Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: Capacitance measurements in saccular hair cells

Neuron,

Vol. 13,875-883,

October,

1994, Copyright

0 1994 by Cell Press

Calcium-Triggered Exocytosis and Endocytosis in an Isolated Presynaptic Cell: Capacitance Measurements in Saccular Hair Cells Thomas D. Parsons,* David Lenzi,+ Wolfhard Almers,* and William M. Roberts+ *Max-Planck-lnstitut fur medizinische Forschung Abteilung Molekulare Zellforschung 69120 Heidelberg Federal Republic of Germany tlnstitute of Neuroscience University of Oregon Eugene, Oregon 97403

Summary Depolarization of isolated frog saccular hair cells caused Caz+-dependent increases in membrane capacitance that we interpret as the fusion of synaptic vesicles with the plasma membrane. During a maintained depolarization to -10 mV, the capacitance increased at a rate corresponding to the fusion of - 500 vesicles per second at each active zone. Release continued at this high rate for up to 2 s, long enough to exhaust >5 times the number of vesicles initially in close apposition to the plasma membrane at active zones. We therefore propose that hair cells are specialized for rapid replenishment of vesicles at release sites. Upon repolarization to -70 mV, the capacitance returned exponentially (time constant, - 14 s) to near the prestimulus level in perforated-patch recordings, but not in whole-cell recordings, suggesting that a mobile intracellular factor is required for membrane retrieval. Introduction The vesicle hypothesis of chemical synaptic transmission proposes that neurotransmitters are released from synaptic vesicles at specialized sites in nerve terminals (de1 Castillo and Katz, 1956). Although this hypothesis has formed the foundation of our understanding of synaptic transmission for almost four decades, the mechanisms of neurotransmitter release and vesicle recycling have remained controversial (Fesce et al., 1994). Technical innovations such as the measurement of changes in cell capacitance to monitor changes in plasma membrane surface area (Neher and Marty, 1982) have recently opened the way to a detailed understanding of both vesicle fusion (exocytosis) and membrane retrieval (endocytosis). Capacitance techniques have proved very successful for studying the secretion of peptides and other hormones from large secretory granules and dense-core vesicles (DCVs) (Lindau, 1991). The techniques have been more difficult to apply to the study of secretion of conventional synaptic transmitters from small clear vesicles because of the complex morphology and remote release sites of most neurons. The difficulty in measuring changes in membrane capacitancewith the resolution needed to study mem-

brane trafficking at conventional synlapses between neurons has led to a search for tractable synaptic preparations. Recently, von Gersdorff and Matthews (1994) have made capacitance measurements in isolated synaptic terminals of retinal bipolar cells, which contain small clear-core vesicles. We have found another sensory cell preparation, hair cells from sacculus of grassfrogs (Rana pipiens), that is well suited to the measurement of capacitance changes associated with synaptic transmission. Synaptic transmission from hair cells on to afferent axons of the eighth cranial nerve has many properties typical of fast synaptic transmission between neurons (for reviews see Roberts et al., 1988; Lenzi and Roberts, 1994). Transmission is quanta1 and requires Ca2+ in the extracellular medium (Furukawa et al., 1978). Depolarization of the hair cell (Figure IA) increases the release of an excitatory neurotransmitter, probably glutamate, from small (30-50 nm) clear-core vesicles clustered at morphologically distinct presynaptic active zones, which can be identified in transmission electron micrographs by the presence of a 200-400 nm diameter osmiophilic presynaptic body adjacent to densely stained pre- and postsynaptic membranes. A single layer of several hundred synaptic vesicles covers the surface of each prsesynaptic body (Figure IC), apparently tethered to it by thin filaments (Gleisner et al., 1973). Hair cells ,from the sacculus of R. pipiens typically have about 20 such active zones, each associated with an array of voltage-gated Caz+ channels and Ca2+-activated potassium channels (Roberts et al., 1990). Depolarization (opens some of these CaH channels, raising the local cytoplasmicconcentration of free Ca2+ to tens or hundreds of micromolar (Roberts, 1993, 1994), which is thought to trigger synaptic exocytosis (Augustine et al., 1991). We report here dynamic measurements of small changes in the surface area of hair celIls from the sacculus of grassfrogs that are consistent with the Ca*+dependent fusion of synaptic vesicles with the plasmalemmaand the subsequent reuptakeof membrane after stimulation (Neher and Marty, 1982). Results Using patch voltage clamp methods (Lewis and Hudspeth, 1983), we have studied capacitance changes caused by depolarization of isolated hair cells. These hair cells tolerated recording through large pipettes (Figure IB), generally yielding access resistances to the cell interior of 4-8 MQ in both whole-cell and perforated-patch experiments. Such low access resistances permitted the rapid voltage control needed for capacitance measurements and for the rapid exchange of soluble molecules between the cytoplasm and recording pipette during whole-#cell recording. To reduce noise and uncover the voltage-gated Ca*+

