from OHCs isolated from 4- to 12-day-old basal turn cochleae as
well as from adult OHCs (30 DAB). On rupturing of the cell membrane, the pipette often recorded a zero current potential of 10 to
20 mV,
which rapidly became more negative as the pipette contents equilibrated
with the cell interior over a period of 30-60 s. After stabilization,
the mean zero current potential recorded under whole cell condition was
45 ± 10 mV (mean ± SD, n = 155) with a
range of
30 to
64 mV.
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Whole cell currents in maturing hair cells
Whole cell currents were recorded from maturing hair cells to aid
distinquishing OHCs from IHCs. This also provided some information about the development of voltage-and Ca2+-dependent
K+ channels, the major conductances in OHCs. Figure
3 shows some typical examples of whole
cell currents recorded from 4- and 11-day-old as well as adult
(30-day-old) basal turn OHCs. The main features of the current records
are apparent in the figure. Responses to hyperpolarizing steps ranging
from 70 to
120 mV reflected, in most cases, only a linear leak
conductance, although a transient inward current was sometimes
observed. Depolarization to potentials more positive than
40 mV
elicited slow, sigmoidal outward currents, reaching maxima in ~70 ms
at
40 mV to <20 ms at +60 mV. The currents increased up to
depolarizations of +60 to +70 mV, reaching ~1.5 nA for 4 DAB and 3.0 nA for 11 DAB and adult. The currents decreased at more depolarized
potentials, and the response became much faster. A partial inactivation
of the currents was often seen with large depolarizations before the
currents reached steady state. Although no pharmacological studies were
done to identify the currents, they are known to be potassium currents
(Housley and Ashmore 1992
; Santos-Sacchi and
Dilger 1988
). The currents clearly resemble the K+
currents previously described in adult guinea pig OHCs (Housley and Ashmore 1992
; Santos-Sacchi and Dilger
1988
).
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The maximum size of the current was significantly different between 4- and 11-day-old OHCs, although the kinetics of the responses were very
similar. The current at 4 DAB was ~50% smaller than that at 11 DAB
for the two examples. For all the cells (n = 16) that
we measured at 4 DAB, the maximum current was 1.4 ± 0.3 nA, in
contrast to 2.7 ± 0.4 nA in 12 day old (n = 14)
and 2.8 ± 0.4 nA in adult OHCs (n = 7). Current
size increased systematically from 4 to 11 DAB, and the kinetics and
size of the current at 11 DAB resembled those of adult OHCs. An example
of the whole cell current of an adult OHC is shown in Fig.
3C for comparison. The K+ currents in adult
gerbil OHCs resemble those found in guinea pig OHCs (Housley and
Ashmore 1992).
Although immature IHCs appeared similar to OHCs, the characteristics of
their whole cell current responses were different from those of OHCs.
As an example, the whole cell current responses obtained from 5- and
11-day-old IHCs are shown in Fig. 3, E and F. The
difference between the 5- and 11-day-old responses is apparent: outward
currents on depolarization from a holding potential of 70 mV are
smaller and slower at 5 DAB than at 11 DAB. For the 5-day-old cell, the
currents reach a maximum in ~24 ms at
40 mV, in contrast to <10 ms
for the same level for the 11-day-old IHC. The K+ current
response at 11 DAB is similar to that of adult guniea pig IHCs, whose
two different potassium conductances (a large conductance with
principal time constant of activation of 0.15-0.35 ms and a smaller
conductance with principal time constant of activation of 2-10 ms)
were characterized (Kros and Crawford 1990
). Kros et al. (1998)
have studied the development of time- and
voltage-dependent conductances of IHCs in mice. They showed that the
current size increased significantly during development. Apparently,
our results are in agreement with their findings.
The differences in kinetics and magnitude of the currents between IHCs and OHCs at different developmental stages are apparent in Fig. 3. The onset of the time- and voltage-dependent currents upon depolarization were much faster in IHCs than in OHCs. The largest current elicited from the IHCs could be two to four times larger than that of OHCs. These differences were used to identify immature OHCs from IHCs when visual identification was equivocal.
