Facilitating efferent inhibition of inner hair cells in the cochlea of the neonatal rat
1 The Cochlear Neurotransmission Laboratory, Center for Hearing and Balance, Department of Otolaryngology–Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Abstract
Cholinergic brainstem neurones make inhibitory synapses on outer hair cells (OHCs) in the mature mammalian cochlea and on inner hair cells (IHCs) prior to the onset of hearing. We used electrical stimulation in an excised organ of Corti preparation to examine evoked release of acetylcholine (ACh) onto neonatal IHCs from these efferent fibres. Whole-cell voltage-clamp recording revealed that low frequency (0.25–1 Hz) electrical stimulation produced evoked inhibitory postsynaptic currents (IPSCs) at a relatively high fraction of failures (65%) and with mean amplitudes of about –20 pA at –90 mV, corresponding to a quantum content of 1. Evoked IPSCs had biphasic waveforms at –60 mV, were blocked reversibly by -bungarotoxin and strychnine and are most likely mediated by the 9/10 acetylcholine receptor, with subsequent activation of calcium-dependent potassium (SK2) channels. Paired pulse stimulation with intervals of 10–100 ms caused facilitation of 200–300% in the mean IPSC amplitude. A train of 10 pulses with an interpulse interval of 25 ms produced increasingly larger IPSCs with maximum amplitudes greater than –100 pA due to facilitation and summation throughout the train. Repetitive efferent stimulation at 5 Hz or higher hyperpolarized IHCs by 5–10 mV and could completely prevent the generation of calcium action potentials normally evoked by depolarizing current injection.
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Introduction
Mechanosensory hair cells of the vertebrate ear are subject to feedback regulation by cholinergic efferent neurones originating in the brainstem. Electrical stimulation of these axons in the floor of the fourth ventricle caused suppression of the compound afferent action potential (Galambos, 1956). Similar methods were used to describe the inhibitory effect on single afferent axons (Wiederhold, 1970; Wiederhold & Kiang, 1970) where the efficacy of inhibition was shown to depend on the rate of electrical stimulation, implying some plasticity in efferent synaptic transmission. Synaptic facilitation of efferent inhibition was shown directly by intracellular recording from hair cells in the turtle's inner ear (Art et al. 1984); however, equivalent effects have not been demonstrated in the mammalian cochlea until now.
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Medial olivocochlear efferents innervate outer hair cells (OHCs) in the first postnatal week and maintain these synapses through adulthood. In contrast, inner hair cells (IHCs) have contacts with medial efferent fibres before birth, but these presumptive synapses disappear after the onset of hearing in the second postnatal week (Simmons et al. 1996; Katz et al. 2004). These transient efferent synapses can release ACh onto neonatal IHCs (Glowatzki & Fuchs, 2000; Katz et al. 2004; Marcotti et al. 2004). It has been suggested that this efferent feedback may be involved in directing IHC maturation, including the formation of afferent synapses (Simmons, 2002).
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Spontaneous IPSCs in OHCs (Oliver et al. 2000; Lioudyno et al. 2004), and in neonatal IHCs (Glowatzki & Fuchs, 2000) are served by 910-containing ACh receptors, allowing calcium influx that activates calcium-dependent SK2 potassium channels (Elgoyhen et al. 1994; Dulon et al. 1998; Yuhas & Fuchs, 1999; Elgoyhen et al. 2001; Lustig & Peng, 2002). However, the random timing of these spontaneous events prevents any direct assessment of efferent release mechanics or plasticity. Further study of the efficacy of hair cell inhibition requires the ability explicitly to evoke release from the efferent endings. Here we report the results of experiments using electrical stimulation to cause inhibitory postsynaptic currents in IHCs of the neonatal rat cochlea. As previously described for efferent inhibition in turtle hair cells (Art et al. 1984), the resting probability of release at the IHC efferent synapse was low, but facilitated markedly during repetitive stimulation of the efferent axons. The resulting large inhibitory postsynaptic currents (IPSCs) could essentially clamp the IHC membrane potential to the potassium equilibrium potential, and prevent the generation of calcium action potentials in neonatal IHCs.
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Methods
The preparation
Procedures for preparing and recording from the postnatal rat organ of Corti were essentially identical to those published previously (Glowatzki & Fuchs, 2000). Animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. Sprague-Dawley rat pups, 7–11 days old, were anaesthetized using pentobarbital and decapitated. The organ of Corti was exposed and the apical turn removed for recording. IHCs in the apical turn of the organ of Corti were visualized using an Axioscope microscope (Zeiss, Oberkochem, Germany) with a 40x water immersion DIC objective, 4x magnification and a NC70 Newvicon camera (Dage MTI, Michigan City, IN, USA) for display. Whole-cell, tight-seal voltage-clamp recordings were made with Sylgard-coated 1 mm borosilicate glass micropipettes (WPI, Sarasota, FL, USA) ranging from 8 to 10 M resistance. Electrodes were advanced through the tissue under positive pipette pressure to avoid extensive dissection. In this way it was possible to maintain the integrity of synaptic contacts on the IHCs.
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The pipette solution was (mM): 150 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 Hepes, 2.5 Na2ATP; pH 7.2 (KOH). The extracellular solution was (mM): 5.8 KCl, 155 NaCl, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 glucose, 10 Hepes; pH 7.4 (NaOH). Mg2+ was excluded from the extracellular solution to maximize current flow through the hair cell ACh receptor (Weisstaub et al. 2002; Gomez-Casati et al. 2005). In control experiments it was shown that quantum content and facilitation were not altered by the absence of external Mg2+, although quantal size was approximately 40% smaller in (0.9 mM) Mg2+, as predicted.
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Recordings were made at room temperature (22–25°C). Solutions, including experimental drugs, were applied by gravity flow into the bath chamber (1.5 ml volume) at a rate of 2–3 ml min–1. All drugs were purchased from Sigma and diluted from frozen stocks.
Electrical stimulation and recording
Bipolar electrical stimulation of efferent axons was provided by a theta glass micropipette (20–30 μm tip diameter) positioned about 20 μm modiolar to the base of an IHC, near the course of the inner spiral bundle. The position of this pipette was adjusted until current passage through it caused synaptic currents in the IHC under study. An electrically isolated constant current source (model DS3, Digitimer Ltd, Welwyn Garden City, UK) was triggered via the data-acquisition computer to generate pulses up to 50 μA, 50–500 μs long to stimulate efferent axons. With some experience in positioning the stimulation pipette, 50 μs long pulses were sufficient for stimulation. With this method we were able to stimulate IPSCs in every IHC recorded.
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Currents and voltages were recorded with pCLAMP9.2 software and an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), low-pass filtered at 2–10 kHz and digitized at 10–50 kHz with a Digidata 1322A board.
Indicated holding potentials were not corrected for liquid junction potentials (–4 mV). Series resistance compensation of 50–70% was used, and the remaining series resistance error for the recorded synaptic currents was calculated to be smaller than 5 mV. Synaptic currents and potentials were analysed with Minianalysis (Synaptosoft, Decatur, GA, USA). IPSCs were identified manually using a search routine for event detection with a threshold of 5 times the RMS noise (7–12 pA). Rise times (10–90%) and decay time constants were analysed only in IPSCs that were not compromised in shape by the stimulus artifact or following events. The decay of IPSCs was fitted with a monoexponential. For further analysis we used Origin 7.5 (OriginLab Corp., Northampton, MA, USA). A two-sample Student's t test assuming unequal variances and a one-tailed t test were used for statistical analysis. Mean values are presented ± standard deviation (S.D.) unless stated otherwise.