Neuron 876

Figure 1. Anatomy

of Saccular

Hair Cells

(A) Transmission electron micrograph of a section through the saccular macula. Each of the two hair cells shown has a bundll presynaptic body 1 (arrow), mechanosensory stereocilia at its apical surface. Afferent synapses, char racterized by an osmiophilic distributed across the basolateral membrane. Scale bar, 5 pm. On the lower right, a recor rding pip (B) Differential interference contrast micrograph of a hair cell during pate :h-clamp recording. is shown sealed to the cell. Scale bar, 8 urn. (C) Higher magnification view of a 120 nm section showing an afferent acti ve zone with osmiophilic membrar pre- and postsynaptic the electron-dense synaptic body, and its halo of lucent synaptic vesicle! s. Scale bar, 200 nm.

current, all cells were studied with Cs+ in the recording pipette to block the large Cap-activated potassium conductance. In both whole-cell and perforated-patch experiments, application of depolarizing voltage steps caused surprisingly large increases in cell capacitance (Figure 2A), which we interpreted to indicate the Ca2+dependentfusionof manysmallsynapticvesicleswith the plasmalemma. The fusion of large DCVs with the plasma membrane cannot account for the observed capacitance increases because DCVs are rare in these hair cells and are not associated with the plasma membrane. From transmission electron micrographsof the saccular epithelium (see Figure IA), we estimated that there are fewer than 60 DCVs per hair cell (see Experimental Procedures). Since the diameter of these candidate DCVs was -100 nm, they could add -20 fF

e of are ette ies,

to the cell capacitance if they all fused with the plasma membrane. During depolarizations lasting 1 s to voltages near 0 mV, the cellular surface area sometimes increased by >500 fF (>5% increase from an initial capacitance of -10 pF; Figure 3A). Twelve of 19 cells studied using whole-cell voltage clamp and 11 of 14 cells studied using perforatedpatch exhibited >lOOfF increases in membrane capacitance following depolarization to -10 mV for 1 s (see Figure 2A). The average magnitude of the capacitance change (measured 450-1000 ms after the stimulus ended) was 303 + 79 fF (mean f SEM; n = 19) in whole-cell recordings and 435 + 109 fF (n = 14) in perforated-patch recordings. We did not measure capacitance changes during the stimulus, when the large Ca2+ conductance interfered with the measurement (Lindau et al., 1992), or immediately after the

Exocytosis and Endocytosis

in Hair Cells

877

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Time after Depolarization (sac) Figure 2. Increases in Membrane Area and Subsequent brane Retrieval Following Stimulation of Hair Cells

Mem-

(A) Changes in capacitance elicited by a 1 s voltage step from -70 mV to -10 mV, utilizing either the whole-cell (WC) or perforated patch-clamp (PP) configuration. In whole-cell, the initial capacitance (C,) was 13.1 pF and the initial access resistance (R,) was 4.1 Ma, whereas in perforated patch-clamp, C, was 11.4 pF and R, was 9.4 MQ. The capacitance trace is blanked during the voltage step (here and in subsequent figures) because the measurement is unreliable at that time (Lindau et al., 1992). (B) Whole-cell (WC)dialysis abolishes membrane retrieval in hair cells. Comparison of the fraction of the added membrane that remained at different times after the end of 1 s or 2 s depolarizations to -10 mV under whole-cell and perforated-patch (PP) conditions. A value of 1.0 on the ordinate corresponds to the initial capacitance increase, measured soon after the end of the depolarization (t = 450-800 ms). To accurately document the time course of membrane retrieval, we selected only cells with an initial capacitance increase >250 pF.