Maturation of ACh-evoked currents
ACh activation of the cholinergic receptors on isolated OHCs
induces an outward K+ current triggered by Ca2+
influx through cholinergic ionotropic receptors (Housley and Ashmore 1991). The functional maturation of ACh receptors was studied by examining when ACh-induced currents could first be recorded
and how they developed between 4 and 12 DAB. For comparison, the
ACh-induced current was also measured from adult gerbil (~30 days
old) OHCs. The ACh-induced current response was measured under whole
cell voltage-clamp mode. To obtain maximal ACh-induced outward current,
the membrane potential was usually held between
10 and
30 mV
(Blanchet et al. 1996
; Evans 1996
). To
maximize the ACh effect, 100 µM of ACh was applied. A positive
ACh-evoked response was defined as any measurable outward current that
was repeatable and time registered with the application of ACh. As examples, Fig. 4 shows responses of basal
turn OHCs isolated from 5- to 12-day-old cochleae. No ACh-induced
inward and/or outward current is detected at 5 DAB. At 6 DAB, a small
outward current (~19 pA) is observed after ACh application. Between 8 and 12 DAB, robust ACh-induced outward currents are recorded. The
induced outward current peaked within 100-200 ms, depending on the
distance of the delivery pipette and ejection pressure. The detailed
time table of onset of ACh-induced outward currents is presented in Table 1 and Fig.
5A.
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In Table 1, the number of responsive cells and the total number of cells tested are given. The number of responsive cells versus the total cells is converted into percentage and plotted in Fig. 5A as a function of age. ACh-induced currents were first examined in 16 cells at 4 DAB and 17 cells at 5 DAB. As the table shows, none of these cells responded positively to the ACh application. At 6 DAB, 2 of 16 cells (12%) exhibited detectable ACh-induced current response. Three of 15 cells tested (20%) showed ACh-induced response at 7 DAB. Over the next 4 days, the number of cells responding positively to ACh application increased. By 11 DAB, the percentage of responsive cells was comparable with that of adult cells tested.
Another conspicuous feature shown in Fig. 4 is the change in size of ACh-induced currents between 6 and 12 DAB. To compare the size of the outward current at different ages, the maximal magnitude of the current was measured. The means and standard deviations of the current maxima are plotted as a function of developmental age in Fig. 5B. As shown, the evoked current response is small when the ACh-induced currents just commenced at 6 DAB. The size of the currents increased as more cells exhibited the induced response. We compared the difference in means of magnitude of the currents of the developmental groups with that of the mature group (30 days old). No statistical significance (P < 0.05, t-test) was found between the mature group and the developmental groups after 11 DAB, indicating that the size of ACh-induced currents reaches mature level around 11 DAB.
Biphasic ACh-induced current or voltage responses were observed in some
OHCs when the cells were held at potentials more negative than 50 mV
(Blanchet et al. 1996
; Evans 1996
). In
those cells, ACh induced a large outward current preceded by a rapid
early inward current. The early inward current is believed to be
carried by Ca2+ influx through cholinergic ionotropic
receptors (Eróstegui et al. 1994a
,b
;
Housley and Ashmore 1991
). We also attempted to record either the biphasic responses or the early inward current by holding the cells between
50 and
70 mV to determine whether the inward current could be observed earlier in development than the onset of the
outward current. We recorded from 65 cells between 4 and 7 DAB, none of
them showed this ACh-induced inward current expressed either as a sole
inward current or as a biphasic response. At 8 DAB, one of five
responsive cells showed a biphasic response. In the adult group, 3 of
12 cells showed an ACh-induced biphasic response. Figure
6 shows two examples of the biphasic
current responses recorded from an 8-day-old and an adult OHC. It is
not surprising that the early inward current can only be seen in a small proportion of cells because the small inward current is usually
masked by the dominant outward current.