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Quantal analysis
The amplitude distributions of spontaneous IPSCs were fitted by a single Gaussian equation. To test whether amplitude distributions for evoked IPSCs were shaped by distinct peaks at integral multiples of the first peak, as established for the neuromuscular junction (Del Castillo & Katz, 1954), we used the following equation (Sahara & Takahashi, 2001):
where N(x) was the number of events with x amplitude, Nt was the total number of evoked events, r was the number of quanta released, m was the quantum content, and μ and 2 were the mean and variance of the distribution. We used this equation under the assumption that low values for the quantum content were observed, with k, the maximum vesicle number, taking a value of 3 in most cases. This function uses for each peak a mean and variance given by a Gaussian distribution, whereas the relative amplitude of each maximum is estimated by Poisson statistics governing the probability of release of one vesicle, two vesicles, or more. This approach allowed us to calculate the quantum content ‘m’. The Poisson distribution also could be used for a separate estimate of quantum content based on the fraction of failures (del Castillo & Katz, 1954).
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Best fits for amplitude distributions were evaluated by finding the least squared difference from the data using Origin 7.5.
Results
Electrically evoked IPSCs in IHCs
To investigate the effects of cholinergic efferent input on neonatal IHC activity, we did whole-cell patch clamp recordings from 43 IHCs in excised apical turns of organ of Corti from 7- to 11-day-old rats. In these recordings we electrically stimulated efferent fibres that directly synapse onto the IHCs. These fibres originate in the brainstem in the medial olivocochlear system and during development reach the IHC area before birth (Simmons, 2002). Medial efferent axons were stimulated electrically with a theta glass micropipette positioned close to the IHC under investigation, about 20 μm modiolar to the IHC's base (for details see Methods) (Fig. 1A).
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A, for bipolar electrical stimulation of efferent fibres some supporting cells were removed and a theta glass micropipette, 10–15 μm in diameter, was positioned 10–20 μm below the base of the IHC. Scale bar 10 μm. Positions of 2 IHCs and recording pipette outlined. B, evoked IPSCs were eliminated reversibly by 1 μM tetrodotoxin (TTX) (n = 2). C, IPSCs were reversibly blocked by 100 nM strychnine, which blocks 910 AChR (n = 2). D, IPSCs were reversibly inhibited by 300 nM -bungarotoxin, an 910 AChR antagonist (n = 2). B–D, traces for each control, drug application and washout are averages of 50–100 sweeps (stimulation at 1 Hz). Each new solution was applied for about 5 min before data collection. Vh = –90 mV. E, IPSC waveform varied with holding potential. IPSCs reversed from completely inward at –90 mV to a biphasic inward (arrowheads) and outward (stars) waveform at membrane potentials positive to the potassium equilibrium potential. F, synaptic potentials mirrored synaptic currents and varied similarly with membrane potential: an initial depolarization (arrowheads) was followed by a hyperpolarization (stars). Arrow indicates time of electrical shock in E and F.
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When timed current pulses (of 50 μA in size, 50 μs long) were delivered via the stimulating pipette, transient inward currents (evoked IPSCs) at a holding potential of –90 mV were evoked in every IHC tested (Fig. 1B). Application of 1 μM tetrodotoxin to the preparation produced a reversible loss of the synaptic response, confirming that these currents resulted from propagation of action potentials in presynaptic axons, presumably to release ACh onto IHCs. Consistent with this interpretation, the IPSCs were reversibly blocked by 100 nM strychnine (Fig. 1C) and 300 nM -bungarotoxin (Fig. 1D), like the 910-containing nAChR (Elgoyhen et al. 1994; Verbitsky et al. 2000; Elgoyhen et al. 2001), and spontaneous synaptic currents recorded in IHCs (Glowatzki & Fuchs, 2000).
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Evoked IPSCs were similar in shape to spontaneous IPSCs and to IPSCs produced by elevated extracellular potassium as previously described in both IHCs (Glowatzki & Fuchs, 2000; Katz et al. 2004) and OHCs (Oliver et al. 2000; Lioudyno et al. 2004). The mean 10–90% rise time of the evoked IPSCs was 9.6 ± 3.8 ms (mean ± S.D., n = 298 IPSCs from 3 IHC recordings) and the mean time constant of decay was 50.2 ± 17.4 ms (n = 244 IPSCs from 3 IHC recordings) at a holding potential of –90 mV.
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Like the previously described spontaneous IPSCs, evoked IPSCs reversed from completely inward at –90 mV to a biphasic inward and outward waveform at membrane potentials positive to the potassium equilibrium potential (EK = –82 mV) (Fig. 1E). The initial inward current of the biphasic IPSC is thought to result from sodium and calcium flux through the 910-containing nAChR (Elgoyhen et al. 1994; Verbitsky et al. 2000; Elgoyhen et al. 2001; Gomez-Casati et al. 2005). The subsequent activation of calcium-dependent small conductance SK2 potassium channels underlies the outward current (Dulon et al. 1998; Yuhas & Fuchs, 1999). Negative to EK, at –90 mV, both the AChR current and SK current are inward. Between EK and –40 mV the AChR current is directed inward whereas the SK current is directed outward, resulting in a biphasic response. Positive to –40 mV the much larger outward current obscures the small initial current through the AChRs.
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To characterize the effect of efferent stimulation on IHC membrane potential, we recorded evoked inhibitory postsynaptic potentials (IPSPs) in current clamp (Fig. 1F). The synaptic potentials mirrored the synaptic currents, starting with a short depolarization and followed by a longer and larger hyperpolarization at potentials close to the IHC resting membrane potential at –60 mV. This hyperpolarization makes the effect of efferent synaptic activity on IHCs inhibitory (see also Fig. 5).
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A, current injections of 30–50 pA for 10 s induced repetitive firing of calcium action potentials in IHCs, with larger currents causing higher frequency firing. B, efferent stimulation (onset indicated by downward arrow) at 2 Hz had little effect on IHC excitability. The downward deflections in the records are stimulus artifacts (examples indicated by arrow heads). C, efferent stimulation at 5 Hz caused a hyperpolarization of 5–10 mV and suppressed the generation of calcium action potentials. A few calcium action potentials still occurred during the hyperpolarization (indicated by stars). Time scale for all figures: 2 s.
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Taken together, the pharmacological data and the biphasic IPSC waveform confirm that with electrical stimulation we were able to exert cholinergic efferent synaptic inhibition of postnatal IHCs.
Synaptic strength of efferent contacts
As a measure of synaptic strength at the IHC efferent synapse, we calculated quantum content, ‘m’, the number of vesicles released per stimulation. We analysed our data in the following way. At a stimulation rate of 0.25 or 1 Hz we observed a relatively high fraction of failures of 0.65 ± 0.16 (n = 32 IHC recordings) and a mean amplitude of ‘successes’ of –22 ± 9 pA (n = 32 IHC recordings, 11 900 IPSCs analysed) at a holding potential of –90 mV. Figure 2A illustrates a recording with three successful stimulations and one failure. Additionally, spontaneous IPSCs occurred during the recordings that were not time-coupled with the stimulus (Fig. 2A). In each recording, evoked IPSC amplitudes fluctuated considerably (Fig. 2B). Examples of amplitude distributions for evoked and spontaneous IPSCs are shown in Fig. 2C.
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A, electrical stimulation (arrows) of the efferents at 1 Hz caused 3 IPSCs and 1 failure. Some spontaneous IPSCs were observed as well. B, evoked IPSCs varied in amplitude. C, amplitude distribution of 114 evoked IPSCs produced by 1 Hz stimulation (300 times), and amplitude distribution of spontaneous IPSCs (inset) from the same cell (the noise floor is shown by the 3 bars centred around 0 mV). The spontaneous IPSC amplitude distribution was fit with a single Gaussian providing a mean of –19 ± 5 pA (n = 38 spontaneous IPSCs). Dividing mean evoked IPSC amplitude by the mean spontaneous IPSC amplitude provided a quantum content of 0.64. Quantal parameters also were obtained from the Poisson distribution derived from the fit of eqn (1) (Methods) to the evoked IPSC amplitude distribution. This gave a mean evoked IPSC amplitude of –22 ± 7 pA for the first peak (n = 114 IPSCs) and a value of 0.75 for the quantum content.