stimulus, during which time we observed capacitance transients that varied among cells in both amplitude and sign (see Figure 2A). Only a small part (<2%) of the capacitance increases reported above can be accounted for by extrapolating the slow, usually upward, drift in the baseline capacitance observed during the 5-20 s interval prior to the application of the stimulus in these cells (whole-cell recordings, +4.5 + 2.4 fF/s, n = 19; perforated-patch recordings, +I.4 f 1.6 fF/s, n = 14). Recovery of capacitance following repolarization to -70 mV was seen in perforated-patch recordings but not whole-cell recordings (see Figure 2A). In whole-

cell recordings, in which at least 200 s was allowed for cytoplasmic dialysis before applying the depolarizing step, the capacitance usually continuied to increase after the stimulus (see Figure 26). The average rate of this poststimulus capacitance increase was +3.8 k 1.0 fF/s (114 fF in 30 s). Because this rate was smaller than the average prestimulus drift (+9.4 rf: 2.5 fF/s in these nine cells), it is possible that a partial (-25%) recovery of membrane was masked by the baseline drift in the whole-cell recordings. In perforated-patch recordings, thecapacitance returned to nearthe prestimulus level over an approximately exponential time course. Only 23% 5 8% (n = 7) of the added membrane remained 20 s after a large stimulus, and only 13% k 8% (n = 7) remained after 30 s (see Figure ;!B). These data correspond to a recovery time constalnt of - 14 s for retrieval of vesicular membrane. After adjusting for the prestimulus baseline drift of +I:1 + 1.3 fF/s in these seven cells, the fraction of unrecovered membrane becomes 18% after 20 s and 8% after 30 s, and the time constant is reduced to -12 s. This loss of the ability of the cells to retrieve membrane during whole-cell recording occurred very rapidly. It was evident well before another type of rundown that has previously been observed in these cells, the reduction in the number of functional Ca*+ channels (Roberts et al., 1990). Large Ca2+ currents were present in cells that did not retrieve membrane (Figure 3A) and, as will be shown below, are indeed required for the capacitance increase. One possible explanation for the sensitivity of memlbrane retrieval to the recording conditions is that some low molecular mass cytoplasmic factor necessary for the maintenance of endocytosis was lost during whole-cell recording. We used two approaches to test whether the observed capacitance increases were triggered by the influx of extracellular Ca2+ through voltage-gated Ca2+ channels. First, we varied the amplitude of the voltage steps to alter the size of the Ca2+ current and found that the magnitude of the capacitance change was proportional to the magnitude of the voltage-gated Ca2+ current (Figure 3A). In seven cells, voltage steps to i-60 mV for 1 s (near the suppression potential of the Ca2+ current) elicited only a small Ca*+current and a small average capacitance increase of 55 + 39 fF, whereas subsequent steps of the same duration to 0 mV caused a large Ca*+ current and a large average capacitance increase of 305 + 134fF. A similar voltage dependence was observed with 100 ms voltage steps (-40 mV, 12.1 + 4.4 fF, n = 8; -10 mV, 21.9 + 4.0, n = 17; +60 mV, 1.5 + 0.4fF, n = 4; combined whole-cell and perforated-patch experiments). A second series of experiments tested the Cap dependence of the capacitance increase by blocking the Ca*+ current with IO mM external Co’+, an inhibitor of Ca2+ influx through voltage-gated Ca*+ channels. Co2+ abolished both the Ca2+ current and the increase in capacitance following a 1 s depolarization to -10 mV (Figure 3B). To determine the time course over which mem-

Neuron a78

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Figure 3. Dependence of the Capacitance Increase on the Ca2+ Current

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(A) Voltage dependence of the capacitance increaseand Ca2+current measured in a hair cell underwholecell conditions(C, = 13.0 pF; Rx = 4.1 MQ). Capacitance increases in response to 1 s steps from -70 mV to (from left to right) +60 mV, +40 mV, +20 mV, and 0 mV are shown in (i); representative Ca2+ currents recorded in response to 10 ms depolarizations to (from left to right) +60 mV, +45 mV, +I5 mV, and 0 mV immediately before the capacitance measurements are shown in (ii); and the data are plotted versus membrane potential in (iii). Note the different time scales in (i) and (ii). (B) Blockade of the capacitance increase and Ca2+ current by Co*+. Ca*+ currents and capacitance changes recorded during voltage steps from -70 mV to -10 mV before (top) and after (middle) addition of 10 m M Co*+ to the bath. Bottom traces show the voltage commands. Recordings were made in the perforated-patch mode (C, = 11.4 pF; R, = 9.4 Mhl).

