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1John Curtin School of Medical Research, Australian National University, Canberra; and 2Garvan Institute of Medical Research, Sydney, Australia
Submitted 4 April 2005; accepted in final form 30 June 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Satzler et al. 2002; Smith et al. 1998; Taschenberger et al. 2002). This synapse acts as a sign-inverter, changing excitatory information coming from the AVCN into inhibitory transmission projecting to the LSO and MSO, where it is compared with excitatory transmission to localize sound.
The calyx of Held has been shown to be more than a simple relay because strong inhibitory synapses, found on the principal cell soma, and spillover of glycine onto the presynaptic terminal, can modify synaptic transmission (Awatramani 2004; Kopp-Scheinpflug et al. 2003; Leao et al. 2004; Turecek and Trussell 2001); these mechanisms have the potential to alter auditory signals at the level of the MNTB before their projection to the superior olivary complex. Because of its important role in sound localization, the calyx synapse undergoes a remarkable development to enable successful and high-fidelity transmission. In a variety of species, these developmental adaptations have been shown to include increases in vesicle pool size and exocytotic efficiency, increased calcium current facilitation and calcium buffering, decreased release probability, and changes in the postsynaptic glutamate receptor subunits mediating excitatory postsynaptic currents (EPSCs) (Iwasaki and Takahashi 2001; Joshi et al. 2004; Taschenberger and von Gersdorff 2000; Taschenberger et al. 2002). The postsynaptic MNTB principal cell also has a number of specializations that enable it to fire action potentials at frequencies 
800 Hz with few failures (Elezgarai et al. 2003; Gan and Kaczmarek 1998; Schneggenburger et al. 2002; Taschenberger and von Gersdorff 2000; Wang et al. 1998; Wu and Kelly 1993).
Although much is known about the normal development of the calyx of Held synapse, the role of activity in the modulation and fine-tuning of such a giant connection has not been studied in detail. In the mouse, the ear canal opens around P12, which means that there is an absence of sound-evoked auditory nerve activity before that age. However, spontaneous auditory nerve activity is present during this early postnatal period and has been shown to be crucial for the development of the mammalian cochlear nucleus (Pasic and Rubel 1989; Rubel and Fritzsch 2002). To examine the effect of spontaneous auditory nerve activity on development of the calyx of Held in mice, we used a model of auditory nerve inactivity, the congenitally deaf mouse (dn/dn). The TMC1 gene mutation in chromosome 19 of the dn/dn mouse results in the malfunction, morphological abnormality, and subsequent degeneration of hair cells and spiral ganglion neurons whereas central connections remain intact (Frank et al. 1983; Keats et al. 1995; Pujol et al. 1983). The mutant mouse is functionally deaf at the time of ear canal opening. In vivo 2-deoxyglucose analysis (Durham et al. 1989) and auditory nerve recordings (MR Youssoufian, A Berntson, and A Paolini, unpublished observations) show a complete lack of spontaneous activity in this animal, as compared with normal pups of the same age.