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We estimated quantum content by four different approaches for six IHC recordings that provided enough data for both evoked and spontaneous IPSCs; a summary of the results is provided in Table 1. One approach was to divide the mean evoked IPSC amplitude by the mean quantal size (q). Assuming that the spontaneous IPSCs are uniquantal events, we estimated the mean quantal size q from the amplitude distributions of spontaneous IPSCs fitted by a single Gaussian equation and obtained –18 ± 2 pA (Fig. 2C, inset). Dividing this value into the mean of evoked IPSCs yielded a mean quantum content (m) of 1.0 ± 0.5 vesicles released per stimulus. Alternatively, we estimated an average quantum content of 0.9 ± 0.5 from the ‘fraction of failures’ based on the Poisson distribution (del Castillo & Katz, 1954). The Poisson assumptions also underlie the estimate of m as equivalent to the reciprocal of the coefficient of variation squared (m = 1/CV2) and provided an average value of 1 ± 0.6 for the six cells studied. Finally, the overall evoked IPSC amplitude distributions were fit with eqn (1) (see Methods) (Fig. 2C). The first peak of this fit provided an estimate for the quantal size q of –20 ± 5 pA, quite similar to the value provided by the Gaussian fit of the spontaneous IPSC distribution. The mean quantum content for all IHCs obtained from fitting the evoked IPSC amplitude distributions again was 1 ± 0.4. However, while this average value was quite comparable, it should be noted that the estimates of quantum content obtained from this approach differed somewhat from those obtained by other methods (Table 1).
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Nonetheless, taken together the different estimates of quantum content were similar: the efferent IHC synapse has a low probability of release at low frequency, with a range of 0.4 up to 2 vesicles released per action potential (at 1 Hz).
Two-pulse facilitation of efferent release
Does the efficacy of the IHC efferent synapse change with higher rates of stimulation We used paired pulse protocols to probe facilitation at the IHC efferent synapse. Pulse pairs with intervals ranging from 1 to 500 ms were presented 50 times each, at a rate of 0.25 Hz (Fig. 3A–D). IPSCs or failures could be seen following the first, or the second (Fig. 3A, insets) or both pulses (not shown). The resulting mean IPSC amplitudes, which include failures, were calculated as a ‘facilitation ratio’ (S2 – S1)/S1, with S2 representing the response to the second pulse, and S1 representing the response to the first pulse (Mallart & Martin, 1968). The facilitation ratio can be considered a measure of the percentage mean IPSC amplitude increase due to facilitation. The 1 ms and 5 ms paired pulse intervals were too short to measure IPSC amplitudes in response to the first pulse for S1. Therefore, in addition to the paired pulse protocol, which provided S2, we used a single pulse protocol at 0.25 Hz recorded in the same IHC to assess S1. This was possible, as IPSCs evoked at 0.25 Hz rate did not show facilitation.
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A, average (n = 50) IPSCs evoked with paired pulses 10 ms apart. Inset shows individual records illustrating the variability of response: failure occurring after the first, or second shock (x scale: 50 ms, y scale: 20 pA). B–D, average (n = 50) IPSCs evoked with different pulse intervals. E, facilitation of the second IPSC as a function of pulse interval. The mean IPSC amplitude Sx = PxAx, the probability of occurrence, Px, times the mean amplitude of successful IPSCs, Ax. The facilitation index was computed as the ratio (S2 – S1)/S1. For intervals between 10 and 100 ms IPSCs showed facilitation: S2 was 200–300% bigger than the S1 (*P < 0.01, P < 0.05, one-tailed t test). F, IPSC amplitude Ax (failures not included) and IPSC probability Px of success were plotted separately. Facilitation was mostly due to a 150–250% rise in probability Px of transmitter release; however, there was also a significant, about 30% increase in IPSC amplitude Ax for 10, 25 and 50 ms pulse intervals (* and same as in E). E–F, facilitation for 50 pulse pairs presented at 0.25 Hz for 5–11 IHCs (mean and standard error of the mean shown). G, quantum content (calculated as 1/CV2) of the second IPSC compared to that of the first with a 25 ms interval. Each data point represents 1 IHC recording. Nine out of 10 IHCs showed facilitation as an increase in quantum content (range 0.09–0.99 for S1 and 0.13–2.08 for S2).
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Facilitation became evident with paired pulse intervals of 10–100 ms (Fig. 3A and E) with mean IPSC amplitudes S2 increasing 200–300% compared to S1. For example, for a 25 ms interval, S1 was –11 ± 12 pA and S2 was –24 ± 25 pA (n = 11 IHC recordings). In addition, although we have not quantified this effect, we noted that paired pulse protocols with facilitation also showed an increased number of IPSCs that were not time-coupled to the stimulus.
As presented, the mean IPSC amplitude, Sx, reflects two factors, the probability of occurrence, Px, and the mean amplitude of the successful IPSCs, Ax; such that Sx = AxPx. To better understand the mechanism of facilitation, Ax and Px were analysed separately in the original records (Fig. 3F). As seen there, the principal effect of paired pulse stimulation was to increase the probability of IPSC occurrence after the second pulse by 150–250%. That is, IPSCs occurred much more reliably when two pulses were presented with a 10–100 ms interval. The effect on the mean amplitude of successful IPSCs was also significant, though smaller with a 30% amplitude increase of A2. For example, for a double pulse interval of 25 ms, the mean ratio of P2/P1 was 1.78 ± 0.73 (n = 11 IHCs, one-tailed t test, P < 0.01) and the mean ratio A2/A1 was 1.34 ± 0.33 (n = 11 IHCs, one-tailed t test, P < 0.01). We estimated (both by ‘method of failures’ and by 1/CV2) the quantum content for the facilitated IPSC S2 and compared it with that of S1 (Fig. 3G). For paired pulse intervals of 25 ms, quantum content rose 150–550% (0.09–0.99 for S1 and 0.13–2.08 for S2) in 9 of 10 IHCs.
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Facilitation and summation during shock trains
Inhibition in the intact cochlea has been most often studied using extended trains of electrical shocks to activate the efferent axons (Galambos, 1956; Wiederhold & Kiang, 1970). The time course of efferent facilitation in the excised organ of Corti was characterized further using trains of 10 pulses with an interpulse interval of 25 ms; 30–50 such trains were presented at a 20 s interval, with the IHC at a holding potential of –90 mV. Facilitation as well as summation augmented the average compound IPSC amplitudes to a value of –128 ± 117 pA (n = 14 IHC recordings), about 6 times the size of the mean IPSC amplitude in response to single pulses. The biggest average compound IPSC reached a value of –450 pA (Fig. 4A). We analysed the facilitation ratio in 14 IHC recordings and compared each mean IPSC amplitude S2 to S10 in the pulse train to the mean amplitude of the first IPSC S1 in the train (Fig. 4B). In these instances, the amplitude of the facilitated IPSC is slightly underestimated, since the true preceding ‘baseline’ was not available. Nonetheless, every mean IPSC amplitude S2 to S10 in the pulse train was significantly larger than S1 showing facilitation (one-tailed t test, P < 0.05, n = 14 IHC recordings, 30–50 pulse trains per IHC averaged). Peak values for an increase in mean IPSC amplitude were found for S4 and S5 (200%), then declined to values of 150% for S6 and S7 and stayed at 100% increase for S8 through S10. This decline of increase of mean IPSC amplitudes during the stimulation train could be due to saturation of nAChRs, or other pre- and postsynaptic processes, and therefore this phenomenon will need further examination. In addition to facilitation of single IPSCs, summation also contributed to the amplitude of the compound IPSCs.