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Strychnine is known to be a potent specific antagonist of cholinergic
responses of chick hair cells and mammalian OHCs (Elgoyhen et
al. 1994; Fuchs and Murrow 1992a
; Housley
and Ashmore 1991
). We tested whether the ACh-induced currents
in neonatal gerbil OHCs could be blocked by strychnine. Figure
7 gives an example of the effect of
strychnine on the ACh-induced currents obtained from basal turn OHCs
isolated from 8- and 12-day-old gerbils. The cells were held at
30
mV, and 100 µM ACh or 100 µM ACh with two different doses of
strychnine was pressure ejected to the synaptic pole of the cell. As
shown, the magnitude of outward current decreases ~50% with 0.01 µM strychnine coapplied with 100 µM ACh. Between 80 and 90% of the
induced outward currents was eliminated at a strychnine concentration
of 0.1 µM. The outward currents partially recovered after wash out.
This result is consistent with the effect of strychnine on
9
homomers as reported by Elgoyhen et al. (1994)
.
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In Fig. 8, the dose dependence of
ACh-activated outward currents is compared for two groups of cells (8 and 12 DAB). Representative responses obtained from two cells (held at
30 mV) at four different doses are plotted in the top
panel. The results obtained from 6 cells at 8 DAB and 6 cells at
12 DAB are plotted in the bottom panel for comparison. As
shown, the responses of cells in both groups increase as the ACh
concentration increases until the maximal response is reached at a
concentration around 100 µM. The maximal response is different for
the two groups of cells; the average maximal response is 53 pA for the
8-day-old group and 110 pA for the 12-day-old group. The data
were fit by the Hill equation {IACh = Imax/[1 + (EC50/[ACh])n]}. The smooth curves
represent the curve fitting. The half-activating concentration
(EC50) is 16.8 µM (n = 1.8) and 20.3 µM
(n = 1.9) for the 8- and 12-day-old groups,
respectively.
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DISCUSSION |
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Efferents, originating in the superior olivary complex,
preferentially synapse with OHCs in mammals and short hair cells in birds. The effect of efferent action, mediated by the release of ACh,
is generally shown to be inhibitory (Amaro et al. 1966; Art et al. 1982
, 1984
; Galambos
1956
; Murugasu and Russell 1996
; Wiederhold and Kiang 1970
) and mediated by a
hyperpolarizing K+ current (Art et al. 1984
;
Eróstegui et al. 1994
; Fuchs and Murrow 1992a
,b
; Housley and Ashmore 1991
;
Shigemoto and Ohmori 1991
). This hyperpolarization
arises from an increase in K+ conductance in the cells. The
postsynaptic mechanism that leads to activation of the outward
K+ current (IK(ACh)) is somewhat
unusual, involving direct gating by ACh of a cation channel through
which Ca2+ enters the cell (Fuchs and Murrow
1992a
,b
; McNiven et al. 1996
). The influx of
Ca2+ then leads to the opening of the nearby
Ca2+-dependent K+ channels
(KCa(ACh)). It has been suggested that this
KCa(ACh) channel is not the same as the big conductance
Ca2+-dependent K+ channel (KCa) in
nonmammalian vertebrate hair cells and mammalian OHCs. The channels
that carry IK(Ca) have a large conductance, are
blocked by internal Cs+ ions, and have a sharply rectifying
current-voltage (I-V) relation. In contrast, the channels
that carry IK(ACh) have a smaller conductance, are not blocked by either internal Cs+ or charybdotoxin but
by apamin, and have a linear I-V relation (Fuchs
1992
; Nenov et al. 1996
). It is likely
that the two types of Ca2+-activated K+
channels are spatially segregated so that the intracellular
Ca2+ signals can activate differential functions of
KCa(K) and KCa(ACh) channels (Blanchet
et al. 1996
). It is unclear whether the subsynaptic cistern
adjacent to efferent endings on hair cells serves to restrict ACh-gated
Ca2+ influx to the immediate postsynaptic cytoplasm or
whether the cistern serves as a store that releases Ca2+ in
response to activation of ACh receptors.