Previously it was shown that congenital deafness had appreciably different effects on synaptic transmission in the AVCN and MNTB at P12 (Oleskevich and Walmsley 2002; Oleskevich et al. 2004). In deaf mice, the large endbulbs of Held in the AVCN exhibited a greater evoked EPSC (eEPSC) amplitude and presynaptic release probability, as well as larger tetanic depression and posttetanic asynchronous release. The calyx of Held, meanwhile, showed no apparent differences. However, these studies were performed after ear canal opening (P12–14). In the present study, we have compared the early maturation of the calyx of Held (P5–P12) between normal and deaf mice to investigate the effect of auditory nerve activity on synapse development. Our results show that a complete absence of auditory nerve activity during the first two postnatal weeks does not affect the development of the calyx of Held; surprisingly, our results also indicate that the maturation of an important property of this synapse—release probability—occurs in the opposite direction for mice compared with other species such as the rat.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Whole cell recordings were made from MNTB principal neurons at postnatal day 5 (P5) (n = 42), P7-8 (n = 7) P12–14 (n = 70), and P18-19 (n = 7). Deafness (dn/dn) mutant and CBA mice were decapitated in accordance with local ethics guidelines; the brains were rapidly removed and 200-µm coronal brain stem slices were prepared in ice-cold standard artificial cerebrospinal fluid (ACSF) containing (in mM): 3 KCl, 26.2 NaHCO3, 1.25 NaH2PO4, 5 MgCl2, 1 CaCl2, 10 glucose, and 218 sucrose (only for cutting). Slices were incubated for 40 min in sucrose-free ACSF with CaCl2:MgCl2 at 2:1 at 36°C. Neurons were visualized with infrared differential interference contrast (IR-DIC) optics and all electrophysiological recordings were performed at room temperature (22–25°C). Slices were continually perfused during recordings with sucrose-free ACSF (oxygenated with 95% O2 – 5% CO2). Patch-clamping electrodes had a tip resistance of 3–6 M
and were filled with an internal solution containing (in mM): 120 CsCl, 4 NaCl, 4 MgCl2, 0.001 CaCl2, 10 HEPES, and 10 EGTA, and adjusted to pH 7.2 and an osmolarity of 300–310 mmol/kg. Series resistance during recordings was <10 M and compensated by 
80%. All EPSCs were recorded and filtered at 10 kHz with an Axopatch 200B or 1D amplifier (Axon Instruments, Union City, CA) then digitized at 20 kHz.
In voltage-clamp mode (holding potential = –60 mV), EPSCs were evoked using a bipolar tungsten microelectrode (0.1 ms, 0.2 Hz) placed at the midline of the brain stem. The stimulation voltage was set to 1.5 x the stimulation threshold for each cell and threshold was periodically checked during recording. Variance in EPSC amplitude was minimized by using a CsCl-based internal solution with QX-314, to block potassium and sodium currents. N-Methyl-D-aspartate (NMDA)–mediated evoked EPSCs were recorded at +60 mV in cells from normal and deaf animals at three developmental ages (P7/8, P12–14, and P18/19). Miniature EPSCs (mEPSCs) mediated by 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors were recorded in the presence of tetrodotoxin (TTX, 1 µM) at –60 and +60 mV, respectively. Data acquisition and subsequent analysis were performed with AxoGraph 4.9 (Axon Instruments).
Variance-mean analysis was used to determine the average release probability of a vesicle from a release site (Pr), average amplitude of the response to a single quantum of neurotransmitter (Qav), and number of independent release sites (N) (Clements and Silver 2000). EPSCs were recorded continuously in the presence of various extracellular calcium concentrations ranging from 0.5 to 3 mM to induce different release probabilities. During a stable period for each calcium concentration, the variance of the EPSC amplitude was calculated and plotted against the mean EPSC amplitude in that epoch to obtain the variance-mean parabola with which the Pr, Qav, and N are estimated. For tetanic stimulation, trains of stimuli consisted of 15 pulses at 100 Hz, repeated at 30s intervals. The readily releasable pool size (RRP) was calculated by plotting the cumulative amplitude of the EPSC responses, fitting a line through responses numbered 10–15 (S10–15), and extrapolating the line through the y-axis. The y-intercept value (i.e., cumulative amplitude) is equal to the product of the RRP and average quantal amplitude (Qav), and thus the RRP size can be estimated using a known value for Qav (obtained either from direct mEPSC recordings or using the variance-mean analysis) (Elmqvist and Quastel 1965; Schneggenburger et al. 1999). For current–voltage relationships, single EPSC amplitudes were recorded at holding potential steps from –60 to +60 mV and plotted against voltage to determine rectification.
Bicuculline methochloride (10 µM, Tocris, Ellisville, MO), (+)-2-amino-5-phosphonopentanoic acid (D-AP5, 30 µM, Tocris), TTX (1 µM, Alomone, Jerusalem, Israel), and strychnine hydrochloride (1 µM, Sigma, St. Louis, MO) were added to the bath ACSF perfusion as indicated. Lidocaine N-ethyl bromide (QX-314, 2 mM, RBI) was added to the internal patch electrode solution for all recordings. Spermine (100 µM–1 mM, Sigma) was added to the internal solution as indicated.
