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. 1998 Sep 15;511(Pt 3):861–869. doi: 10.1111/j.1469-7793.1998.861bg.x

Developmental changes in EPSC quantal size and quantal content at a central glutamatergic synapse in rat

Mark C Bellingham 1, Rebecca Lim 1, Bruce Walmsley 1
PMCID: PMC2231152  PMID: 9714866

Abstract

  1. Developmental changes in amplitude and time course of single-fibre-evoked and spontaneous EPSCs mediated by AMPA and NMDA receptors at the endbulb-bushy cell synapse of rats from 4 to 22 days of age were recorded using whole-cell patch-clamp methods in in vitro slices of cochlear nucleus.

  2. The mean conductance of the AMPA component of evoked EPSCs increased by 66 %, while that of the NMDA component decreased by 61 %, for 12- to 18-day-old rats cf. 4- to 11-day-old rats.

  3. The mean AMPA spontaneous EPSC conductance increased by 54 %, while mean NMDA spontaneous EPSC conductance decreased by 83 %, for 12- to 22-day-old rats cf. 4- to 11-day-old rats. The mean number of quanta contributing to peak evoked AMPA conductance also increased by 78 % in the older age group, after correction for the asynchrony of evoked quantal release.

  4. The decay time constant of spontaneous AMPA EPSCs showed a small decrease in older animals, while the decay time constant of spontaneous NMDA EPSCs was markedly decreased in older animals. The decay time constants of evoked NMDA EPSCs showed a quantitatively similar decrease to that of spontaneous NMDA EPSCs. This suggests that AMPA receptor subunit composition is unlikely to undergo developmental change, while NMDA receptor subunit composition may be substantially altered during synaptic maturation.

  5. These data are consistent with a developmentally increased efficacy of AMPA receptor-mediated synaptic transmission at the endbulb-bushy cell synapse, due to an increase in underlying AMPA-mediated quantal size and content during the same period as a transient co-localization of NMDA receptors.

During development, regulation of the strength of synaptic transmission between neurones plays a central role in the formation of neural maps in the mammalian brain (Kandel & O'Dell, 1992; Goodman & Schatz, 1995). The strength of a synaptic connection between central neurones is governed by the number of neurotransmitter release sites and the properties of quantal synaptic transmission at each release site (Walmsley, 1993). Developmental modifications of synaptic transmission may be achieved by alterations in the total number of release sites, the probability of quantal transmitter release at each release site, and/or the amplitude and time course of the quantal synaptic currents generated at each release site. Quantitative measurements of developmental changes in these properties are central to our understanding of how synaptic strength is regulated during the establishment and modification of neuronal networks.

Little is known about changes in the quantal parameters of synaptic transmission during development. At glutamatergic synapses, the principal excitatory synapses of the central nervous system, there have been few studies of developmental changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (Wu et al. 1996; Chuhma & Ohmori, 1998) or N-methyl-D-aspartate (NMDA) (Hestrin, 1992; Carmignoto & Vicini, 1992; Burgard & Hablitz, 1993; Wu et al. 1996; Shi et al. 1997) receptor-mediated excitatory postsynaptic currents (EPSCs); none of these studies unambiguously demonstrate changes at the quantal level.

A major disadvantage in studying quantal EPSCs in most central neurones is that there may be considerable electrotonic distortion of synaptic currents due to the variable and usually unknown somatic-dendritic location of the synaptic contacts, and so the true amplitude and time course of quantal synaptic currents cannot be directly measured (Walmsley, 1993). However, these problems are avoided at the endbulb of Held, a powerful synapse made between individual auditory nerve fibres and bushy cells in the anteroventral cochlear nucleus (AVCN), since all glutamatergic release sites are on the bushy cell soma (Neises et al. 1982; Ryugo et al. 1996). It has been shown that quantal EPSCs generated at endbulb release sites during single-fibre-evoked transmission, and spontaneous EPSCs recorded in the same bushy cell, are represented by the same population of events (Isaacson & Walmsley, 1995a). Since the amplitude distributions of spontaneous EPSCs arising from an endbulb are unaffected by the presence of tetrodotoxin, to block active presynaptic Na+ currents, and are not influenced by postsynaptic receptor desensitization (Isaacson & Walmsley, 1996), spontaneous EPSC amplitude is an accurate and direct measure of quantal EPSC amplitude.