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A, response of an IHC at Vh = –90 mV to a 10 pulse, 25 ms interval, train. The pulse train was presented at a 20 s interval and repeated 30–50 times, the average compound IPSC is shown. Inset: an individual record showing marked fluctuations on the falling phase of the compound IPSC. B, facilitation relative to the first response during a 10 pulse, 25 ms interval, train. Data from 14 IHC recordings are included, each with 30–50 repeats of the 10 pulse train. Mean IPSC amplitude was significantly increased throughout the train (mean ± S.E.M., *P < 0.01, P < 0.05, one-tailed t test).
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Following the last pulse, the compound IPSC decayed slowly, over the course of nearly 1 s. This is considerably slower than the 50 ms time constant of decay for single IPSCs. Several processes could contribute to this slow decay. One possibility is suggested by the inset of Fig. 4A, which shows an individual record in which marked fluctuations are observed on the falling phase of the compound IPSC that were not seen in the averaged records. Some of these fluctuations were similar in waveform to ‘spontaneous’ IPSCs that occasionally followed the paired pulse protocols. It was already mentioned that in double pulse protocols that induced facilitation, the number of IPSCs increased that were not time-coupled to the pulse. This effect also may occur after a train of pulses and could reflect delayed release from the efferent terminal. A second possibility is that there is enhanced and delayed calcium-induced calcium release from the subsynaptic cistern of the hair cell (Lioudyno et al. 2004), causing spontaneous transient currents through the postsynaptic SK channels. Like in OHCs (Saito, 1980), a subsynaptic cistern that could serve as a calcium store has been found postsynaptically facing neonatal IHC efferent synapses (Ginzberg & Morest, 1984; Sobkowicz, 1992; Bruce et al. 2000). This phenomenon therefore needs further investigation.
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In summary, the experiments of Figs 3 and 4 show that compound IPSCs can grow much stronger due to facilitation and summation at the efferent synapse compared to the effect of a single IPSC.
Inhibition of IHC excitability
Neonatal IHCs generate calcium action potentials spontaneously or in response to injected current (Kros et al. 1998; Marcotti et al. 2003a, b). We used depolarizing current steps through the recording electrode to cause the activation of repetitive action potentials in rat IHCs during the second postnatal week. Three levels of current injection were used (30–50 pA) to depolarize the IHC from –80 to levels between –70 and –55 mV. The latter two steps evoked repetitive activity at frequencies between 2 and 10 Hz (Fig. 5). When these excitatory stimuli were combined with efferent pulse trains at frequencies of 5 Hz and greater, action potentials were essentially eliminated and the steady membrane potential hyperpolarized by 5–10 mV (Fig. 5C). Thus, efferent inhibition becomes potent when transmitter release has been facilitated by higher frequency firing.
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Discussion
Cholinergic efferent fibres innervate adult OHCs and neonatal IHCs in the mammalian cochlea. It has been shown that the cholinergic effect on both these types of hair cells is essentially identical, involving nicotinic AChRs composed of 9 and 10 subunits (Elgoyhen et al. 1994; Elgoyhen et al. 2001; Lustig & Peng, 2002), and subsequently activated calcium-dependent SK channels (Evans, 1996; Dulon et al. 1998; Yuhas & Fuchs, 1999). It remains to be seen whether a postsynaptic calcium store (the synaptoplasmic cistern) supports inhibition of IHCs, as has been found for OHCs (Evans et al. 2000; Lioudyno et al. 2004). There is substantial evidence that cholinergic inhibition of hair cells in various end-organs of other vertebrates results from similar, if not identical, synaptic mechanisms (Flock & Russell, 1973; Art et al. 1984; Fuchs & Murrow, 1992; Sugai et al. 1992). When functional measures of efferent inhibition have been made, it has been found that relatively high frequency trains of efferent action potentials (assumed to result from the electrical shocks used for activation) were required to produce consistent, measurable effects (Wiederhold & Kiang, 1970; Flock & Russell, 1973; Art et al. 1984). Studies in turtle hair cells demonstrated that the probability of release to single action potentials was significantly less than unity, ranging from 0.08 to 0.3, and that transmitter release from efferent endings facilitated strongly during repetitive firing (Art et al. 1984).
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The present study demonstrates that efferent axons in the mammalian cochlea show resting levels of release, and plasticity similar to that found in the turtle. Single shocks at frequencies less than 2 Hz evoked only small IPSCs in IHCs with a mean probability of occurrence of 0.35. However, with repeated stimulation, IPSCs were both larger and more likely. For trains of 10 shocks the summed, facilitated IPSCs reliably reached more than –100 pA in amplitude. Likewise, calcium action potentials in IHCs were only inhibited by efferent activity at rates of 5 Hz or greater. This frequency nearly corresponds to the interpulse interval (100 ms) at which facilitation first becomes evident in two-pulse protocols. The implication of this result is that efferent inhibition only is effective when occurring repetitively, and at sufficiently high frequencies so that facilitation of transmitter release can occur. In this way feedback from the central nervous system operates in a kind of failsafe mode, having consequence only when strongly driven, and spontaneous, inadvertent activity will not alter cochlear function significantly. Given the strong conservation of efferent synaptic mechanisms among vertebrate hair cells, it seems likely that the behaviour described here for neonatal IHCs will also occur in OHCs of the mature mammalian cochlea. The ability to stimulate the efferents electrically and produce strongly facilitated inhibition of defined duration, will enable studies of the mechanisms of fast and slow inhibition seen in vivo (Sridhar et al. 1997), as well as further investigation of possible longer-term effects on OHC electromotility (Dallos et al. 1997).
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The present study also reaffirms the conclusion that functional inhibitory synapses are present on neonatal IHCs prior to the onset of hearing. What purpose do these synapses serve Clearly they can suppress the excitability of the IHCs, and presumably prevent evoked and spontaneous transmitter release from ribbon synapses known to be functional at this stage (Glowatzki & Fuchs, 2002). Previous studies have suggested that this transient efferent innervation may play a role in the ultimate functional maturation of cochlear hair cells (Simmons, 2002). Most impressively, surgical lesion of the efferent nerve supply caused kittens to fail to develop normal hearing (Walsh et al. 1998). The pattern of loss was much like that seen with OHC damage. Also, in transgenic mice lacking the principal voltage-gated calcium channel of cochlear hair cells, the normal maturation of IHC excitability failed to occur and efferent synapses remained in place long after the normal period (Brandt et al. 2003). Thus, a neonatal period of synaptic cross-talk between efferent and afferent neurones may be necessary to the ultimate innervation pattern, and the final functional state of both IHCs and OHCs.
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It is unknown at present what, if any, endogenous firing takes place among hair cells and efferent neurones before the onset of hearing in vivo. A few studies documented spontaneous, or even rhythmic, activity in afferent nerve fibres prior to the onset of hearing (Lippe, 1994; Gummer & Mark, 1994). What remains uncertain is whether that activity depends on hair cell transmitter release, or arises from intrinsic excitability of the afferent neuron, as can occur in vitro (Lin & Chen, 2000). Likewise, several studies have shown that IHCs are capable of firing spontaneous calcium action potentials in preparations in vitro (Marcotti et al. 2003a,b); it remains to be determined whether they do so in vivo. Finally, neonatal medial olivocochlear neurones recorded in a brainstem slice preparation, identified by retrograde labelling from the cochlea, have the ability to discharge tonically, when stimulated with current injection (Fujino et al. 1997). Ultimately it will be necessary to address these questions in the intact, developing ear to learn if mechanisms of spontaneous activity will be essential to activity-dependent maturation, as shown for the retina (for review see Shatz, 1996).