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brane is added to the plasmalemma during a maintained depolarization, we terminated depolarizations at various times and measured the increase in capacitance as a function of stimulus duration (Figure 4). During

depolarizations

to

-10

mV,

the

cells

main-

tained a high rate of capacitance increase for at least 2 s (322 + 70 fF/s; see legend to Figure 4). The rate of capacitance increase corresponds to the fusion of -10,000 vesicles per second throughout the cell (see Experimental Procedures), - 500 vesicles per second at each of the cell’s -20 active zones, and to one vesicle fusion per active zone every 2 ms. Maintaining exocytosis at such a rate for 1 s requires >I0 times the number of vesicles reported to be apposed to the plasma membrane at active zones of similar dimensions in goldfish saccular hair cells (Hama and Saito, 1977; Hama, 1980).

Discussion Evidence That Capacitance Increases Are Due to Exocytosis of Synaptic Vesicles While there can be little doubt that the increased membrane capacitance that we observed following depolarization represents an increase in the area of membrane that is electrically accessible from the cell body, there are several potential sources of this added membrane that must be considered. The possibility that changes in the resistance of the narrow necks of the stereocilias could modulate the apparent membrane capacitance of the cell by influencing the charging of the stereociliary membrane is ruled out by patch recordings that show rapid and completecharging of the stereociliary membrane by voltage steps applied to the cell body (Roberts et al., 1990).

Exocytosis and Endocytosis 879

in Hair Cells

(ii)

tween these tubules and the plasmalemma were Ca2+ dependent, perhaps through their participation in endocytosis of vesicular membrane, it is (conceivable that the tubules could have contributed to the observed capacitance increases. However, thistubular network has not been observed in anatomical studies of hair cells from the frog sacculus (Jacobs and Hudspeth, 1990) or crista ampullaris (Gleisner el: al., 1973). The fourth and most likely source of the large increases in membrane capacitance following depolarization is the population of small clearr-core synaptic vesicles in the hair cell. These vesicles are highly concentrated at presynaptic sites, close to the cluster of -90 large-conductance Caz+ channels at each active zone (Roberts et al., 1990, 1991; Roberts, 1993, 1994). The voltage dependence and Cd+ sensitivity of the capacitance increase indicate that it depends upon Ca*+ entering through these presynaptic Ca*+ channels.

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0:5 1 .o 1.5 2.0 Duration of Voltage Step (SC)

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Capacitance

Increases

2.5

with

Increasing

(A) The capacitance change shown in (i) is the average of nine consecutive responses to 100 ms depolarizations applied at 1 Hz obtained from one cell (C, = 13.6 pF; R, = 7.2 MM. Single responses to 1 s (ii) and 2 s (iii) depolarizations from a different cell (C, = 10.4 pF; RX = 4.1 MD) are also shown. (B) Mean capacitance increases after IO ms, 100 ms, 1 s, and 2 s voltage steps from -70 mV to -10 mV recorded in the whole-cell mode. Error bars represent the SEM of averages from n cells; to avoid possible effects of vesicle depletion, only the first response from each cell was included in the average. The line shows the weighted linear regression tothefourmeansand standarderrors shown (slope = 322 +_ 70 fF/s). The baseline drift in these cells (+7.4 * 1.9 fF/s) accounts for only a small fraction of this increase. The right ordinate shows the approximate number of synaptic vesicles that would need to fuse with the plasma membrane to yield the observed capacitance changes (see Discussion for estimate of the capacitance of a single vesicle).

A second possible source of added membrane are DCVs, which are known to undergo Ca2+-dependent fusion with the cell membrane of many cells. However, DCVs have not been reported in previous electron microscopic studies of hair cells (Gleisner et al., 1973; Hama and Saito, 1977; Jacobs and Hudspeth, 1990). We found too few candidate DCVs in the electron micrographs that we examined to account for more than -5% of the large capacitance increases observed in this study. A third possible source of added membrane is a subsurface membrane system, such as the anastomosing tubules that have been reported in chick hair cells (Hama and Saito, 1977). If the connection be-