Immunohistochemistry
P5 and P12 deafness (dn/dn) mutant and CBA mice were decapitated and the brains rapidly removed and fixed for 20–30 min in 4% paraformaldehyde in phosphate-buffered saline (PBS) with 30% sucrose. Brains were washed two to three times with 30% sucrose in PBS and stored overnight at 4°C. Coronal sections (20 µm) were made using the cryostat and mounted on silane slides. Sections were blocked with 10% normal serum and 0.3% Triton X-100 in 0.1 M PBS then incubated in blocking solution containing primary antibodies directed against the GluR1, 2, 2/3, and 4 subunits (Chemicon, Temecula, CA). Antibodies were used at a concentration of 1–2 µg/mL. After being washed in PBS, an Alexa-555 conjugated goat anti-rabbit IgG secondary antibody was added to the sections (1:1,000, Chemicon). Neurons were subsequently labeled with a fluorescent Nissl stain (NeuroTrace 500/525, Molecular Probes, Eugene, OR). Slices were coverslipped and viewed with a confocal microscope (Zeiss LSM 510, Thornwood, NY) and images obtained using 10x, 20x, and 40x Plan-Neofluar and Plan-Apochromat objectives (Zeiss). Z-stacks of each MNTB were collapsed into a single plane for analysis of immunoreactivity. Because certain proteins in the MNTB are known to be tonotopically organized, the GluR subunit immunoreactivity of the MNTB was assessed both across the whole nucleus and in the three distinct tonotopic compartments (medial, intermediate, and lateral). As controls, slices were processed without the primary antibody incubation and also after primary antibody incubation with control peptides in a ratio of 1:1. No staining was observed in either control experiment.
Results are expressed as means ± SE. Statistical significance was determined using Student's t-test and ANOVA. Some data in this study (P12 MNTB variance-mean and tetanic depression) formed part of a previous study (Oleskevich et al. 2004) and have been combined here with additional data for reanalysis and comparison with younger animals (P5) to minimize the number of mice killed and increase the reliability of the data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Stimulus-evoked EPSCs were collected from MNTB principal cells at P5 and P12 in both normal and deaf animals. EPSCs were large (amplitude >1 nA), fast (decay <2 ms), and blocked by CNQX, indicating that they were AMPA receptor mediated. In accordance with previous studies (Chuhma and Ohmori 1998; Futai et al. 2001; Joshi and Wang 2002) the mean peak EPSC amplitude was about 1 nA at P5 and significantly increased with age to 5 nA at P12 (P < 0.0005) (Fig. 1A and Table 1). The P5 EPSCs were more variable in amplitude than their mature counterpart, exhibiting a wide range in EPSC amplitudes in response to stimulation (Fig. 1C). There was a significant change in the decay kinetics of the EPSC with age, consistent with previous reports (Chuhma and Ohmori 1998; Futai et al. 2001; Joshi and Wang 2002). From P5 to P12 the decay time, which was fit with a single exponential, significantly decreased (decay at P5 = 1.9 ± 0.09 ms in normal, n = 21; decay at P12 = 0.86 ± 0.06 ms, n = 19, P < 0.0005) (Fig. 1A). On average, EPSCs were larger, faster, and less variable at P12. Cells from congenitally deaf animals at P5 and P12 also showed this developmental pattern, with a fivefold increase in EPSC amplitude (P < 0.0005) and a significant decrease in decay time with age (decay at P5 = 1.7 ± 0.1 ms in deaf, n = 9; decay at P12 = 1.1 ± 0.2, n = 5, P < 0.01) (Fig. 1A).
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). At the calyx of Held there was a significant change in PPR from predominantly PPF at P5 to PPD at P12 (Fig. 1B). The average PPR at P5 was about 1.5, whereas at P12 it was 0.8 in normal and deaf mice (P < 0.0005 difference with age for normal and P < 0.005 for deaf) (Table 1). In total, PPF occurred in 19 out of 21 cells from normal animals and eight out of nine cells from deaf animals at P5. At P12, PPD occurred in 11 out of 16 cells from normal animals and 11 out of 11 cells from deaf animals. These data provide an indication that the release probability may be changing at this synapse with age, and that this developmental change can occur in the absence of all auditory nerve activity.