In the mature rat, synaptic transmission at the endbulb synapse occurs exclusively at somatic release sites and is mediated by AMPA receptors, which generate an extremely brief but large synaptic current, consistent with transmission of the precise timing information required for sound localization (Isaacson & Walmsley, 1995b). However, in the neonatal rat, both the nerve-evoked EPSC and spontaneous EPSCs exhibit AMPA and NMDA receptor-mediated components, which are temporally distinct from each other (Isaacson & Walmsley, 1995b). In this study, we have taken advantage of this to investigate the developmental time course of changes in the AMPA and NMDA receptor-mediated components of single-fibre-evoked EPSCs and their relationship to developmental changes in spontaneous quantal EPSCs.

METHODS

Preparation and recording methods

Recordings (n = 121 cells) were made from parasagittal slices of the anteroventral cochlear nucleus from 4- to 22-day-old Wistar rats, prepared as described previously (Isaacson and Walmsley, 1995a, b). Animal care and handling in experiments were in accordance with local university and national guidelines. In brief, animals were deeply anaesthetized with sodium pentobarbitone (20 mg kg−1, i.p.), then killed by decapitation after their responses to noxious stimuli (withdrawal and corneal reflexes) had ceased. The brainstem was then rapidly removed, and slices made in ice-cold Ringer solution as given below, with the exception that MgCl2 and CaCl2 concentrations were 5 and 1 mM respectively. Slices were then transferred to a similar solution at 34-37°C for 1 h, then held in the Ringer solution given below until recording took place. During recording, the slices were superfused with a Ringer solution containing (mM): 119 NaCl, 2.5 KCl, 1 MgCl2 or 1.3 Mg2SO4, 2.0 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, with 10–20 μm strychnine, equilibrated with 95 % O2-5 % CO2. Patch electrodes (1.5-3 MΩ resistance) contained (mM): 117.5 caesium gluconate, 17.5 CsCl, 4 NaCl, 10 Hepes, 3 Mg-ATP, 0.2 Na2-GTP, and 10 EGTA (pH 7.3). Membrane potential values reported here have not been corrected for junction potential, which was -10 mV. Series resistance (< 8 MΩ) was routinely compensated by > 80 %. Experimental results were obtained using either thin slices (150 μm) viewed under infrared Nomarski optics (Zeiss Axioskop FS, Carl Zeiss, Germany; Newvicon C2400-ER video camera, Hamamatsu, Japan; infrared filter, 790–750 nm band pass, Omega Optical, Brattleboro, MA, USA), or thick slices (200-400 μm) viewed under a stereo microscope.

For visualized patch clamping in thin slices, EPSCs were evoked via a Ringer solution-filled pipette (tip diameter, 10–20 μm) placed over a fascicle of the auditory nerve which coursed towards the recorded cell. In other experiments, the stump of the auditory nerve was stimulated directly via a bipolar tungsten stimulating electrode. Single-fibre inputs showed evoked EPSCs with stable amplitude without intermediate levels, minimal latency variation, stable peak amplitude over a range of at least 5 V suprathreshold and all-or-none responses at threshold stimulus levels. Endbulb EPSCs were distinguished by their all-or-none response to stimulation and fast kinetics at -70 mV (Isaacson & Walmsley, 1995a, b). Synaptic currents were recorded, amplified and low-pass filtered at 5–10 kHz with an Axopatch-1D amplifier (Axon Instruments) before being digitized at 20–100 kHz (Digidata 1200, Axon Instruments, or Data Translation 2821, Data Translation, Marlboro, MA, USA). Data were also recorded on videotape with a pulse code modulated video cassette recorder (A. R. Vetter, Rebersberg, PA, USA) and digitized later off-line. Experiments were performed at room temperature (22-25°C). Whole-cell membrane capacitance was determined from the dial setting of the amplifier after compensation of the initial transient generated by a brief voltage step, and showed an age-related decrease from an average value of 24.4 ± 1.7 pF (4-11 days, n = 30) to 18.6 ± 0.6 pF (12-18 days, n = 74; P = 0.0035).