Endocytosis Depends on a Small, Mobile Intracellular Factor Endocytosis of synaptic vesicle membrane following exocytosisisobligatoryforthemaintenanceof normal synaptic transmission (Koenig et al., 1989). Several rates have been reported for endocytosis following Ca2+-dependent exocytosis. In pituitary melanotrophs, membrane retrieval following fusion of DCVs is reported to have two phases with time constants of 350 ms and 4 s (Thomas et al., 1994). Slowler endocytosis has been observed at presynaptic terminals in dissociated hippocampal neurons (time constant, -60 s; Ryan et al., 1993) and at neuromuscular junctions (two endocytotic processes with time courses of seconds and tens of seconds; Miller and Heuser, 1984). Fast and slow components of endocytosis have also been observed in bipolar cell terminals frorn the goldfish retina (time constants, 1.3 s and 31 s; von Gersdorff and Matthews, 1994). Although we have measured only a slow endocytotic time constant of -14 s, a faster endocytotic process could act after short bouts of exocytosis as it does in bipolar cells (von Gersdorff and Matthews, 1994). In the hair cells studied in our experiments, the competence for endocytosis was maintained for the duration of perforated-patch recordings but lost within 200 s during whole-cell dialysis, which suggests that some mobilecytoplasmiccomponent is required. Alternatively, a component of the whole-cell pipette solution could have inhibited endocytosis. It is unlikely that the Ca2+ buffer in the pipette (I mM EGTA) could have been responsible for the loss of endocytosis, given that the native Ca2+ buffer of the hair cell present in perforated-patch experiments captures Ca2+ much more effectively than 1 mM EGTA (Roberts, 1993). If the loss of a cytoplasmic factor is responsible, one can use the data from Figure 2B and the empirical formula of Pusch and Neher (1988) (see Experimental Procedures) to estimate its molecular mass to be less than 5 kDa. Although this factor could be a peptide, other candidates include K+ and GTP.

Neuron 880

Small GTP-binding proteins such as Rab5 are thought to regulate other endocytotic processes (Bucci et al., 1992). Alternatively, the formation of clathrin-coated pits in fibroblasts has been shown to depend on intracellular K+ (Larkin et al., 1983), which was rapidly replaced by Cs+ in our whole-cell recordings but was perhaps replaced more slowly during perforatedpatch recordings. The ability to modulate endocytosis in this time-resolved assay should prove to be a powerful approach to study the regulation of membrane retrieval in hair cells. Relating Capacitance Increases to the Number of Synaptic Vesicles Synapticvesicles of vestibular hair cells have reported outer diameters between 25 nm and 50 nm (R. catesbeiana sacculus, 40 nm Uacobs and Hudspeth, 19901; goldfish sacculus, 40-50 nm [Hama and Saito, 1977; R. temporaria crista ampullaris, 25-35 nm [Gleisner et al., 19731). The dielectric (capacitive) element of the vesicle is expected to have a significantly smaller diameter because it occupies onlythe - 4 nm thick lipid core of the much thicker membrane seen in electron micrographs (Singer and Nicolson, 1972; Dilger et al., 1982). For our calculation of vesicle capacitance, we used a diameter of 32 nm, halfway between the average outer diameter (39.3 nm) and inner diameter (24.3 nm) of synaptic vesicles in saccular hair cells of this species (measured from Figure 12C in Roberts et al., 1990; see Experimental Procedures). This value may underestimate the diameter by -20% owing to shrinkage during aldehyde fixation (Tatsuoka and Reese, 1989). Assuming a specific membrane capacitance of 1 uF/cm* (see Hille, 1992), we estimate the capacitance of a single vesicle to be 32 aF (atto = 10-18). Consequences of a Prolonged High Rate of Exocytosis The most striking finding in our studies of exocytosis is the ability of hair cells to maintain for 1 s or longer an exocytotic rate equivalent to -10,000 vesicles per second (Figure 4). This rate is comparable to that of endocrine cells and isolated nerve terminals during their exocytotic burst (Neher and Zucker, 1993; Thomas et al., 1993a, 1993b; von Gersdorff and Matthews, 1994), but is maintained in hair cells for at least ten times longer (>I s versus -100 ms). Such prolonged, rapid exocytosis implies that hair cell synapses must draw vesicles from a much larger pool than could be in close proximity to the plasma membrane at active zones. There is room for at most 80 vesicles in the narrow cytoplasmic space between the presynaptic body and the plasma membrane at each active zone (see Experimental Procedures). This calculation probably overestimates the number of membrane-associated vesicles at each active zone, which in hair cells from the goldfish sacculus are arranged in five to six rows of five to six vesicles each, with adjacent rows separated by a dense bar (Hama and Saito, 1977; Hama, 1980). Even a pool of 80 releaseready vesicles at each active zone would be exhausted

