Development of Acetylcholine-Induced Responses in Neonatal Gerbil Outer Hair Cells

David Z. Z. He and Peter Dallos

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha 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).


    METHODS
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INTRODUCTION
METHODS
RESULTS
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REFERENCES

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 MOmega . The access resistance, which is the actual electrode resistance obtained on establishment of whole cell configuration, typically ranged from 6 to 10 MOmega . 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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Fig. 1. Video images showing the experimental setup for recording ACh-evoked current response from gerbil outer hair cells (OHCs) with whole cell voltage-clamp techniques. The preparations were placed on an inverted microscope (Zeiss) with Hoffmann optics. The video images were captured by a frame-grabber and processed with Adobe Photoshop. A: recording was made from a 4-day-old OHC in a cluster of hair cells and supporting cells. B: recording was made from an 8-day-old isolated OHC. ACh was delivered to the synaptic pole of the OHC with pressure ejection from a puffer pipette positioned ~10-15 µm away from the synaptic pole. Bars represent 20 and 10 µm for A and B, respectively.

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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2. A: survey microphotograph of the basilar membrane-organ of Corti complex obtained from 7-day-old gerbil basal turn cochlea. The picture was taken with an inverted microscope (Leitz) and bright-field illumination. Arrows indicate hair cell region. Scale bar represents 120 µm. B: an OHC isolated from 7-day-old basal turn cochlea. The microphotograph was taken from the same inverted microscope with Hoffmann optics. Bar represents 10 µm.

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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Fig. 3. Whole cell currents recorded from neonatal gerbil hair cells. Cells were voltage clamped to a holding potential of -70 mV. Membrane currents were recorded in response to voltage commands varying from -100 to 140 mV in 20-mV steps. The current-voltage functions (I-V curves), derived from the steady-state responses, are plotted. In this and all subsequent figures, outward currents are plotted upward. A: whole cell currents of a 4-day-old OHC. Cell length: 16 µm; cell capacitance: 6.3 pF. B: whole cell currents of an 11-day-old OHC. Cell length: 20 µm; cell capacitance: 9.6 pF. C: whole cell currents for an adult OHC. Cell length: 20 µm; cell capacitance: 9.4 pF. The scale bar for current amplitude applies to A-C. D: I-V curves of OHCs measured from the steady-state responses of A-C. E: whole cell currents for a 5-day-old inner hair cell (IHC). Cell length: 19 µm; cell capacitance: 8.4 pF. F: whole cell currents for an 11-day-old IHC. Cell length: 24 µm; cell capacitance: 9.8 pF. The scale bar for current amplitude applies to E and F. Note that the scale bars for current amplitude are different for the OHCs and IHCs. G: waveform of the voltage command. H: I-V curves of IHCs measured from the steady-state responses of E and F. Access resistance was ~75% compensated, and cell capacitance was fully compensated in the recordings. The data were corrected for voltage errors due to uncompensated series resistance.

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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Fig. 4. ACh-evoked outward currents recorded from 5- to 12-day-old basal turn OHCs. The membrane potentials were held at -30 mV to obtain maximal ACh-induced outward current. The amount of ACh used was 100 µM. A: noise floor of currents recorded from a 5-day-old basal turn cell. No measurable current response that was repeatable and time registered with the application of ACh is seen. B: ACh-induced outward currents recorded from a 6-day-old basal turn cell. A small outward current (µ19 pA) is observed after ACh application. C-E: ACh-induced outward currents recorded from 8-, 10-, and 12-day-old basal turn OHCs. Robust ACh-induced outward currents are seen. Note that the size of the currents increases between 6 and 12 DAB. F: membrane potential change after ACh application in the same cell shown in E. A hyperpolarization of ~5 mV in membrane potential after ACh treatment is seen.


                              
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Table 1. Maturation of ACh-induced currents in neonatal gerbil OHCs



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Fig. 5. A: onset of ACh-evoked outward K+ currents. The fraction of ACh-responsive cells vs. the total number of cells tested is plotted as a function of developmental ages. Percentage of ACh-responsive cells from adult group is also shown for comparison. B: means ± SD of magnitude of ACh-evoked outward currents at different ages. Analysis (t-test) indicates that the means of current magnitudes of the mature group and the developmental groups after 10 DAB were not statistically different.

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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Fig. 6. Examples of biphasic ACh-evoked currents obtained from OHCs isolated from 8-day-old and adult gerbils. The cells, held at -55 mV, were stimulated by pressure ejection of 100 µM ACh from micropipettes positioned ~15 µm away from the synaptic poles. The zero current potential for the 2 cells were -59 (8 DAB) and -62 (adult) mV. Note that the evoked currents are composed of a small early inward current and a prominent outward current.

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 alpha 9 homomers as reported by Elgoyhen et al. (1994).



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Fig. 7. Examples of inhibition of ACh-induced currents by strychnine in OHCs isolated from 8- and 12-day-old gerbils. The cells were held at -30 mV, and 100 µM ACh was pressure ejected to the synaptic pole of the cell to obtain the control response. Top panels: control responses. Middle panels: 0.01 and 0.1 µM strychnine was coapplied with 100 µM ACh to OHCs. Bottom panels: the ACh-induced responses after wash out. Note that the magnitude of outward currents decreases as the strychnine concentration increases, and the currents are partially recovered after wash out.

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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Fig. 8. Dose dependence of ACh-activated outward currents for 2 groups of cells (8 and 12 DAB). Top panel: representative responses obtained from 2 cells (holding at -30 mV) at 4 different doses. Bottom panel: results obtained from 6 cells at 8 DAB and 6 cells at 12 DAB. The smooth curves were fit by the Hill equation {IACh = Imax/[1 + (EC50/[ACh])n]} with half-activating concentrations (EC50) of 16.8 (8 DAB) and 20.3 (12 DAB) µM and n-coefficients of 1.8 and 1.9. Statistical significance was found between the 2 EC50.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 9 nAChR subunit is expressed in hair cells of developing animals. A study by Luo et al. (1998) showed that mRNAs of alpha 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 alpha 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 alpha 9 nAChR subunit during development, it is likely that the expression of alpha 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 alpha 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 alpha 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society