The NMDA component of calyceal EPSCs has been shown to significantly decrease with development, particularly from P7 to P15, and this process can be blocked by auditory nerve lesion in mice during the first two postnatal weeks (Futai et al. 2001). To investigate this developmental decrease in the deaf mice, evoked EPSCs were recorded at +60 mV at P7/8, P12–14, and P18/19. Although there was no change in NMDA amplitude from P7/8 to P12–14 in both normal and deaf animals, there was a significant decrease over the next developmental week (NMDA amplitude at P7/8 was 2.2 ± 0.1 nA in normal, n = 4, and 2.3 ± 0.3 nA in deaf, n = 3; amplitude at P12–14 was 1.7 ± 0.3 nA in normal, n = 4 and 2.3 ± 0.3 nA in deaf, n = 3; amplitude at P18/19 was 0.24 ± 0.1 nA in normal, n = 3, and 0.64 ± 0.1 nA in deaf, n = 3, P < 0.05 difference with age) (Fig. 2C). There were also no significant differences in NMDA amplitude between normal and deaf at any age. Thus whereas the AMPA component increases rapidly from P5 to P12, the NMDA-mediated EPSC gradually decreases until it is almost nonexistent by P19. Importantly, this developmental change is maintained in congenitally deaf mice.
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Development of release parameters revealed by variance-mean analysis is unaffected by congenital deafness
Variance-mean analysis was used to investigate the development of Pr, N, and Qav at the calyx of Held synapse with age and congenital deafness. The width or spread of the variance-mean parabola relates to the number of release sites, the initial slope of the parabola gives an estimate of average quantal amplitude, and the degree of curvature is directly proportional to the release probability (Fig. 3A). Visual examination of the variance-mean parabolas from P5 and P12 reveals a developmental change in quantal parameters (Fig. 3B). Both the degree of curvature as well as the size of the parabola is affected by age, whereas the slope of the initial segment remains the same. These data illustrate that Qav is stable during development, whereas N and Pr alter with age (Fig. 3C). The Qav at P5 and P12 was not significantly different, about 30–50 pA (Table 1). However, the number of release sites at P12 was five- to tenfold greater than that at P5, and the Pr almost doubled with age (P < 0.001 for difference with age in N and P < 0.0005 for difference with age in Pr) (Table 1). This increase in Pr is in line with the observed decrease in intracell variability in EPSC amplitude at P12 and the switch from PPF to PPD with age. Moreover, a plot of the EPSC amplitude distribution at P5 and P12 (not shown) illustrates that there is a trend toward larger-amplitude events at P12, which also suggests an increased Pr with development. However, the increase in Pr with age at the calyx of Held was unexpected because most previous studies at this synapse describe a decrease in Pr with age (Sakaba et al. 2002) (but see DISCUSSION).
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Readily-releasable pool significantly increases with development in deaf and normal animals
Trains of high-frequency stimuli (15 stimuli at 100 Hz) can cause tetanic depression by affecting both the presynaptic terminal and the postsynaptic cell (Fig. 4A). Tetanus causes depletion of the readily releasable pool as well as postsynaptic receptor desensitization, with vesicle depletion playing the major role at the calyx of Held at this frequency (von Gersdorff and Borst 2002). Here, tetanic depression was used to obtain an estimate of RRP, a method first used at a nerve–muscle synapse (Elmqvist and Quastel 1965; Schneggenburger et al. 1999) (see METHODS). From the cumulative amplitude plot and subsequent fitted line, it was estimated that the RRP increased seven- to eightfold with age from <100 vesicles at P5 to 550–650 vesicles at P12 (P < 0.05 for difference with age in normal and P < 0.06 for deaf weakly significant) (Fig. 4, B and C and Table 1). This value for RRP is considered to represent the pool of vesicles that are immediately ready and available for release during high-frequency stimulation (Schneggenburger et al. 2002). This robust increase was present in both normal and deaf mice.