within the first 160 ms of a depolarization to -10 mV. Nevertheless, a membrane area corresponding to >5 times this number of vesicles could be added to the plasma membrane with no apparent decrement in release rate (Figure 4). Possible Role for the Synaptic Body in ExocytoSis Models of secretion based upon results from neuroendocrinecells predict adecline in the exocytotic rate as the pool of readily releasable vesicles is exhausted (Heinemann et al., 1993; Neher and Zucker, 1993; Thomas et al., 1993a, 1993b; von Rtiden and Neher, 1993). Our observation that the release rate did not decline during sustained exocytosis could argue against the presence of a small, exhaustible pool of release-ready vesicles in hair cells. Such a releaseready pool of vesicles has been suggested to be the initial supply of membrane-associatedvesicles (Neher and Zucker, 1993; Thomas et al., 1993b). This apparent inconsistency with observed exocytosis in other cell types and morphological evidence from hair cells (see above) can be explained in two ways. Either a brief and rapid burst of exocytosis from a small, releaseready pool went undetected in our experiments, or the membrane associated vesicles were replenished at a rate faster than exocytosis so that the releaseready pool was never exhausted. Further capacitance measurements with higher sensitivity and better temporal resolution will be needed before the former hypothesis can be excluded. Whether or not hair cells possess a small, rapidly exhausted pool of release-ready vesicles, our work implies that hair cells can rapidly move vesicles to the membrane to replace those that have fused with the plasmalemma. It is possible that the synaptic bodies in hair cells (and the synaptic ribbons in other cells) are an essential part of this process. Experiments using a membrane impermeant-extracellular tracer to visualize membrane retrieval in cochlear inner hair cells have shown that a large fraction (48%) of the synaptic vesicles associated with presynaptic bodies become labeled within 3 min after addition of horseradish peroxidase to the perilymph and that this labeling is blocked by perfusion with low Ca2+lhigh Mg2+ saline (Siegel and Brownell, 1986). This rapid labeling is consistent with these vesicles being part of the releasable pool. There is room for -520 vesicles to be tethered by 20 nm filaments (Cleisner et al., 1973) around a 400 nm diameter presynaptic body (see Experimental Procedures).Thus,the20presynaptic bodies could provide a pool of - 10,400 vesicles ready for rapid delivery to release sites. The synaptic bodies could also serve as large targets for the diffusionlimited capture of vesicles from a cytoplasmic pool. Other more elaborate processes that involve the synaptic bodies in active vesicle transport are also possible. Although there is currently no evidence directly linking the synaptic bodies to the resupply of vesicles at release sites, the intriguing observation that hair cells and other cells with synaptic ribbons lack synap-

Exocytosis and Endocytosis

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in Hair Cells

sins (Favre et al., 1986; Scarfone et al., 1988,199l; Mandell et al., 1990) suggests that the presynaptic bodies may serve some related function in regulating the availability of synaptic vesicles for secretion. We therefore propose the hypothesis, to be tested in future experiments, that the synaptic body confers on the hair cell an exceptional ability to replenish its release-ready pools, allowing its synapses to sustain high rates of transmitter release for prolonged periods. Experimental