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Abstract
Cholinergic brainstem neurones make inhibitory synapses on outer hair cells (OHCs) in the mature mammalian cochlea and on inner hair cells (IHCs) prior to the onset of hearing. We used electrical stimulation in an excised organ of Corti preparation to examine evoked release of acetylcholine (ACh) onto neonatal IHCs from these efferent fibres. Whole-cell voltage-clamp recording revealed that low frequency (0.25–1 Hz) electrical stimulation produced evoked inhibitory postsynaptic currents (IPSCs) at a relatively high fraction of failures (65%) and with mean amplitudes of about –20 pA at –90 mV, corresponding to a quantum content of 1. Evoked IPSCs had biphasic waveforms at –60 mV, were blocked reversibly by -bungarotoxin and strychnine and are most likely mediated by the 9/10 acetylcholine receptor, with subsequent activation of calcium-dependent potassium (SK2) channels. Paired pulse stimulation with intervals of 10–100 ms caused facilitation of 200–300% in the mean IPSC amplitude. A train of 10 pulses with an interpulse interval of 25 ms produced increasingly larger IPSCs with maximum amplitudes greater than –100 pA due to facilitation and summation throughout the train. Repetitive efferent stimulation at 5 Hz or higher hyperpolarized IHCs by 5–10 mV and could completely prevent the generation of calcium action potentials normally evoked by depolarizing current injection.
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Introduction
Mechanosensory hair cells of the vertebrate ear are subject to feedback regulation by cholinergic efferent neurones originating in the brainstem. Electrical stimulation of these axons in the floor of the fourth ventricle caused suppression of the compound afferent action potential (Galambos, 1956). Similar methods were used to describe the inhibitory effect on single afferent axons (Wiederhold, 1970; Wiederhold & Kiang, 1970) where the efficacy of inhibition was shown to depend on the rate of electrical stimulation, implying some plasticity in efferent synaptic transmission. Synaptic facilitation of efferent inhibition was shown directly by intracellular recording from hair cells in the turtle's inner ear (Art et al. 1984); however, equivalent effects have not been demonstrated in the mammalian cochlea until now.
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Medial olivocochlear efferents innervate outer hair cells (OHCs) in the first postnatal week and maintain these synapses through adulthood. In contrast, inner hair cells (IHCs) have contacts with medial efferent fibres before birth, but these presumptive synapses disappear after the onset of hearing in the second postnatal week (Simmons et al. 1996; Katz et al. 2004). These transient efferent synapses can release ACh onto neonatal IHCs (Glowatzki & Fuchs, 2000; Katz et al. 2004; Marcotti et al. 2004). It has been suggested that this efferent feedback may be involved in directing IHC maturation, including the formation of afferent synapses (Simmons, 2002).
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Spontaneous IPSCs in OHCs (Oliver et al. 2000; Lioudyno et al. 2004), and in neonatal IHCs (Glowatzki & Fuchs, 2000) are served by 910-containing ACh receptors, allowing calcium influx that activates calcium-dependent SK2 potassium channels (Elgoyhen et al. 1994; Dulon et al. 1998; Yuhas & Fuchs, 1999; Elgoyhen et al. 2001; Lustig & Peng, 2002). However, the random timing of these spontaneous events prevents any direct assessment of efferent release mechanics or plasticity. Further study of the efficacy of hair cell inhibition requires the ability explicitly to evoke release from the efferent endings. Here we report the results of experiments using electrical stimulation to cause inhibitory postsynaptic currents in IHCs of the neonatal rat cochlea. As previously described for efferent inhibition in turtle hair cells (Art et al. 1984), the resting probability of release at the IHC efferent synapse was low, but facilitated markedly during repetitive stimulation of the efferent axons. The resulting large inhibitory postsynaptic currents (IPSCs) could essentially clamp the IHC membrane potential to the potassium equilibrium potential, and prevent the generation of calcium action potentials in neonatal IHCs.
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Methods
The preparation
Procedures for preparing and recording from the postnatal rat organ of Corti were essentially identical to those published previously (Glowatzki & Fuchs, 2000). Animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. Sprague-Dawley rat pups, 7–11 days old, were anaesthetized using pentobarbital and decapitated. The organ of Corti was exposed and the apical turn removed for recording. IHCs in the apical turn of the organ of Corti were visualized using an Axioscope microscope (Zeiss, Oberkochem, Germany) with a 40x water immersion DIC objective, 4x magnification and a NC70 Newvicon camera (Dage MTI, Michigan City, IN, USA) for display. Whole-cell, tight-seal voltage-clamp recordings were made with Sylgard-coated 1 mm borosilicate glass micropipettes (WPI, Sarasota, FL, USA) ranging from 8 to 10 M resistance. Electrodes were advanced through the tissue under positive pipette pressure to avoid extensive dissection. In this way it was possible to maintain the integrity of synaptic contacts on the IHCs.
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The pipette solution was (mM): 150 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 Hepes, 2.5 Na2ATP; pH 7.2 (KOH). The extracellular solution was (mM): 5.8 KCl, 155 NaCl, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 glucose, 10 Hepes; pH 7.4 (NaOH). Mg2+ was excluded from the extracellular solution to maximize current flow through the hair cell ACh receptor (Weisstaub et al. 2002; Gomez-Casati et al. 2005). In control experiments it was shown that quantum content and facilitation were not altered by the absence of external Mg2+, although quantal size was approximately 40% smaller in (0.9 mM) Mg2+, as predicted.
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Recordings were made at room temperature (22–25°C). Solutions, including experimental drugs, were applied by gravity flow into the bath chamber (1.5 ml volume) at a rate of 2–3 ml min–1. All drugs were purchased from Sigma and diluted from frozen stocks.
Electrical stimulation and recording
Bipolar electrical stimulation of efferent axons was provided by a theta glass micropipette (20–30 μm tip diameter) positioned about 20 μm modiolar to the base of an IHC, near the course of the inner spiral bundle. The position of this pipette was adjusted until current passage through it caused synaptic currents in the IHC under study. An electrically isolated constant current source (model DS3, Digitimer Ltd, Welwyn Garden City, UK) was triggered via the data-acquisition computer to generate pulses up to 50 μA, 50–500 μs long to stimulate efferent axons. With some experience in positioning the stimulation pipette, 50 μs long pulses were sufficient for stimulation. With this method we were able to stimulate IPSCs in every IHC recorded.
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Currents and voltages were recorded with pCLAMP9.2 software and an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), low-pass filtered at 2–10 kHz and digitized at 10–50 kHz with a Digidata 1322A board.
Indicated holding potentials were not corrected for liquid junction potentials (–4 mV). Series resistance compensation of 50–70% was used, and the remaining series resistance error for the recorded synaptic currents was calculated to be smaller than 5 mV. Synaptic currents and potentials were analysed with Minianalysis (Synaptosoft, Decatur, GA, USA). IPSCs were identified manually using a search routine for event detection with a threshold of 5 times the RMS noise (7–12 pA). Rise times (10–90%) and decay time constants were analysed only in IPSCs that were not compromised in shape by the stimulus artifact or following events. The decay of IPSCs was fitted with a monoexponential. For further analysis we used Origin 7.5 (OriginLab Corp., Northampton, MA, USA). A two-sample Student's t test assuming unequal variances and a one-tailed t test were used for statistical analysis. Mean values are presented ± standard deviation (S.D.) unless stated otherwise.