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). Although variance-mean analysis estimates a weighted average of release probabilities across a number of heterogeneous release sites (Pr), tetanic depression provides the average probability of release of a vesicle from the pool of readily releasable vesicles (Pves). Because the y-intercept of the line through response number 10–15 of the cumulative amplitude plot is equal to NQav, and that the amplitude of an EPSC is equal to NPvesQav, then dividing the average EPSC amplitude from a cell by its NQav obtained during tetanic depression yields an estimate of release probability (or here, Pves). The values for Pves from tetanic depression show a significant increase with age similar to the results obtained with the variance-mean analysis (Fig. 5A and Table 1). At P5, Pves was about 0.25 and increased to 0.5 at P12 (P < 0.005 for difference with age in normal and similar trend in deaf, although P < 0.09 for deaf because n = 3 at P12). The estimates of release probability derived from both tetanic depression and variance-mean analysis show a similar increase with age and lack of difference between normal and deaf animals.
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The degree of tetanic depression after a high-frequency train of action potentials is thought to be related to vesicle depletion and subsequent replenishment as well as postsynaptic receptor desensitization. At the calyx of Held, high-frequency stimuli delivered at 100 Hz resulted in substantial depression of EPSCs by S15 in both normal and deaf mice at P5 and P12 (Fig. 5B). At P5, the S1 response averaged 2 nA, whereas the S15 EPSC was 80 pA, resulting in a depression of 95% at the young calyx of Held synapse. At P12, the S1 amplitude was 5 nA and the S15 EPSC was 1 nA, showing a total depression of 76% (Fig. 5C and Table 1). The developmental decrease in the degree of depression after tetanus was significant in both normal and deaf mice (P < 0.001 for difference with age in normal and P < 0.005 in deaf).
Immunohistochemistry of postsynaptic AMPA receptor subunit composition illustrates a predominance of GluR1 and 4
Postsynaptic AMPA receptors at the calyx of Held have been shown to undergo significant changes with development (Joshi et al. 2004; Koike-Tani et al. 2005). AMPA receptors are composed of different combinations of four possible subunits: GluR1, 2, 3, and 4. These subunits each lend a receptor complex different characteristics; in particular, GluR2 renders a receptor channel impermeable to calcium as the result of an arginine residue in the second transmembrane domain (Sato et al. 1993). To establish the early postnatal development of AMPA receptor subunit composition and to ascertain its susceptibility to deafness, immunohistochemistry for GluR1–4 was performed at P5 and P12.
At P5 in both normal and deaf animals, antibodies directed against GluR1–4 all showed significant amounts of staining above background level in the MNTB (Fig. 6A). GluR1 and GluR4 appeared to have the most intense staining, at approximately 0.005–0.006 units of fluorescence per square micrometer (f/µm2) (GluR1 = 0.006 ± 0.0006 f/µm2 in normal, n = 6, and 0.006 ± 0.0007 f/µm2 in deaf, n = 6; GluR4 = 0.005 ± 0.002 f/µm2 in normal, n = 6, and 0.005 ± 0.0006 f/µm2 in deaf, n = 5). There were also consistent amounts of GluR2 and GluR2/3 labeling, and at P5 GluR2 and GluR2/3 staining was approximately equivalent, indicating that there is very little GluR3 in the MNTB at this age (GluR2 = 0.003 ± 0.0005 f/µm2 in normal, n = 6, and 0.003 ± 0.0003 f/µm2 in deaf, n = 6; GluR2/3 = 0.003 ± 0.0002 f/µm2 in normal, n = 6, and 0.003 ± 0.0002 f/µm2 in deaf, n = 6). In MNTB from normal and deaf animals, GluR1 and 4 immunoreactivity were not significantly different, and GluR1 was significantly greater than GluR2 and GluR2/3 (P < 0.005 for both normal and deaf). GluR4 was not significantly greater than GluR2 and 2/3 in the MNTB of normal animals, whereas the difference reached significance in the deaf animals (P < 0.01 for GluR2 and P < 0.05 for GluR2/3).
