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1 Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, Kennett Square, 19348; 2 Department of Otorhinolaryngology, Head and Neck Surgery, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 24 November 2003; accepted in final form 13 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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1 ms) while sustaining neurotransmitter release for prolonged periods of time (minutes). Here we probed the properties and possible anatomical substrate of prolonged neurotransmitter release by using electrical measures of cell surface area as a proxy for neurotransmitter release to study hair cell exocytosis evoked by repetitive stimuli. We observed marked depression of exocytosis by chick tall hair cells. This exocytic depression cannot be explained by calcium current inactivation, presynaptic autoinhibition by metabotropic glutamate receptors, or postsynaptic receptor desensitization. Rather, cochlear hair cell exocytic depression resulted from the exhaustion of a functional vesicle pool. This releasable vesicle pool is large, totaling approximately 8,000 vesicles, and is nearly 10 times greater than the number of vesicles tethered to synaptic ribbons. Such a large functional pool suggests the recruitment of cytoplasmic vesicles to sustain exocytosis, important for maintaining prolonged, high rates of neural activity needed to encode sound. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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; Parsons and Sterling 2003; von Gersdorff 2001), which anchors to the presynaptic membrane only nanometers from the clustered, voltage-gated calcium channels (Issa and Hudspeth 1994; Nachman-Clewner et al. 1999; Roberts et al. 1990). The ribbon tethers synaptic vesicles by short filaments in an apparent depot close to the sites of calcium influx (Lenzi et al. 1999). Vesicles attached to the synaptic ribbon of the rod bipolar cells define a functional pool of "readily releasable vesicles" (von Gersdorff et al. 1996), and depletion of these vesicles from the ribbon is thought to contribute to synaptic depression (von Gersdorff and Matthews 1997).
The cochlea must encode information about the amplitude, duration, and frequency of the acoustic stimulus. The continuous nature of acoustic signals requires that the hair cell synapse sustain the release of neurotransmitter for prolonged periods of time. Such high-frequency firing can persist unabated for several minutes (Kiang 1965). The temporal demands of the auditory system are also remarkable. Two examples of the hair cell's exquisite dependency on phasic signaling occur with sound localization and periodicity pitch. Minute differences (10's of µs) in the arrival of acoustic waves at each ear are used by higher centers in the brain for azimuthal sound localization (Moiseff and Konishi 1981). Pitch recognition of certain frequency sounds such as "the missing fundamental" (de Boer 1976; Schouten 1940) is limited by the ability the hair cell synaptic output to maintain a preferred phase angle between individual cycles of an acoustic stimuli and afferent fiber discharges. Depending on the species, the ability of an afferent fiber to phase-lock to an acoustical signal fails between 0.9 kHz (frog: Hillery and Narins 1987) and 9.0 kHz (barn owl: Koppl 1997; Sullivan and Konishi 1984). Thus hearing requires the hair cell synapse to maintain notable temporal fidelity (1 ms) while sustaining neurotransmitter release for prolonged periods of time (minutes).
Auditory nerve discharge rates adapt in response to continuous pure tone stimulation (Kiang 1965). A decrement in the release of neurotransmitter by the hair cell has been suggested to underlie auditory nerve adaptation (Furukawa et al. 1982; Moser and Beutner 2000). Our studies in the chick basilar papilla (avian equivalent of the mammalian cochlea) of both hair cell exocytosis and eighth nerve single-unit recordings support this notion (Spassova et al., unpublished observations). Sound-evoked afferent synaptic activity adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. Comparison of the readily releasable pool size and number of vesicles tethered to the synaptic ribbon suggested that the readily releasable pool is defined by vesicles tethered to the synaptic ribbons. However, longer depolarizations yielded a second, slower, sustained kinetic component of exocytosis.
Here we probed the properties and possible anatomical substrate of prolonged neurotransmitter release by using electrical measures of cell surface area as a proxy for neurotransmitter release to study hair cell exocytosis evoked by repetitive stimuli. We observed marked depression of exocytosis by chick auditory hair cells. We attribute this depression to the exhaustion of a functional vesicle pool. Such a large functional pool suggests the recruitment of cytoplasmic vesicles to sustain exocytosis and maintain prolonged, high rates of neural activity needed to encode sound.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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) and will be described in brief. Tissue preparation
Cochlear capsules were harvested (Zidanic and Fuchs 1995) from 7- to 12-day-old white leghorn chicks (Gallus domesticus) following a protocol approved by the IACUC of the University of Pennsylvania. The sensory epithelium was removed, and a section of the sensory epithelium approximately 400 µm long, starting 0.5 mm from the apical tip of the basilar papilla, was mounted in a recording chamber. To minimize any possible confounding effects of tonotopy (Martinez-Dunst et al. 1997), our patch-clamp recordings were limited to the neural-most tall hair cells located approximately 0.5 to 1.0 mm from the apical end of the basilar papilla [corresponding to frequencies around 200 Hz (Manley et al. 1996)]. The basolateral surface of the neural-most individual tall hair cells was visualized with an upright, fixed-stage compound microscope. The recording chamber contained the control external saline (in mM: 154 NaCl, 6 KCl, 5 HEPES, 8 glucose, 2 MgCl2, 5 CaCl2, pH balanced to 7.4 with NaOH) (Fuchs et al. 1988).