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Quantal analysis
The amplitude distributions of spontaneous IPSCs were fitted by a single Gaussian equation. To test whether amplitude distributions for evoked IPSCs were shaped by distinct peaks at integral multiples of the first peak, as established for the neuromuscular junction (Del Castillo & Katz, 1954), we used the following equation (Sahara & Takahashi, 2001):
where N(x) was the number of events with x amplitude, Nt was the total number of evoked events, r was the number of quanta released, m was the quantum content, and μ and 2 were the mean and variance of the distribution. We used this equation under the assumption that low values for the quantum content were observed, with k, the maximum vesicle number, taking a value of 3 in most cases. This function uses for each peak a mean and variance given by a Gaussian distribution, whereas the relative amplitude of each maximum is estimated by Poisson statistics governing the probability of release of one vesicle, two vesicles, or more. This approach allowed us to calculate the quantum content ‘m’. The Poisson distribution also could be used for a separate estimate of quantum content based on the fraction of failures (del Castillo & Katz, 1954).
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Best fits for amplitude distributions were evaluated by finding the least squared difference from the data using Origin 7.5.
Results
Electrically evoked IPSCs in IHCs
To investigate the effects of cholinergic efferent input on neonatal IHC activity, we did whole-cell patch clamp recordings from 43 IHCs in excised apical turns of organ of Corti from 7- to 11-day-old rats. In these recordings we electrically stimulated efferent fibres that directly synapse onto the IHCs. These fibres originate in the brainstem in the medial olivocochlear system and during development reach the IHC area before birth (Simmons, 2002). Medial efferent axons were stimulated electrically with a theta glass micropipette positioned close to the IHC under investigation, about 20 μm modiolar to the IHC's base (for details see Methods) (Fig. 1A).
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A, for bipolar electrical stimulation of efferent fibres some supporting cells were removed and a theta glass micropipette, 10–15 μm in diameter, was positioned 10–20 μm below the base of the IHC. Scale bar 10 μm. Positions of 2 IHCs and recording pipette outlined. B, evoked IPSCs were eliminated reversibly by 1 μM tetrodotoxin (TTX) (n = 2). C, IPSCs were reversibly blocked by 100 nM strychnine, which blocks 910 AChR (n = 2). D, IPSCs were reversibly inhibited by 300 nM -bungarotoxin, an 910 AChR antagonist (n = 2). B–D, traces for each control, drug application and washout are averages of 50–100 sweeps (stimulation at 1 Hz). Each new solution was applied for about 5 min before data collection. Vh = –90 mV. E, IPSC waveform varied with holding potential. IPSCs reversed from completely inward at –90 mV to a biphasic inward (arrowheads) and outward (stars) waveform at membrane potentials positive to the potassium equilibrium potential. F, synaptic potentials mirrored synaptic currents and varied similarly with membrane potential: an initial depolarization (arrowheads) was followed by a hyperpolarization (stars). Arrow indicates time of electrical shock in E and F.
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When timed current pulses (of 50 μA in size, 50 μs long) were delivered via the stimulating pipette, transient inward currents (evoked IPSCs) at a holding potential of –90 mV were evoked in every IHC tested (Fig. 1B). Application of 1 μM tetrodotoxin to the preparation produced a reversible loss of the synaptic response, confirming that these currents resulted from propagation of action potentials in presynaptic axons, presumably to release ACh onto IHCs. Consistent with this interpretation, the IPSCs were reversibly blocked by 100 nM strychnine (Fig. 1C) and 300 nM -bungarotoxin (Fig. 1D), like the 910-containing nAChR (Elgoyhen et al. 1994; Verbitsky et al. 2000; Elgoyhen et al. 2001), and spontaneous synaptic currents recorded in IHCs (Glowatzki & Fuchs, 2000).
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Evoked IPSCs were similar in shape to spontaneous IPSCs and to IPSCs produced by elevated extracellular potassium as previously described in both IHCs (Glowatzki & Fuchs, 2000; Katz et al. 2004) and OHCs (Oliver et al. 2000; Lioudyno et al. 2004). The mean 10–90% rise time of the evoked IPSCs was 9.6 ± 3.8 ms (mean ± S.D., n = 298 IPSCs from 3 IHC recordings) and the mean time constant of decay was 50.2 ± 17.4 ms (n = 244 IPSCs from 3 IHC recordings) at a holding potential of –90 mV.
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Like the previously described spontaneous IPSCs, evoked IPSCs reversed from completely inward at –90 mV to a biphasic inward and outward waveform at membrane potentials positive to the potassium equilibrium potential (EK = –82 mV) (Fig. 1E). The initial inward current of the biphasic IPSC is thought to result from sodium and calcium flux through the 910-containing nAChR (Elgoyhen et al. 1994; Verbitsky et al. 2000; Elgoyhen et al. 2001; Gomez-Casati et al. 2005). The subsequent activation of calcium-dependent small conductance SK2 potassium channels underlies the outward current (Dulon et al. 1998; Yuhas & Fuchs, 1999). Negative to EK, at –90 mV, both the AChR current and SK current are inward. Between EK and –40 mV the AChR current is directed inward whereas the SK current is directed outward, resulting in a biphasic response. Positive to –40 mV the much larger outward current obscures the small initial current through the AChRs.
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To characterize the effect of efferent stimulation on IHC membrane potential, we recorded evoked inhibitory postsynaptic potentials (IPSPs) in current clamp (Fig. 1F). The synaptic potentials mirrored the synaptic currents, starting with a short depolarization and followed by a longer and larger hyperpolarization at potentials close to the IHC resting membrane potential at –60 mV. This hyperpolarization makes the effect of efferent synaptic activity on IHCs inhibitory (see also Fig. 5).
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A, current injections of 30–50 pA for 10 s induced repetitive firing of calcium action potentials in IHCs, with larger currents causing higher frequency firing. B, efferent stimulation (onset indicated by downward arrow) at 2 Hz had little effect on IHC excitability. The downward deflections in the records are stimulus artifacts (examples indicated by arrow heads). C, efferent stimulation at 5 Hz caused a hyperpolarization of 5–10 mV and suppressed the generation of calcium action potentials. A few calcium action potentials still occurred during the hyperpolarization (indicated by stars). Time scale for all figures: 2 s.
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Taken together, the pharmacological data and the biphasic IPSC waveform confirm that with electrical stimulation we were able to exert cholinergic efferent synaptic inhibition of postnatal IHCs.
Synaptic strength of efferent contacts
As a measure of synaptic strength at the IHC efferent synapse, we calculated quantum content, ‘m’, the number of vesicles released per stimulation. We analysed our data in the following way. At a stimulation rate of 0.25 or 1 Hz we observed a relatively high fraction of failures of 0.65 ± 0.16 (n = 32 IHC recordings) and a mean amplitude of ‘successes’ of –22 ± 9 pA (n = 32 IHC recordings, 11 900 IPSCs analysed) at a holding potential of –90 mV. Figure 2A illustrates a recording with three successful stimulations and one failure. Additionally, spontaneous IPSCs occurred during the recordings that were not time-coupled with the stimulus (Fig. 2A). In each recording, evoked IPSC amplitudes fluctuated considerably (Fig. 2B). Examples of amplitude distributions for evoked and spontaneous IPSCs are shown in Fig. 2C.
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A, electrical stimulation (arrows) of the efferents at 1 Hz caused 3 IPSCs and 1 failure. Some spontaneous IPSCs were observed as well. B, evoked IPSCs varied in amplitude. C, amplitude distribution of 114 evoked IPSCs produced by 1 Hz stimulation (300 times), and amplitude distribution of spontaneous IPSCs (inset) from the same cell (the noise floor is shown by the 3 bars centred around 0 mV). The spontaneous IPSC amplitude distribution was fit with a single Gaussian providing a mean of –19 ± 5 pA (n = 38 spontaneous IPSCs). Dividing mean evoked IPSC amplitude by the mean spontaneous IPSC amplitude provided a quantum content of 0.64. Quantal parameters also were obtained from the Poisson distribution derived from the fit of eqn (1) (Methods) to the evoked IPSC amplitude distribution. This gave a mean evoked IPSC amplitude of –22 ± 7 pA for the first peak (n = 114 IPSCs) and a value of 0.75 for the quantum content.