Outward K+ currents have been observed in mammalian OHCs
(Ashmore 1987; Housley and Ashmore
1992
; Santos-Sacchi and Dilger 1988
). There is
no clear consensus as to their indentity, although recent evidence
suggests that at least part of the current is carried by
Ca2+-dependent K+ channels (Housley and
Ashmore 1992
; Mammano et al. 1995
),
whereas most of them are carried by voltage-dependent K+
channels (Santos-Sacchi et al. 1997
). The development of
time- and voltage-dependent ion channels has never been examined in OHCs. By recording whole cell currents, we find that the size of
outward K+ currents increases significantly between 4 and
11 DAB (Fig. 3). The current size reaches a mature level at 11 DAB. The
increase in size (or conductance) might suggest that the density of
K+ channels increases during this time window.
Woolf and Ryan (1984) reported that cochlear
microphonics could first be measured at 12 DAB. Recordings of neural
responses from cochlear spiral ganglion cells (Echteler et al.
1989
) and from the cochlear nucleus (Ryan et al.
1982
) also suggest that the onset of auditory function in
gerbils is between 12 and 14 DAB. By measuring ACh-induced currents, we
have shown that the first detectable ACh-evoked current occurs in 12%
of gerbil basal turn OHCs at 6 DAB. By 11 DAB, the number of cells
responding positively to ACh application is comparable with that of the
adult cells tested. These results indicate that the development of
ACh-induced current response begins around 6 DAB and that responses are
adultlike around 11 DAB, at the time of the onset of auditory function. He et al. (1994)
demonstrated that OHCs developed motile
responses between 7 and 12 DAB in gerbils. Apparently, the maturation
ACh-induced current responses occurs in the same time period when OHCs
acquire electromotile behavior. From the fact that motile responses,
ion conductances, and ACh receptor function are all developed before 12 DAB, it is concluded that OHCs are functionally mature at the onset of hearing.
Dulon and Lenoir (1996) studied the ACh-induced currents
in neonatal rat OHCs. Although they reached the same conclusion as in
this work, that the onset of ACh-evoked response occurs at 6 DAB in
neonatal OHCs, there are two differences between the two sets of
results. They reported that the majority of OHCs displayed inward
nicotinic-like currents near the resting membrane potential at 8 DAB
(11 displayed inward whereas only 4 showed outward current). At 12 DAB,
the ACh-induced current response switched to outward current. They
suggested that the change in polarity of the current response during
development was likely due to either the maturation of K+
channels or progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca2+ and nearby
Ca2+-activated K+ channels at the synaptic pole
of OHCs. In contrast, we did not observe any inward current (manifested
as either inward current alone or biphasic responses) at holding
potentials more negative than
55 mV from 4 to 7 DAB. Neither did we
see any polarity change in the ACh-induced responses throughout the
time window examined, although biphasic responses were seen at a later
stage in some cells. We know that the fast inward Ca2+
current is directly related to the ionotropic receptor while the large
outward K+ current is carried by the nearby
KCa(ACh) channels. If the functional maturation of the ACh
receptor occurred earlier than that of the KCa(ACh)
channel, we would have observed the inward current with no outward
K+ current as Dulon and Lenoir (1996)
reported. The fact that the first ACh-induced response could be
observed with outward K+ current at 6 DAB indicates that
the functional onset of the ACh receptor, the nearby
KCa(ACh) channels, and the coupling between them occurs at
the same time.
Although Dulon and Lenoir (1996) observed a sudden
change in response polarity between 8 and 12 DAB, they did not examine whether the ACh-induced outward current increased between 8 and 12 DAB.
We demonstrate that the magnitude of ACh-induced currents grows between
6 and 11 DAB. There are several possibilities for the cause of the
growth of the ACh-induced outward current. Apparently, an increase in
the number of ACh receptors can account for this. This is in line with
the findings of Simmons and Morley (1998)
, who
demonstrated that the peak levels of expression of the
9 nAChR
subunit occurred around 6-10 DAB in rat OHCs. Alternatively, an
increase in the number KCa(ACh) channels can also result in the growth of ACh-induced outward current, if the ACh receptors are
fully developed (their number no longer increases) at 6 DAB. However,
from the present study, one is unable to determine whether such growth
is due to the increase in number of ACh receptors or the number of
KCa(ACh) channels.