Whole cell patch-clamp recording
Individual tall hair cells were patch clamped using tight seal recordings in whole cell configuration (Hamill et al. 1981). Cells were voltage-clamped at a holding potential of –81 mV with either an Axopatch 200B (Axon Instruments, Foster City, CA) or an EPC9 patch-clamp amplifier (HEKA Electronics GmbH, Lambrecht, Germany). A mean resting membrane capacitance of 6.1 ± 0.2 pF (n = 56) was recorded from tall hair cells by a mean access resistance 8.9 ± 0.3 M
(n = 56). Pipettes (KIMAX 51, World Precision Instruments) were coated with purple ski wax (SWIX, Lillehammer, Norway) to reduce their capacitance, and filled with the control internal solution [in mM: 115 glutamic acid, 115 CsOH, 20 tetraethylammonium (TEA) chloride, 13 NaCl, 10 HEPES, 3 MgCl, 5 NaATP, 0.3 GTP, 0.2 EGTA, pH balanced with CsOH to 7.4]. A liquid junction potential of +11 mV was measured for our cesium glutamate internal solution (Spassova et al. 2001), and all voltages are corrected by subtraction of this liquid junction potential.
Changes in cell membrane capacitance (
Cm) were used to monitor fusion of neurotransmitter-containing vesicles during exocytosis, and measured with either the "piecewise linear" technique with a dual lock-in amplifier (H. Meyer, Goettingen, Germany) (Gillis 1995; Lindau and Neher 1988) or by the "sine+DC" mode of the EPC-9 software lock-in amplifier (Gillis 2000). Either an 800-Hz (40 mV peak-to-peak) or a 1.25-kHz (25 mV peak-to-peak) sinusoidal command voltage was superimposed on the holding potential of –81 mV. Hair cell excytosis was elicited by trains of 4–10 depolarizing voltage-clamp steps to –21 mV that ranged in duration from 0.3 to 3.0 s at 6-s intervals. Calcium currents were sampled at 50-µs intervals, filtered at 2.9 kHz. Capacitance data were filtered and analyzed off-line using IgorPro software (Wavemetrics, Lake Oswego, OR) or Microsoft Excel (Microsoft, Redwood, WA).
Data analysis
All experiments were done at 20–22° C, and all measurements are given as mean values ± 1 SE unless otherwise stated. Values of Cm elicited by each depolarization were quantified as the difference between the average value of a 200-ms Cm segment immediately before the depolarization and one measured 400 ms after the end of the depolarizing pulse. This avoided any contamination of the exocytotic response by capacitive transients not associated with exocytosis (Debus et al. 1995; Horrigan and Bookman 1994).
An alternative to the repeated-measures ANOVA, the generalized estimating equations, was used to compare the successive Cm values of the CPPG-treated versus control cells because it accounts for the dependency of successive Cm values for a given cell (Zeger and Liang 1986). The Cm data were fitted to the finite pool model with a least-squares algorithm (TableCurve 2D, Systat Software, Richmond, CA), and how well the model explained data variance was assessed with the R2 statistic. The number of available capacitance data points ranged from 4 to 10 per cell and all the available data for each cell were used in the fitting procedure. The dependency of B0 on pulse length was tested with a simple linear regression model (Sigmaplot, SPSS, Chicago, IL).