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We estimated quantum content by four different approaches for six IHC recordings that provided enough data for both evoked and spontaneous IPSCs; a summary of the results is provided in Table 1. One approach was to divide the mean evoked IPSC amplitude by the mean quantal size (q). Assuming that the spontaneous IPSCs are uniquantal events, we estimated the mean quantal size q from the amplitude distributions of spontaneous IPSCs fitted by a single Gaussian equation and obtained –18 ± 2 pA (Fig. 2C, inset). Dividing this value into the mean of evoked IPSCs yielded a mean quantum content (m) of 1.0 ± 0.5 vesicles released per stimulus. Alternatively, we estimated an average quantum content of 0.9 ± 0.5 from the ‘fraction of failures’ based on the Poisson distribution (del Castillo & Katz, 1954). The Poisson assumptions also underlie the estimate of m as equivalent to the reciprocal of the coefficient of variation squared (m = 1/CV2) and provided an average value of 1 ± 0.6 for the six cells studied. Finally, the overall evoked IPSC amplitude distributions were fit with eqn (1) (see Methods) (Fig. 2C). The first peak of this fit provided an estimate for the quantal size q of –20 ± 5 pA, quite similar to the value provided by the Gaussian fit of the spontaneous IPSC distribution. The mean quantum content for all IHCs obtained from fitting the evoked IPSC amplitude distributions again was 1 ± 0.4. However, while this average value was quite comparable, it should be noted that the estimates of quantum content obtained from this approach differed somewhat from those obtained by other methods (Table 1).
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Nonetheless, taken together the different estimates of quantum content were similar: the efferent IHC synapse has a low probability of release at low frequency, with a range of 0.4 up to 2 vesicles released per action potential (at 1 Hz).
Two-pulse facilitation of efferent release
Does the efficacy of the IHC efferent synapse change with higher rates of stimulation We used paired pulse protocols to probe facilitation at the IHC efferent synapse. Pulse pairs with intervals ranging from 1 to 500 ms were presented 50 times each, at a rate of 0.25 Hz (Fig. 3A–D). IPSCs or failures could be seen following the first, or the second (Fig. 3A, insets) or both pulses (not shown). The resulting mean IPSC amplitudes, which include failures, were calculated as a ‘facilitation ratio’ (S2 – S1)/S1, with S2 representing the response to the second pulse, and S1 representing the response to the first pulse (Mallart & Martin, 1968). The facilitation ratio can be considered a measure of the percentage mean IPSC amplitude increase due to facilitation. The 1 ms and 5 ms paired pulse intervals were too short to measure IPSC amplitudes in response to the first pulse for S1. Therefore, in addition to the paired pulse protocol, which provided S2, we used a single pulse protocol at 0.25 Hz recorded in the same IHC to assess S1. This was possible, as IPSCs evoked at 0.25 Hz rate did not show facilitation.
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A, average (n = 50) IPSCs evoked with paired pulses 10 ms apart. Inset shows individual records illustrating the variability of response: failure occurring after the first, or second shock (x scale: 50 ms, y scale: 20 pA). B–D, average (n = 50) IPSCs evoked with different pulse intervals. E, facilitation of the second IPSC as a function of pulse interval. The mean IPSC amplitude Sx = PxAx, the probability of occurrence, Px, times the mean amplitude of successful IPSCs, Ax. The facilitation index was computed as the ratio (S2 – S1)/S1. For intervals between 10 and 100 ms IPSCs showed facilitation: S2 was 200–300% bigger than the S1 (*P < 0.01, P < 0.05, one-tailed t test). F, IPSC amplitude Ax (failures not included) and IPSC probability Px of success were plotted separately. Facilitation was mostly due to a 150–250% rise in probability Px of transmitter release; however, there was also a significant, about 30% increase in IPSC amplitude Ax for 10, 25 and 50 ms pulse intervals (* and same as in E). E–F, facilitation for 50 pulse pairs presented at 0.25 Hz for 5–11 IHCs (mean and standard error of the mean shown). G, quantum content (calculated as 1/CV2) of the second IPSC compared to that of the first with a 25 ms interval. Each data point represents 1 IHC recording. Nine out of 10 IHCs showed facilitation as an increase in quantum content (range 0.09–0.99 for S1 and 0.13–2.08 for S2).
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Facilitation became evident with paired pulse intervals of 10–100 ms (Fig. 3A and E) with mean IPSC amplitudes S2 increasing 200–300% compared to S1. For example, for a 25 ms interval, S1 was –11 ± 12 pA and S2 was –24 ± 25 pA (n = 11 IHC recordings). In addition, although we have not quantified this effect, we noted that paired pulse protocols with facilitation also showed an increased number of IPSCs that were not time-coupled to the stimulus.
As presented, the mean IPSC amplitude, Sx, reflects two factors, the probability of occurrence, Px, and the mean amplitude of the successful IPSCs, Ax; such that Sx = AxPx. To better understand the mechanism of facilitation, Ax and Px were analysed separately in the original records (Fig. 3F). As seen there, the principal effect of paired pulse stimulation was to increase the probability of IPSC occurrence after the second pulse by 150–250%. That is, IPSCs occurred much more reliably when two pulses were presented with a 10–100 ms interval. The effect on the mean amplitude of successful IPSCs was also significant, though smaller with a 30% amplitude increase of A2. For example, for a double pulse interval of 25 ms, the mean ratio of P2/P1 was 1.78 ± 0.73 (n = 11 IHCs, one-tailed t test, P < 0.01) and the mean ratio A2/A1 was 1.34 ± 0.33 (n = 11 IHCs, one-tailed t test, P < 0.01). We estimated (both by ‘method of failures’ and by 1/CV2) the quantum content for the facilitated IPSC S2 and compared it with that of S1 (Fig. 3G). For paired pulse intervals of 25 ms, quantum content rose 150–550% (0.09–0.99 for S1 and 0.13–2.08 for S2) in 9 of 10 IHCs.
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Facilitation and summation during shock trains
Inhibition in the intact cochlea has been most often studied using extended trains of electrical shocks to activate the efferent axons (Galambos, 1956; Wiederhold & Kiang, 1970). The time course of efferent facilitation in the excised organ of Corti was characterized further using trains of 10 pulses with an interpulse interval of 25 ms; 30–50 such trains were presented at a 20 s interval, with the IHC at a holding potential of –90 mV. Facilitation as well as summation augmented the average compound IPSC amplitudes to a value of –128 ± 117 pA (n = 14 IHC recordings), about 6 times the size of the mean IPSC amplitude in response to single pulses. The biggest average compound IPSC reached a value of –450 pA (Fig. 4A). We analysed the facilitation ratio in 14 IHC recordings and compared each mean IPSC amplitude S2 to S10 in the pulse train to the mean amplitude of the first IPSC S1 in the train (Fig. 4B). In these instances, the amplitude of the facilitated IPSC is slightly underestimated, since the true preceding ‘baseline’ was not available. Nonetheless, every mean IPSC amplitude S2 to S10 in the pulse train was significantly larger than S1 showing facilitation (one-tailed t test, P < 0.05, n = 14 IHC recordings, 30–50 pulse trains per IHC averaged). Peak values for an increase in mean IPSC amplitude were found for S4 and S5 (200%), then declined to values of 150% for S6 and S7 and stayed at 100% increase for S8 through S10. This decline of increase of mean IPSC amplitudes during the stimulation train could be due to saturation of nAChRs, or other pre- and postsynaptic processes, and therefore this phenomenon will need further examination. In addition to facilitation of single IPSCs, summation also contributed to the amplitude of the compound IPSCs.