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AMPA EPSCs are inwardly rectifying in the presence of intracellular spermine
To corroborate the immunohistochemical data, electrophysiological experiments were performed with intracellular spermine (100 µM–1 mM), which selectively blocks AMPA receptor complexes that contain GluR1, 3, and 4 at positive potentials. A significant spermine-induced inward rectification of AMPA-mediated EPSCs implies that the majority of synaptic AMPA receptor complexes contain GluR1, 3, and/or 4. At both ages, control AMPA I–V curves were approximately linear, with a reversal potential between 5 and 15 mV (Fig. 7, A and B). The degree of rectification was determined by the rectification index (RI), or the ratio of EPSC amplitude at +60 to that at –60 mV. At both P5 and P12, the RI in the absence of exogenous intracellular spermine was consistently around 0.4, and there were no significant differences in RI between neurons from normal and deaf animals (Fig. 7C). This natural inward rectification is likely the result of endogenous intracellular spermine that is not completely diluted by the internal patch pipette solution.
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At P12 in normal and deaf animals, intracellular spermine again caused a significant increase in inward rectification of evoked AMPA EPSCs compared with control conditions (normal, n = 5, P < 0.005; deaf, n = 6, P < 0.05). Overall, the spermine-induced inward rectification was significantly greater at P12 than at P5; RI in the presence of spermine was about 0.2 at P5 and about 0.07 at P12 (P < 0.0005 for difference with age in normal and P < 0.001 in deaf). These results indicate that there is a further reduction in GluR2-containing postsynaptic AMPA receptors with age, which is in accordance with the immunohistochemistry data showing a significant decrease in GluR2 labeling from P5 to P12.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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500 Hz. It is clear from experiments with congenitally deaf animals that neither spontaneous nor evoked auditory nerve activity is necessary for these pre- and postsynaptic developmental specializations.
The majority of our results agree with the previous literature with respect to the calyx of Held. In mice, it is accepted that EPSC amplitude increases with age (reports vary from around 0.5–5 nA at P5 to 1.5–10 nA at P12), whereas decay time significantly decreases to 1 ms (Chuhma and Ohmori 1998; Futai et al. 2001; Joshi and Wang 2002). The increase in the readily releasable pool at the calyx of Held is also well documented in mice and rats, with reports showing a two- to 2.5-fold increase in the number of vesicles from P5 to P12 (Iwasaki and Takahashi 2001; Taschenberger and von Gersdorff 2000). It has been shown previously that the active zones and postsynaptic densities are large at P5 and break up into multiple smaller ones with age (Taschenberger et al. 2002), which is consistent with the significant increase in number of release sites we observed in this study. The substantial twofold increase in release probability with age, on the other hand, was unexpected. Most of the literature reports a significant decrease in release probability from about 0.8 to 0.4 with development (Sakaba et al. 2002), whereas our results quite clearly support an increase (for further discussion see following text). The degree of depression after tetanus has been reported to be greater at P5 than at P12 (Joshi and Wang 2002; Taschenberger and von Gersdorff 2000). Interestingly, there are reports of an inverse correlation between release probability and tetanic depression, with a high release probability synapse showing less tetanic depression than a low Pr synapse (Brenowitz and Trussell 2001; Brenowitz et al. 1998; Oleskevich and Walmsley 2002). This correlation is in line with our variance-mean and tetanic depression results for Pr and Pves, which showed an increase in release probability and decrease in tetanic depression with age. Decreased tetanic depression has also been shown to correlate with a larger RRP size at the calyx (Iwasaki and Takahashi 2001; Taschenberger and von Gersdorff 2000), in keeping with our observations.
Reports of AMPA receptor subunit expression in the MNTB are varied. Using in situ hybridization, immunohistochemistry, RT-PCR, and Western blots, most studies report the presence of all four subunits, GluR1–4, in the mature MNTB (Sato et al. 2000). However it is evident that GluR1 and GluR4 play a major role with development at the MNTB (Caicedo and Eybalin 1999; Caicedo et al. 1998). A more recent study looking specifically at the postsynaptic AMPA receptor subunit composition at the calyx of Held showed clearly that calcium-permeable AMPA receptors (i.e., those containing GluR1, 3, and 4 with no GluR2) dominate in postsynaptic densities of mature principal cells, and that there is a progressive decline in the expression of all subunits with age (Joshi et al. 2004). Furthermore, a study looking at the relative contribution of the splice variants "flip" and "flop" showed a significant presence of GluR2 and GluR4 flop at both young and older ages, with almost no expression of GluR1–4 flip variants (Koike-Tani et al. 2005). The combination of GluR3/4 and flop splice variants seems to be responsible for the increased kinetics of AMPA receptor gating (and therefore decrease in decay kinetics) with development. These results are in accordance with the data we have shown at the calyx in both normal and deaf mice at P5 and P12, indicating that auditory nerve activity is not necessary for normal development of postsynaptic AMPA receptor subunits.