Estimate of pool size
The number of vesicles in the releasable pool was calculated by dividing Cm by the estimated single-vesicle capacitance reported in an anatomical study on the bullfrog saccular hair cell (Lenzi et al. 1999). This single-vesicle capacitance of 37 aF was derived using a specific capacitance of membrane as 1 µF/cm2 and a mean vesicle diameter of 34.3 nm. A similar mean vesicle diameter was found when we measured a sample of the vesicles found in the published electron micrograph of chick cochlear hair cells taken from the same approximate location on the basilar papilla as cells in our experiments (Martinez-Dunst et al. 1997).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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Hair cell depolarization induces a membrane capacitance jump attributed to the calcium-dependent increase in cell surface area associated with fusion of synaptic vesicles preceding neurotransmitter release (Beutner et al. 2001; Moser and Beutner 2000; Parsons et al. 1994; Spassova et al. 2001). Repeated stimulation of the chick cochlear hair cell with identical depolarizations spaced 6 s apart results in a series of steplike capacitance increases (Fig. 1A). However, each subsequent exocytic response in the train is smaller than the previous. The extent of the exocytic depression in Fig. 1A is quantified by measuring each subsequent capacitance jump (Fig. 1B). The amplitude of subsequent Cm decays over the course of 2 to 4 stimuli and reaches a constant, but nonzero, value during the train of depolarizing stimuli. On average, the Cm response to depolarizing stimulus trains behaved similarly (Fig. 1C). The first pulse in the train elicited a mean Cm of 194 ± 20 fF (n = 29). Subsequent Cm progressively decreased and ultimately approached a constant value of 15 ± 2 fF per pulse. This 13-fold activity-dependent decrement of exocytosis is reminiscent of synaptic depression, a common form of synaptic plasticity after repeated activation of the nerve terminal (Betz 1970; Zucker 1989).
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Direct recording of calcium influx by voltage-dependent calcium channels (Fuchs et al. 1990; Hudspeth and Lewis 1988) allow us to test whether ICa inactivation is the cause of the observed exocytic depression. Figure 2A shows ICa current activated by a train of 7 depolarizations to –21 mV. In this cell, no decrease in the amplitude of the current was observed between subsequent pulses as ICa remained constant throughout the train at –97.1 ± 0.8 pA (n = 7 pulses). As in other cells studied, no significant calcium channel inactivation develops during the individual test pulse of the train (Fig. 2B). Consistent with the observations here, hair cell ICa is characterized as both rapidly activating and deactivating, and noninactivating (Fuchs et al. 1990; Hudspeth and Lewis 1988; Rodriguez-Contreras et al. 2002; Zidanic and Fuchs 1995), and thus does not account for the exocytic depression.
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; von Gersdorff et al. 1997). These receptors are present and functional in some hair cells (Guth et al. 1993; Hendricson and Guth 2002a,b), and thus repeated one-second depolarizations were given in the presence and absence of (RS)-a-cyclopropyl-4-phosphono-phenylglycine (CPPG), a potent antagonist of presynaptic mGlu receptors (Jane et al. 1996) (Fig. 2C). If presynaptic mGluR activation was responsible for declining Cm, then exposure to a mGLuR antagonist is predicted to alleviate the exocytic depression. However, statistical analysis of exocytic depression in the presence of 300 µM CPPG (n = 5 cells) showed no difference from that in control cells (n = 7 cells; P = 0.17).
The exocytic depression could be secondary to the washout of a factor or factors mediating exocytosis over the time period of the recording (Hay and Martin 1992; Moser and Beutner 2000; Neves and Lagnado 1999). To test this notion, we examined the ability of the hair cell exocytic depression to recover. The standard train of stimuli was applied to depress exocytosis, and then a variable interval was allowed before a single test stimulus was delivered. The ratio of the test pulse to the first pulse of the conditioning train is plotted in Fig. 2D. Over 60% of the initial Cm is recovered within 1.5 min of the end of the train. This slow recovery time course after trains of depolarization is similar to the retinal bipolar cell ribbon synapse where 100 s or longer is required for recovery (von Gersdorff and Matthews 1997). The lack of complete recovery may be attributable to the rundown or washout of exocytic capacity over a time course of many minutes. However, a major portion of exocytosis recovers after a depressing stimulus train and suggests that exocytic rundown does not explain the exocytic depression observed during an approximately 50-s stimulus train.
Large pool of releasable vesicles
Having ruled out several possible mechanisms of depression, we hypothesized that the diminishing exocytic response results from the exhaustion of a finite releasable vesicle population (Neher 1998; Schneggenburger et al. 2002). A simple model was developed to test this hypothesis. Given a finite releasable vesicle pool with size B0 and 
, the fraction of vesicles available for release, then Cm is predicted as B0 x . Alternatively, pairs of successive Cm values can be used to determine B0 if is similar from stimulation to stimulation (Gillis et al. 1996).