Data analysis

Data were recorded and analysed using pCLAMP 6 (Axon Instruments) and WCP (Dr J. Dempster, Strathclyde University, UK). Data are shown as means ±s.e.m. Unless indicated otherwise, the statistical test used was Student's unpaired two-tailed t test (with Welch's correction when variance of the two data groups was unequal); all statistical tests were done with Prism 2 (Graphpad, Sorrento, CA, USA).

Spontaneous EPSCs were detected and measured using WCP; detection was triggered by crossing an amplitude threshold, which was set at a level close to that of the recording noise for individual neurones so that at least 30 % of detected events were subsequently rejected as noise by manual inspection of records. In order to ascertain that this detection procedure did not miss smaller amplitude events, for several representative cells, spontaneous EPSCs were also detected using AxoGraph 3.5 (Axon Instruments), which uses a sliding template algorithm capable of detecting all spontaneous EPSCs with amplitudes more than 3 times the standard deviation of the experimental noise (Clements & Bekkers, 1997). Population parameters (amplitude, rise time and decay time constant) of EPSCs detected with AxoGraph or WCP during the same epoch of data were not significantly different, indicating that small EPSCs were not being missed with our standard detection procedure.

The amplitude of the fast AMPA component was determined by subtracting the mean baseline amplitude over a period (0.25-1 ms) immediately preceding the event from the peak value. The amplitude of the slow NMDA component of EPSCs was determined by averaging over a 5 ms window centered around the peak of the NMDA component and subtracting a similar baseline amplitude. EPSC amplitude was converted to equivalent conductance, g, to allow direct comparison between NMDA and AMPA components recorded at positive and negative membrane potentials, respectively. Reversal potential used for calculation of AMPA and NMDA conductance was +10 mV (Isaacson & Walmsley, 1995b); measurement of reversal potential for evoked EPSCs confirmed that this value was an accurate approximation (range, +8 to +13 mV) and did not change for cells from animals aged from 5 up to 18 days (M. C. Bellingham, unpublished observations).

The number of quantacontributing to the peak amplitude of the AMPA component of evoked EPSCs was initially calculated by dividing the mean peak evoked AMPA conductance by the mean peak AMPA conductance of spontaneous EPSCs from the same cell. However, this underestimates the number of quanta, as it has previously been demonstrated that the evoked EPSC time course is slower than that of the spontaneous EPSC, due to the asynchrony of evoked quantal release at this synapse (Isaacson & Walmsley, 1995a). The time course of the evoked EPSC is given by the convolution of the quantal asynchrony probability density function, described by the function:

graphic file with name tjp0511-0861-m1.jpg (1)

where a, b and c are constants, and the time course of the averaged spontaneous EPSC (Isaacson & Walmsley, 1995a). Since we have measured the time course of evoked and spontaneous EPSCs, we convolved the spontaneous EPSC at -70 mV with eqn (1), adjusting the free parameters b and c until the measured time course of the evoked EPSC at -70 mV was reproduced (Igor Pro 3, Wavemetrics, Lake Oswego, OR, USA), thus allowing us to derive the probability density function describing the asynchrony of release. This function was then used to calculate a correction factor which quantified the actual number of quanta contributing to the peak conductance of the evoked EPSC relative to the minimal number of quanta that could account for peak conductance, i.e. assuming synchronous release of quanta and dividing peak evoked EPSC conductance by peak quantal EPSC conductance.