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A, response of an IHC at Vh = –90 mV to a 10 pulse, 25 ms interval, train. The pulse train was presented at a 20 s interval and repeated 30–50 times, the average compound IPSC is shown. Inset: an individual record showing marked fluctuations on the falling phase of the compound IPSC. B, facilitation relative to the first response during a 10 pulse, 25 ms interval, train. Data from 14 IHC recordings are included, each with 30–50 repeats of the 10 pulse train. Mean IPSC amplitude was significantly increased throughout the train (mean ± S.E.M., *P < 0.01, P < 0.05, one-tailed t test).
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Following the last pulse, the compound IPSC decayed slowly, over the course of nearly 1 s. This is considerably slower than the 50 ms time constant of decay for single IPSCs. Several processes could contribute to this slow decay. One possibility is suggested by the inset of Fig. 4A, which shows an individual record in which marked fluctuations are observed on the falling phase of the compound IPSC that were not seen in the averaged records. Some of these fluctuations were similar in waveform to ‘spontaneous’ IPSCs that occasionally followed the paired pulse protocols. It was already mentioned that in double pulse protocols that induced facilitation, the number of IPSCs increased that were not time-coupled to the pulse. This effect also may occur after a train of pulses and could reflect delayed release from the efferent terminal. A second possibility is that there is enhanced and delayed calcium-induced calcium release from the subsynaptic cistern of the hair cell (Lioudyno et al. 2004), causing spontaneous transient currents through the postsynaptic SK channels. Like in OHCs (Saito, 1980), a subsynaptic cistern that could serve as a calcium store has been found postsynaptically facing neonatal IHC efferent synapses (Ginzberg & Morest, 1984; Sobkowicz, 1992; Bruce et al. 2000). This phenomenon therefore needs further investigation.
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In summary, the experiments of Figs 3 and 4 show that compound IPSCs can grow much stronger due to facilitation and summation at the efferent synapse compared to the effect of a single IPSC.
Inhibition of IHC excitability
Neonatal IHCs generate calcium action potentials spontaneously or in response to injected current (Kros et al. 1998; Marcotti et al. 2003a, b). We used depolarizing current steps through the recording electrode to cause the activation of repetitive action potentials in rat IHCs during the second postnatal week. Three levels of current injection were used (30–50 pA) to depolarize the IHC from –80 to levels between –70 and –55 mV. The latter two steps evoked repetitive activity at frequencies between 2 and 10 Hz (Fig. 5). When these excitatory stimuli were combined with efferent pulse trains at frequencies of 5 Hz and greater, action potentials were essentially eliminated and the steady membrane potential hyperpolarized by 5–10 mV (Fig. 5C). Thus, efferent inhibition becomes potent when transmitter release has been facilitated by higher frequency firing.
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Discussion
Cholinergic efferent fibres innervate adult OHCs and neonatal IHCs in the mammalian cochlea. It has been shown that the cholinergic effect on both these types of hair cells is essentially identical, involving nicotinic AChRs composed of 9 and 10 subunits (Elgoyhen et al. 1994; Elgoyhen et al. 2001; Lustig & Peng, 2002), and subsequently activated calcium-dependent SK channels (Evans, 1996; Dulon et al. 1998; Yuhas & Fuchs, 1999). It remains to be seen whether a postsynaptic calcium store (the synaptoplasmic cistern) supports inhibition of IHCs, as has been found for OHCs (Evans et al. 2000; Lioudyno et al. 2004). There is substantial evidence that cholinergic inhibition of hair cells in various end-organs of other vertebrates results from similar, if not identical, synaptic mechanisms (Flock & Russell, 1973; Art et al. 1984; Fuchs & Murrow, 1992; Sugai et al. 1992). When functional measures of efferent inhibition have been made, it has been found that relatively high frequency trains of efferent action potentials (assumed to result from the electrical shocks used for activation) were required to produce consistent, measurable effects (Wiederhold & Kiang, 1970; Flock & Russell, 1973; Art et al. 1984). Studies in turtle hair cells demonstrated that the probability of release to single action potentials was significantly less than unity, ranging from 0.08 to 0.3, and that transmitter release from efferent endings facilitated strongly during repetitive firing (Art et al. 1984).
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The present study demonstrates that efferent axons in the mammalian cochlea show resting levels of release, and plasticity similar to that found in the turtle. Single shocks at frequencies less than 2 Hz evoked only small IPSCs in IHCs with a mean probability of occurrence of 0.35. However, with repeated stimulation, IPSCs were both larger and more likely. For trains of 10 shocks the summed, facilitated IPSCs reliably reached more than –100 pA in amplitude. Likewise, calcium action potentials in IHCs were only inhibited by efferent activity at rates of 5 Hz or greater. This frequency nearly corresponds to the interpulse interval (100 ms) at which facilitation first becomes evident in two-pulse protocols. The implication of this result is that efferent inhibition only is effective when occurring repetitively, and at sufficiently high frequencies so that facilitation of transmitter release can occur. In this way feedback from the central nervous system operates in a kind of failsafe mode, having consequence only when strongly driven, and spontaneous, inadvertent activity will not alter cochlear function significantly. Given the strong conservation of efferent synaptic mechanisms among vertebrate hair cells, it seems likely that the behaviour described here for neonatal IHCs will also occur in OHCs of the mature mammalian cochlea. The ability to stimulate the efferents electrically and produce strongly facilitated inhibition of defined duration, will enable studies of the mechanisms of fast and slow inhibition seen in vivo (Sridhar et al. 1997), as well as further investigation of possible longer-term effects on OHC electromotility (Dallos et al. 1997).
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The present study also reaffirms the conclusion that functional inhibitory synapses are present on neonatal IHCs prior to the onset of hearing. What purpose do these synapses serve Clearly they can suppress the excitability of the IHCs, and presumably prevent evoked and spontaneous transmitter release from ribbon synapses known to be functional at this stage (Glowatzki & Fuchs, 2002). Previous studies have suggested that this transient efferent innervation may play a role in the ultimate functional maturation of cochlear hair cells (Simmons, 2002). Most impressively, surgical lesion of the efferent nerve supply caused kittens to fail to develop normal hearing (Walsh et al. 1998). The pattern of loss was much like that seen with OHC damage. Also, in transgenic mice lacking the principal voltage-gated calcium channel of cochlear hair cells, the normal maturation of IHC excitability failed to occur and efferent synapses remained in place long after the normal period (Brandt et al. 2003). Thus, a neonatal period of synaptic cross-talk between efferent and afferent neurones may be necessary to the ultimate innervation pattern, and the final functional state of both IHCs and OHCs.
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It is unknown at present what, if any, endogenous firing takes place among hair cells and efferent neurones before the onset of hearing in vivo. A few studies documented spontaneous, or even rhythmic, activity in afferent nerve fibres prior to the onset of hearing (Lippe, 1994; Gummer & Mark, 1994). What remains uncertain is whether that activity depends on hair cell transmitter release, or arises from intrinsic excitability of the afferent neuron, as can occur in vitro (Lin & Chen, 2000). Likewise, several studies have shown that IHCs are capable of firing spontaneous calcium action potentials in preparations in vitro (Marcotti et al. 2003a,b); it remains to be determined whether they do so in vivo. Finally, neonatal medial olivocochlear neurones recorded in a brainstem slice preparation, identified by retrograde labelling from the cochlea, have the ability to discharge tonically, when stimulated with current injection (Fujino et al. 1997). Ultimately it will be necessary to address these questions in the intact, developing ear to learn if mechanisms of spontaneous activity will be essential to activity-dependent maturation, as shown for the retina (for review see Shatz, 1996).
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