Two recent studies attempted to pinpoint when the 9 nAChR subunit is
expressed in hair cells of developing animals. A study by Luo et
al. (1998)
showed that mRNAs of
9 are expressed before birth
and peak around 6 DAB in rat OHCs. Another study by Simmons and
Morley (1998)
, using [35S]-labeled cRNA in situ
hybridization techniques, showed that an expression of
9 nAChR
subunit was found after birth (0 DAB) and the highest levels of
expression occurred around 10 DAB in rat OHCs. Although no studies have
been done in gerbils to detect the expression of the
9 nAChR subunit
during development, it is likely that the expression of
9 follows a
similar time course as in rats, because the maturation of the auditory
function in both species occurs around 10-12 DAB. It needs to be
pointed out that the first expression of mRNA of
9 and the onset of
receptor function are two different matters. The expression of mRNA at birth does not necessarily mean that a functional receptor protein molecule is already present in the membrane. On the other hand, the
finding of a first detectable ACh-induced current at 6 DAB cannot rule
out that the receptor proteins actually emerge in the membrane well
before the onset of their function. However, the in situ hybridization
results suggest that the peak expression of
9 is between 6 and 10 DAB. This is temporally coincident with the onset and development of
ACh-induced currents revealed in the present studies.
It is generally accepted that there are longitudinal and radial
gradients in density of efferent innervation of OHCs (Liberman et al. 1990). Basal turn OHCs possess more efferent endings
than their apical turn counterparts, and efferent terminals on row 1 OHCs outnumber those on row 3 OHCs. Therefore, in this study we only
measured responses from basal turn OHCs. Developmental studies in mouse
and rat indicate that efferent innervation arrives at the apical OHCs
at least 2 days later than at the basal turn OHCs (Cole and
Robertson 1992
; Sobkowicz and Emmerling 1989
). Maturation of OHC electromotility is also delayed by 1-2 days in the
apical cells (He et al. 1994
). Because the organ of
Corti develops in the basal turn first and maturation then proceeds toward the apex, it is reasonable to assume that the maturation of the
ACh-induced responses in the apical turn may occur a few days later
than in the basal turn cells.
As in other altricial rodents such as mouse, rat, and hamster, OHCs in
gerbils are exclusively innervated by afferents in the first few days
after birth (Echteler 1992). As development progresses,
inappropriate afferent connections to OHCs withdraw as efferent fibers
approach. Although it is not clear when efferent neurons form
functional synapses with OHCs in the gerbil, the timing of functional
efferent innervation can be inferred from that in the mouse and
hamster, whose onset of auditory function is very similar to that of
the gerbil. Labeling experiments in rat and hamster indicate that
efferent fibers contact OHCs around 4-8 DAB (Cole and Robertson
1992
; Simmons et al. 1990
; Sobkowicz and
Emmerling 1989
). If in gerbils the efferent fibers contact OHCs
around 4-8 DAB as in rat and hamster, the appearance of ACh-induced currents is temporally overlapping with the formation of efferent synapses. This is noteworthy because it is well-known that the number
and distribution of nACh receptors in the neuromuscular junction are regulated by innervation during development. A remaining question is whether the number and distribution of AChRs in OHCs are
regulated by efferent innervation.
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ACKNOWLEDGMENTS |
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We thank X. Lin, X. Hu, and G. Emadi for technical assistance, and M. A. Cheatham and the late B. Evans for helpful discussions.
This work was supported by National Institute of Deafness and Other Communication Disorders Grants DC-00708 and DC-02764, and by a McKnight Senior Fellowship to P. Dallos.
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FOOTNOTES |
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Address for reprint requests: D.Z.Z. He, Auditory Physiology Laboratory, Northwestern University, Frances Searle Building, 2299 North Campus Drive, Evanston, IL 60208.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 April 1998; accepted in final form 6 November 1998.
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REFERENCES |
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