After subtraction of baseline current, single-exponential functions were fitted to the decay phase of AMPA-mediated EPSCs at -70 mV, and of NMDA-mediated EPSCs at +50 mV (WCP). The synaptic current was fitted to the region from 90 % of peak current to baseline, with a fixed plateau value of 0, over a period of approximately 5 ms (for AMPA EPSCs) or > 150 ms (for NMDA EPSCs) after the peak of the EPSCs. The duration of the fitted portion of the EPSC, and of the data record analysed, was always at least double the decay time constant of the best-fit exponential function; fitting of this length of data allows reliable estimation of decay time constants to within 5 % (Dempster, 1993). Fits were only accepted if the residual current was less than 2 %; this resulted in the exclusion from further analysis of a higher proportion of NMDA spontaneous EPSCs from the older age group, since the low signal-to-noise ratio of the NMDA receptor-mediated component produced fits with unacceptably high standard errors for fitted parameters. While fitting a double-exponential function produced a statistically significant better fit (one-tailed f test, Prism 2) than a single-exponential function in some cells (15/121), the amplitude of the second time constant component was always < 10 % of the first component, and was thus a minor contributor to the time course of decay. Previous studies of evoked and spontaneous EPSCs at this synapse have also noted that the decay phase of AMPA-mediated EPSCs was well fitted by single-exponential functions (Isaacson & Walmsley, 1995b, 1996). Thus, to facilitate comparison, all cells were fitted with single exponentials.

RESULTS

Developmental changes in single-fibre-evoked EPSC conductance

At early postnatal ages (4-11 days), stimulation of an individual auditory nerve fibre evokes an all-or-none EPSC with a small AMPA component and a large NMDA component at a holding potential of +50 mV (Fig. 1A). In older animals (postnatal days 12-18), the amplitudes of these two components show reciprocal changes (Fig. 1B). Grouped data show that there is a significant 66 % increase in the amplitude of the AMPA component of the evoked EPSC (g = 20.5± 3.2 nS, n = 22, 4–11 days; g = 34.1± 4.8 nS, n = 42, 12–18 days; P = 0.02; Fig. 1C), and a reciprocal significant 61 % decrease in the amplitude of the NMDA component (g = 26.6± 5.4 nS, 4–11 days; g = 10.3± 1.5 nS, 12–18 days; P = 0.007; Fig. 1C). Linear regression of the amplitude of the AMPA (Fig. 1D) or NMDA component (Fig. 1E) of the evoked EPSC at +50 mV in individual neurones against age shows significant departure from zero slope (evoked AMPA conductance, slope 3.2 nS day−1, P = 0.002; evoked NMDA conductance, slope -2.1 nS day−1, P = 0.003; f test for both). This establishes that there is a developmental increase in the amplitude of the AMPA receptor-mediated component of the EPSC evoked by stimulation of individual auditory nerve fibres, which is accompanied by a decrease in the amplitude of the NMDA receptor-mediated component arising from the same endbulb.

Figure 1. Reciprocal developmental changes in AMPA vs. NMDA receptor-mediated components of EPSCs evoked in bushy cells of anteroventral cochlear nucleus by single auditory nerve fibre stimulation.

Figure 1

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A, averaged traces of the nerve-evoked EPSC in a 4-day-old animal. The EPSC exhibits a large NMDA receptor-mediated component and a small AMPA receptor-mediated component (indicated with horizontal arrows), compared with the corresponding dual components of the EPSC in a 16-day-old animal (B). Stimulus artifacts have been removed and the stimulus is marked with a vertical arrow in A and B. C, grouped data (means ±s.e.m.) show that there is a significant increase in the AMPA receptor-mediated component of the evoked EPSC (* P < 0.05) in 12- to 18-day-old rats (Inline graphic), compared with that in 4- to 11-day-old rats (▪). In contrast, there is a significant decrease in the amplitude of the NMDA receptor-mediated component of the evoked EPSC for the same two age groups (** P < 0.01). D and E, plots of evoked EPSC conductance at +50 mV (D, AMPA component; E, NMDA component) for individual cells vs. animal age, with linear regression fits which show significant age-related trends. Amplitude of the evoked EPSC has been converted to conductance, g (nS), in this and all subsequent figures.