The hair cell exocytotic responses to repeated depolarizations followed an apparently exponential decay (see Fig. 3A), consistent with a constant release fraction during successive pulses. Therefore we extended the Gillis–Neher formalism to determine the initial pool size using the Cm of successive depolarizations of the stimulus train. In addition to B0 and , the model also included a constant R, which is interpreted as the amount of pool refilled in between stimuli. Successive Cm magnitudes would be expected to yield a geometric series having the following form (see APPENDIX for derivation)
 | (1) |
Cn is the capacitance change in fF associated with the nth pulse.
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Cm values predicted by the model (solid line in Fig. 3A) correlated well with the experimental observations. In this example, the model fit with an R2 of 0.95 and yielded a value of 0.44 for , whereas B0 = 300 fF and R = 24 fF. Values of varied widely between cells (range: 0.23 and 0.96; mean 0.64 ± 0.04; n = 29). However, the fit of the model to the data remained robust (R2 > 0.75) over this wide variation of (Fig. 3B).
Given the good fitofour finite pool model to the data, we then estimated the pool size of synaptic vesicles that support prolonged exocytosis. The pool of vesicles available before each stimulus was determined by dividing Cn+1 by . The mean value of B0, the initial pool, using this method was 311 ± 33 fF (n = 29 cells) and corresponds to approximately 8,000 synaptic vesicles (see METHODS). B0 was independent of both pulse duration (Table 1) and the fractional release per pulse (Fig. 3C), further validating the appropriateness of the finite pool model. The mean value for the constant R was 18.7 ± 3.3 fF per pulse, or 3.1 fF/s. This was similar to the 15.0 ± 2.0 fF per pulse slope of the asymptote fitted to the average Cm data in Fig. 1C. Given a pool size of approximately 310 fF and a refilling rate of approximately 3 fF/s, then nominally 100 s would be required to refill the large releasable pool. Thus these estimates of R are consistent with the 90- to 120-s exocytic recovery time measured after a train of depolarizations (see Fig. 2D).
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Cm data in Fig. 1C. Furthermore, the original algorithm described by Gillis et al. (1996) to determine with a pair of pulses also yielded a similar value for pool size. Because was not assumed to be constant for successive pulses, an upper limit of B0 (Bmax) was determined, which was equal to B0 when the fractional release of the first pulse equaled that of the second. In the hair cell, the paired-pulse algorithm yielded a value for Bmax of 307 ± 30 fF (n = 42). The data used for the paired-pulse analysis include the first 2 pulses from the 29 cells fitted to the finite pool model. None of these 3 estimates of initial pool size is significantly different from the other (P > 0.05). Thus 3 different estimates of B0 yielded approximately 8,000 vesicles. The correspondence between the first 2 measures of initial pool size with the prediction of paired-pulse algorithm is important, given that the latter estimate does not share any assumptions about or R. Taken together these observations support the hypothesis that a large finite releasable vesicle population mediates prolonged exocytosis after repetitive stimulation of the cochlear hair cell. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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The concept of functional pools of releasable or available vesicles at a synapse is well established (Neher 1998). These pools vary in both size and number, and likely serve different functions at different synapses. Perhaps the best-studied example is the rod bipolar cell of the goldfish retina. There, a readily releasable pool of approximately 6,000 vesicles has been identified (von Gersdorff and Matthews 1994), and mediates the fast transient release of glutamate (von Gersdorff et al. 1998). Because the size of this functional pool corresponds to anatomical measures of the number of vesicles attached to the rod bipolar cell synaptic ribbons, the two are thought to be equivalent (von Gersdorff et al. 1996).
We recently described (Spassova et al., unpublished observations) a readily releasable pool in chick cochlear hair cells that is similarly defined by the vesicles tethered to the synaptic ribbon. This pool contains only about 1,000 vesicles because of the small number and size of synaptic ribbons in chick hair cells (Martinez-Dunst et al. 1997). The experimental protocols used in this report, however, lack sufficient temporal resolution to specifically discern the contribution of readily releasable vesicles to the functional pool identified here that is exhausted by trains of stimuli. This large releasable pool is 8 times the size of the readily releasable pool, and thus is too big to be accounted for by vesicles tethered to the ribbon. Therefore we suggest that the large releasable pool described here consists, in part, of vesicles tethered to the synaptic ribbon, but primarily of vesicles recruited from the cytoplasm.