Procedures

We studied hair ceils from the sacculus of grassfrogs, R. pipiens. Hair cell ultrastructure was viewed by transmission electron microscopyas described (Roberts et al., 1990), using relativelythick (>I20 nm) sections to aid in counting DCVs, which are rare in these cells. In electron micrographs (magnification, 25,000x) of a longitudinal section through seven hair cells, we found only five profiles that could have been DCVs. From the ratio of the section thickness (>I20 nm) to the hair cell diameter (
display system (INDEC Systems, Sunnyvale, CA). When measuring Ca2* currents, traces were leak subtracted ahd averaged before storing to disk. In some cases, digitized data were transferred to a personal computer (Dell Computer, Langen, Germany) for digital filtering and subsequent analysis. The increase in membrane capacitance calused by a voltage step was measured by subtracting the averaged baseline capacitance immediately before the step from the capacitance averaged during a 200 ms sample period beginning 450-800 ms after the end of the step. Responses were often observed to decline during repeated presentation of stimuli lasting 1 s or longer. We did not investigate the effect of the interstimulus interval on this decline or the ability of the response to recover. Instead, we excluded from our analysis all data obtainecl after the first response to a step longer than 100 ms (except as noted). To determine more accurately the magnitude of the evoked capacitance increase, one can subtract the blaseline drift from the measured capacitance change. Such corrections were found to be insignificant for responses to stimuli lasting <2 s but could be significant during measurements of capacitance recovery, which lasted up to 30 s after the stimulus. The magnitude of the drift was estimated from the slope of the baLseline during the 5-20 s interval between the calibration of the capacitance trace and the application of the stimulus. The solurces of the slow drift observed before the stimulus, and after the stimulus in whole-cell recordings, are not understood. Tlhe amplitude and even the direction of these drifts varied considerably between cells, as can be seen by comparing the top trace in Figure 2A with Figure 3Ai. The data are presented in the figures without correction for drift; adjusted values are given1 in the text. To calculate the capacitance of a single vesicle, we needed to obtain an estimate of its diameter, measured in the middle of the lipid bilayer. Since published values of the outer diameters of the vesicles cannot be used for this purpose unless one also knows the inner diameter or membrane thickness, we measured the inner and outer diameters of synaptic vesicles from a published electron micrograph of hair cells from the same species and organ used in our experiments (Figure 12C in Roberts et al., 1990). A photographic negative of the figure w,as digitized using a video camera and Image-l software. Ellipses were fit to the inner and outer edges of 26 vesicles that had clearly delineated membrane profiles. The means and standard deviations of the inner and outer diameters of the vesicles (expressed as the diameter of a sphere having the same surface area as an ellipsoid with the measured major and minor axes) were 24.3 * 1.7 nm for the inner edge and 39.3 f 1.7 nm for the outer edge. The maximum numberof synapticvesicles thlat could possibly be in contact with the plasma membrane at an active zone was determined by counting how many 40 nm diameter vesicles could be hexagonally packed in a single layer within a disk (376 nm in diameter) extending 50 nm beyond the edge of the active zone particle arrays found in saccular hair celUs of the species used for our experiments (R. pipiens; average diameter of the particle array, 276 nm; Roberts et al., 1990; seealso Roberts, 1994). To estimate the maximum number of vesiclles that could be tethered to each presynaptic body, we divided the surface area of a sphere having a radius of 240 nm (the sum of the radii of the presynaptic body and a synaptic vesicle plus the tether length) by the area of a hexagon that encompasses a 40 nm diameter circle. We estimated the size of the cytoplasmic factor required for endocytosis using the formula of Pusch and Neher (1988),

M =

I 1 rK 3 R, (CJ’.5

where M is the molecular mass of the factor bseing dialysed (in Daltons), K is a scaling factor to correct for the size difference between hair cells and chromaffin cells (23.946; Pusch and Neher, 1988), C, is the initial cell capacitance (9.4 pF), R, is the electrical resistance between the pipette and the cytosol (5.05 MO), and ‘c is the time constant of the washout (in seconds). In our experiments, the mean time from patch bsreak to the first

Neuron 882

capacitance measurementwas 2OOs, which weassumed to represent at least two time constants (>85% washout of the soluble factor).

Koenig, J. H., Kosaka, T., and Ikeda, K. (1989). The relationship between the number of synaptic vesicles and the amount of transmitter released. J. Neurosci. 9,1937-1942.

Acknowledgments

Kroese, A. B., Das, A., and Hudspeth, A. J. (1989). Blockage of the transduction channels of hair cells in the bullfrog’s sacculus by aminoglycoside antibiotics. Hear. Res. 37, 203-218.

We thank T. Bork and E. Schabtach for technical expertise with electron microscopy and B. D.Anson, C. J.Augustine, W. J. Betz, R. H. Chow, J. Coorssen, A. K. Lee, M. Lindau, E. Neher, and G. A. Nevitt for their critical reading of a previous version of this manuscript. This work was supported by an HFSPO Fellowship (T. D. P.), by National Institutes of Health grant NS27142 (W. M. R.), and by a McKnight Scholars Award (W. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

23, 1994; revised

July 1, 1994.

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