Developmental changes in spontaneous quantal EPSC conductance

Changes in amplitude of the AMPA and NMDA receptor-mediated components of the evoked EPSC could be due to a change in the underlying quantal current amplitude, a change in quantal content (i.e. the number of quanta released in a single evoked EPSC), or both. To investigate changes in quantal size, the AMPA component of spontaneous EPSCs was measured in isolation at negative holding potentials (-70 mV), and the NMDA component of spontaneous EPSCs was measured at positive holding potentials (+50 mV), to relieve voltage-dependent Mg2+ block of NMDA channels. For each cell, the average of several hundred spontaneous EPSCs was calculated to obtain a mean quantal amplitude. Figure 2A illustrates representative changes in amplitude of AMPA and NMDA components of spontaneous EPSCs at +50 mV. Figure 2C shows that over the first 3 weeks after birth, a significant increase in the mean amplitude of the AMPA component of spontaneous EPSCs occurs. A linear regression fit to these data revealed a significant 3-fold increase in AMPA quantal amplitude between 4 days and 3 weeks after birth (slope 0.04 nS day−1; P < 0.0001, f test), while grouped data show a significant 54 % increase in the mean amplitude of the AMPA component of spontaneous EPSCs (4-11 days, g = 0.46± 0.03 nS, n = 30; 12–18 days, g = 0.70± 0.04 nS, n = 65; P < 0.0001; Fig. 2B).

Figure 2. Quantal EPSCs at the endbulb synapse change significantly during development.

Figure 2

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A, the reciprocal change in amplitude of the fast (AMPA) and slow (NMDA) components of averaged spontaneous EPSCs recorded in a cell from a 7-day-old animal cf. a 16-day-old animal. B, grouped data (means ±s.e.m.) show that there is a significant increase in the mean amplitude of the AMPA receptor-mediated component of spontaneous EPSCs in 12- to 22-day-old animals (Inline graphic) compared with that in 4- to 11-day-old animals (▪). In contrast, the NMDA receptor-mediated component from spontaneous EPSCs in the same age groups shows a significant decrease in mean amplitude. *** P < 0.001. C and D, the mean amplitude of the AMPA and NMDA components, respectively, of spontaneous EPSCs is plotted against postnatal age for individual cells. Between 4 days and 3 weeks after birth, there is a significant increase in the AMPA receptor-mediated component of spontaneous EPSCs, and a simultaneous decrease in the amplitude of the NMDA receptor-mediated component. Data are fitted with a linear regression fit (C) and a single-exponential function (D).

We also found that the amplitude of the NMDA component of spontaneous EPSCs decreased dramatically with postnatal age (Fig. 2D). Grouped data show that there was a significant 83 % decrease in the mean amplitude of the NMDA component of spontaneous EPSCs (g = 0.84± 0.15 nS, 4- 11 days, n = 33; g = 0.13± 0.017 nS, 12–22 days, n = 60; P < 0.0001; Fig. 2B). This finding contrasts markedly with those of a number of previous studies, which reported that there was no developmental change in the amplitude of spontaneous NMDA receptor-mediated EPSCs (Hestrin, 1992; Carmignoto & Vicini, 1992; Burgard & Hablitz, 1993; Wu et al. 1996; Shi et al. 1997). Interestingly, linear regression of the amplitude of the NMDA component of spontaneous EPSCs against age did not produce an adequate fit to the data (runs test, P = 0.0002), while fitting a single-exponential function did (decay time constant, 4.5 days; runs test, P = 0.22). Linear regression of the amplitude of the NMDA component of spontaneous EPSCs against age for data from 12 days and older showed that amplitude did not change significantly with age after this time point (slope -0.006 ± 0.006, n = 60; P = 0.33, f test).