Interestingly, a lack of spontaneous auditory nerve activity from birth also does not seem to affect the NMDA component of calyceal EPSCs. Futai et al. (2001) found that bilateral cochlear ablation at P7 in the mouse resulted in an attenuation of the normal developmental decrease in the NMDA component of the evoked calyceal EPSC in the MNTB. In contrast, we found that the developmental decrease in the NMDA-mediated EPSC proceeded normally in the dn/dn mice. Furthermore, a study by Nakagawa et al. (2000) showed a transient decrease in NMDA expression in adult rats after unilateral cochlear ablation. These results indicate that it is not simply the presence or absence of early auditory nerve activity that dictates the developmental profile of postsynaptic glutamate receptors at the calyx of Held. Other factors, such as the complete loss, after cochlear ablation, of the major excitatory connection (the endbulb of Held) with AVCN bushy cells, and the resulting potential loss of neurotrophic factors, may be involved. The mechanisms underlying the developmental decrease in NMDA-mediated EPSCs, as well as the refinement of AMPA receptors in MNTB neurons, remain to be determined.
Release probability at the calyx increases with age in mice
All lines of evidence from our current study point to an increase in release probability with age. These include 1) the developmental increase in Pr calculated from variance-mean analysis, 2) the correlated increase in Pves calculated from tetanic depression at P5 and P12, 3) the significant switch from predominantly paired-pulse facilitation at P5 to paired-pulse depression at P12, and 4) the predominance of larger amplitude events at P12. Our consistent results contradict other reports in the literature. Studies of the calyx of Held in rats report a two-fold decrease in release probability with age, whereas we report a twofold increase with age in the mouse (Iwasaki and Takahashi 2001; Taschenberger and von Gersdorff 2000; Taschenberger et al. 2002). One possible reason for the contradiction with the literature may relate to the well-established heterogeneous release probability at calyx (Schneggenburger et al. 2002). Variance-mean analysis may overestimate release probability by weighting more heavily the contribution of high release probability release sites. However, this possibility does not explain the consistent trend of an increase in Pr and Pves with development. A second possibility is that release probability peaks around P12 and then decreases to lower levels after mature hearing occurs, reconciling our results with those published in rat. This interesting possibility still does not explain the contradictory trends of release probability in mouse and rat in the first two postnatal weeks of life—which is a crucial developmental period in the absence of sound-evoked auditory nerve activity.
Another possible explanation for the discrepancy between our results in mouse and those published from rat may be that there is a species difference in the development of release probability at the calyx of Held. There is already a well-documented species difference between mouse and rat at the calyx of Held in the development of the AMPA-mediated evoked EPSC amplitude: whereas evoked EPSC size does not change with age in rats, it does increase substantially in mice with development (Iwasaki and Takahashi 2001; Joshi and Wang 2002). Thus although the development of other synaptic parameters (readily releasable pool, average quantal amplitude, and number of release sites) is consistent between mouse and rat, the difference in release probability and evoked EPSC amplitude shows that the same synapse can mature differently in two different species.
Interestingly, the increase in release probability with development at the mouse calyx of Held is distinctly different from development at many other synaptic connections. Synapses from most other areas of the brain, including cerebellum, hippocampus, thalamus, and cortex, show a decrease in the probability of release with age (Bolshakov and Siegelbaum 1995; Pouzat and Hestrin 1997; Takada et al. 2005; Wasling et al. 2004; Yanagisawa et al. 2004). Furthermore, where there are heterogeneous populations of release probability synapses, the developmental decrease in Pr is even specific to high release probability terminals, showing both a selective reduction in Pr at high-probability synapses and a maintenance of low-probability synapses with age (Wasling et al. 2004; Yanagisawa et al. 2004). It may be beneficial for the mature calyx of Held to possess a high release probability. There are an abundance of mechanisms with which the release probability at this synapse can be instantaneously modified, including presynaptic glycinergic, metabotropic glutamate, adenosine, and noradrenaline receptors (Barnes-Davies and Forsythe 1995; Brenowitz and Trussell 2001; Isaacson 1998; Leao and Von Gersdorff 2002; Takahashi et al. 1996; Turecek and Trussell 2001). It is possible that the combination of a high basal release probability and moment-to-moment modulation at the mature calyx of Held allows for a wider range of release probabilities, and thus more context-specific synaptic transmission.