This pool may be the anatomical basis of the slow component of chick hair cell exocytosis that mediates reloading of the ribbon with vesicles (Spassova et al., unpublished observations). However, the large pool described here is still only a fraction of either the 45,000 release competent vesicles reported in mouse cochlear hair cells (Beutner et al. 2001) or the 2–6 x 105 total synaptic vesicles in the frog saccular hair cell (Lenzi et al. 1999). Even given quantitative differences in the presynaptic ultrastructure of different hair cells, the releasable pool defined in this report is sufficiently small compared with these other vesicle counts to argue that the pool consists of only a subset of cytoplasmic vesicles. What defines this subset of hair cell synaptic vesicles as releasable remains unclear. Calcium has been asserted at other synapses to play a role in vesicle recruitment (Sakaba and Neher 2001; Wang and Kaczmarek 1998; Wu and Borst 1999). Mouse cochlear hair cells exhibited a sustained secretory component that depended on intracellular calcium buffering (Moser and Beutner 2000), and may be supported by mechanisms common to our large releasable pool. We used 0.2 mM EGTA as the pipette calcium buffer, which is similar to the equivalent endogenous buffer in the mouse hair cell as well as permissive for the sustained secretory component. Other mechanisms proposed to mobilize or recruit synaptic vesicles to release sites at different synapses remain controversial, but may involve actin cortices (Job and Lagnado 1998; Sakaba and Neher 2003), synapsin (Chi et al. 2001; Farve et al. 1986; Hilfiker et al. 1998), and/or molecular motors (Griesinger et al. 2002; Heidelberger et al. 2003; Mochida et al. 1994; Ryan 1999).
Relevance of large releasable pool to hair cell function
Hair cell afferent synapses exhibit exceptional phasic and tonic neurotransmitter release. Comparison of readily releasable pool kinetics in chick cochlear hair cells with single-unit recordings of cochlear nerve in the same species suggest that adaptation of nerve discharge is mediated by synaptic vesicles tethered to the synaptic ribbons (Spassova et al., unpublished observations). However, the adapted nerve fiber continues to fire for seconds and minutes if the stimulus is maintained, and this ability to sustain prolonged neurotransmitter release requires a large number of available vesicles, presumably supplied by the functional pool described here. The ample supply of synaptic vesicles also ensures that transmitter release is both reliable and timely, even given inherently slow exocytic processes (Almers 1994). Thus the existence of a large releasable pool is likely critical to both phasic and tonic neurotransmitter release by cochlear hair cells.
We estimated this pool size based on membrane capacitance measurement, which is the net sum of exo- and endocytosis. Our previous work on bullfrog saccular hair cells has shown that whole cell patch-clamp recording blocks endocytosis presumably attributable to the washout of critical factors (Parsons et al. 1994). This is in contrast to studies on mouse inner hair cells where whole cell recording conditions were less favorable for cytoplasmic exchange, and endocytosis was observed (Moser and Beutner 2000). Although we see no evidence of endocytosis in our measurements on chick cochlear hair cells, we cannot, however, formally exclude that possibility. If endocytosis is ongoing during the pulse, then our measures are actually underestimates of the size of the large functional pool needed to sustain prolonged exocytosis.
In conclusion, a large functional pool of synaptic vesicles maintains prolonged hair cell exocytosis evoked by repetitive stimulation. This functional pool is almost 10-fold larger than the number of vesicles that are found on the synaptic ribbon, and differs from what has been reported in the retinal bipolar cell. There the releasable pool evoked under similar stimulation conditions is not more than twice the size of the vesicles tethered to the ribbons (von Gersdorff and Matthews 1997; von Gersdorff et al. 1996). Apparently the sustained demands on neurotransmitter release placed by hearing require specialized mechanisms that allow a large number of cytoplasmic vesicles to rapidly participate in exocytosis. We also observed that both the exhaustion and refilling of chick hair cell readily releasable pool is as much as 10 times faster (Spassova et al., unpublished observations) than comparable measures in the retinal bipolar cells. Taken together these observations further reinforce the existence of fundamental differences between ribbon synapses in the auditory and visual systems.
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APPENDIX: DERIVING CALCULATION OF INITIAL RELEASABLE POOL SIZE |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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Cn
Cm per pulse (fF/pulse)
Multiple depolarizations
Any depolarization yields the capacitance change Cn+1 by the equation
 | (A1) |
 | (A2) |
from each side of Eq. A2, and rearranging, yields
 | (A3) |
 | (A4) |
 | (A5) |
, B0, and R. |
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONAPPENDIX: DERIVING CALCULATION...ACKNOWLEDGMENTSREFERENCES |
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GRANTS
This work was funded by National Institute on Deafness and Other Communication Disorders Grant DC-03763 and a Sloan Research Fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. D. Parsons, Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, 382 West Street Rd., Kennett Square, PA 19348 (E-mail: thd{at}vet.upenn.edu).
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