Developmental changes in quantal content

These data established that the time course of developmental changes in quantal EPSC amplitude was similar to changes in evoked EPSC amplitude. However, developmental changes in the AMPA receptor-mediated component of evoked EPSCs were greater than could be accounted for by changes in quantal EPSC amplitude. This suggested that, at a single endbulb, the number of quanta contributing to the peak evoked AMPA receptor-mediated conductance might also undergo developmental regulation. The minimal number of quanta underlying the peak amplitude of the AMPA receptor-mediated component of evoked EPSCs at +50 mV was estimated for each cell, by dividing the evoked EPSC amplitude by the quantal EPSC amplitude, based on the previous demonstration that the underlying quantal EPSCs at the endbulb synapse and the spontaneous EPSCs recorded in the same cell are represented by the same population of events (Isaacson & Walmsley, 1995a). This calculation of the number of quanta contributing to the peak amplitude of evoked EPSCs underestimates the number of quanta, due to the asynchrony of quantal evoked release at the endbulb synapse (Isaacson & Walmsley, 1995a). To account for the effects of asynchrony, this minimal number of quanta was then multiplied by a correction factor of 1.35 (4-11 days) or 1.38 (12-18 days), calculated as described in Methods. In grouped data, the mean number of quanta contributing to the peak AMPA conductance increased significantly by 78 % (P = 0.007), from 40.5 ± 6.7 (n = 20) in 4- to 11-day-old animals to 72.0 ± 8.8 (n = 27) in 12- to 18-day-old animals. The function describing the asynchrony of quantal release is not substantially different for the two age groups during the rising phase of the evoked EPSC, and this is reflected in the minor difference in the correction factor used for the two age groups. These results suggest that the increase with age in evoked AMPA receptor-mediated EPSC amplitude is due to increases in both quantal amplitude and the number of quanta contributing to peak EPSC amplitude. Interestingly, there is a prolonged period of asynchronous release following the peak amplitude in young animals, as indicated both by the longer decay time constant of the evoked EPSC (see below) and from calculation of the asynchony probability density function.

Developmental changes in evoked and spontaneous EPSC kinetics

These changes in amplitude of AMPA and NMDA receptor-mediated EPSCs were not due to developmental alteration of the voltage dependence of AMPA or NMDA receptor-mediated EPSCs (M. C. Bellingham and B. Walmsley, unpublished observations). Changes in receptor-mediated EPSC amplitude may be due to alteration of the receptor subunit composition and/or the number of receptors present in the postsynaptic density. Different receptor subunit compositions are often associated with substantial changes in AMPA (Geiger et al. 1995; Angulo et al. 1997) and NMDA receptor-mediated current kinetics (Farrant et al. 1994; Feldmeyer & Cull-Candy, 1996; Flint et al. 1997; Shi et al. 1997). Developmental changes in EPSC kinetics were therefore examined by measurement of the 10–90 % rise time of evoked and spontaneous AMPA EPSCs, and the decay time course of AMPA and NMDA components of evoked and spontaneous EPSCs by fitting a single-exponential function to averaged EPSCs to determine their decay time constant (τdecay).

Both evoked and spontaneous AMPA EPSCs at -70 mV showed significant changes in 10–90 % rise time during development. In grouped data, evoked AMPA EPSC 10–90 % rise time decreased by 33 % (4-11 days, 0.59 ± 0.05 ms, n = 11; 12–18 days, 0.46 ± 0.02, n = 28; P = 0.024), while spontaneous AMPA EPSCs decreased by 13 % (4-11 days, 0.25 ± 0.01 ms, n = 22; 12–18 days, 0.22 ± 0.004 ms, n = 53; P = 0.037).