The unique developmental increase in Pr at the mouse calyx of Held is similar to its unique development of postsynaptic AMPA receptor subunits. Although most areas of the brain show an increase in the expression of GluR2 with age (presumably to decrease calcium influx), the postsynaptic principal cell at the calyx of Held shows a reduction in GluR2 to the point of inexistence, as reported in this study (Caicedo et al. 1998; Pellegrini-Giampietro et al. 1992). The calyx of Held can thus be viewed as a highly specialized synapse that develops in a manner unlike any other synapse in the CNS.
Congenital deafness has no effect on the development of synaptic transmission at the calyx of Held
Unlike the endbulb of Held in the AVCN, we confirm that many crucial aspects of synaptic transmission at the calyx of Held are unaffected by congenital deafness (Oleskevich and Walmsley 2002; Oleskevich et al. 2004). Others have shown that the deafness mouse maintains some functional synaptic connections in the auditory brain stem (Bock et al. 1982), although auditory nerve stimulation resulted in abnormally large potentials in the inferior colliculus. However, it was also shown that neonatal sound deprivation in mice results in the decreased neuronal size of MNTB principal cells and their "incomplete maturation" (Webster and Webster 1979, 1977). Several studies have reported that different types of deafness and sound deprivation have differential effects on auditory brain stem nuclei. Deaf white cats show significant changes in the morphology and structure of cochlear nucleus neurons, including bushy and multipolar cells (Redd et al. 2002; Saada et al. 1996), as well as medial superior olivary neurons (Schwartz and Higa 1982). Deaf white cats also exhibit changes in endbulb synaptic parameters, such as a reduction in synaptic vesicle density, an increase in synapse size, and a thickening of the pre- and postsynaptic densities (Ryugo et al. 1997). Postsynaptic excitatory and inhibitory receptors in the cochlear nucleus and central nucleus of the inferior colliculus are also significantly affected by an early loss of auditory activity (Marianowski et al. 2000). Specifically in the MNTB, a loss of afferent activity in mice has been shown to result in many other changes, including a loss in tonotopicity of certain proteins and changes in potassium currents and calcium-binding proteins (Caicedo et al. 1997; von Hehn et al. 2004). Interestingly, the effect of congenital deafness on synapses can be specific to circuits (i.e., the endbulb of Held synapse in the ILD pathway can be affected differently by auditory deprivation than the modified endbulb synapse in the ITD pathway) (Redd et al. 2000). Here we show conclusively that congenital deafness, leading to a complete lack of auditory nerve activity during the first two postnatal weeks, does not adversely affect the development of synaptic transmission at the calyx of Held. This emphasizes the fact that caution must be used in assuming that different models of deafness will have the same consequences in relation to central auditory pathways.
In conclusion, the mature calyx of Held is a remarkable synapse that combines a high release probability, large number of release sites and readily releasable pool, and specialized postsynaptic AMPA receptors to maintain high-fidelity synaptic transmission at high frequencies. Although it has been shown to affect other auditory brain stem nuclei and their synaptic parameters, congenital deafness has no effect on the development of synaptic transmission at this robust giant synapse. In the absence of all auditory nerve activity during the first two postnatal weeks, the calyx of Held is still able to develop into one of the largest and strongest synapses in the CNS.
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Address for reprint requests and other correspondence: M. Youssoufian, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra ACT 2600, Australia (E-mail: Monique.Youssoufian{at}anu.edu.au)
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P < 0.001; 
P < 0.005 from P5 to P12. Error bars are SE.
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