Evoked AMPA EPSCs showed a substantial and significant developmental decrease in τdecay. In grouped data (Fig. 3B), evoked AMPA EPSC τdecay decreased by 50 % (4-11 days, 1.33 ± 0.18 ms, n = 14; 12–18 days, 0.66 ± 0.20 ms, n = 34; P = 0.0025). In contrast, spontaneous AMPA EPSCs showed smaller changes in τdecay, as illustrated in Fig. 3A. In grouped data (Fig. 3B), spontaneous EPSCs showed a small but statistically significant decrease (21 %) in AMPA τdecay at -70 mV (4-11 days, 0.43 ± 0.04 ms, n = 27; 12–18 days, 0.34 ± 0.04 ms, n = 51; P = 0.03). We found that the larger change in evoked AMPA EPSC τdecay could be accounted for by increased quantal release during the falling phase of the asynchonous release function in the younger age group, together with the measured decrease in spontaneous AMPA EPSC τdecay.

Figure 3. Developmental changes in EPSC kinetics.

Figure 3

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The decay time constant of the AMPA receptor-mediated EPSC shows a small decrease in spontaneous EPSCs and a larger decrease in evoked EPSCs in older animals, while the decay time constant of the NMDA receptor-mediated component of spontaneous and evoked EPSCs shows similar large decreases in older animals. A, averaged spontaneous EPSCs from neurones at -70 mV, from a 4-day-old animal (filled symbols) and a 14-day-old animal (open symbols), are plotted, together with single-exponential fits to the decay phase of the AMPA component of the EPSCs. The decay time constant for the 4-day-old animal is slightly larger than that for the 14-day-old animal (τdecay= 0.47 cf. 0.39 ms). B, grouped data (means ±s.e.m.) showing mean decreases in the AMPA receptor-mediated EPSC τdecay of spontaneous (* P < 0.05) and evoked EPSCs (** P < 0.01) from 4- to 11-day-old rats (▪) cf. 12- to 18-day-old rats (Inline graphic). C, averaged spontaneous EPSCs from neurones at +50 mV from a 4-day-old animal (filled symbols) and a 14-day-old animal (open symbols) are plotted, together with single-exponential fits to the decay phase of the NMDA component of the EPSCs; note that for clarity, the full record length has not been plotted and, after the first 27 ms, each data point plotted is an average of 4 consecutive data points in the original fitted record. The decay time course is slower for the 4-day-old than for the 14-day-old rat (τdecay= 108 cf. 37 ms). D, grouped data (means ±s.e.m.) showing mean decreases in the NMDA receptor-mediated EPSC τdecay of spontaneous and evoked EPSCs from 4- to 11-day-old rats (▪) cf. 12- to 18-day-old rats (Inline graphic). ** P < 0.01.

In contrast, both evoked and spontaneous EPSCs showed similar changes in the τdecay of their NMDA component at +50 mV, exhibiting a significantly larger τdecay (i.e. a slower decay time course) early in development, as illustrated in Fig. 3C for spontaneous EPSCs. Grouped data for the NMDA receptor-mediated component of evoked EPSCs showed a 40 % decrease in τdecay (4-11 days, 101.2 ± 11.5 ms, n = 20; 12–18 days, 60.4 ± 3.4 ms, n = 41; P = 0.003), while grouped data for the NMDA receptor-mediated component of spontaneous EPSCs showed a 33 % decrease in τdecay (4-11 days, 95.0 ± 8.6 ms, n = 25; 12–18 days, 64.0 ± 4.7 ms, n = 31; P = 0.002). However, within each age group, the decay time constants of the NMDA component of spontaneous and evoked EPSCs were not significantly different (4-11 days, P = 0.66; 12- 18 days, P = 0.53).

DISCUSSION

It has proved to be difficult to study developmental changes in quantal synaptic currents in central neurones, due to electrotonic distortion of current amplitude and time course by the variable dendritic location of most synaptic contacts, compounded by significant developmental changes in dendritic morphology or synapse location. Due to the somatic location of synaptic contacts between the endbulb of Held and bushy cells in the cochlear nucleus (Neises et al. 1982; Ryugo et al. 1996), and the previous demonstration that spontaneous EPSCs arising from the endbulb are quantal events (Isaacson & Walmsley, 1995a, 1996), we have been able to investigate absolute changes in quantal conductance and time course of AMPA and NMDA receptor-mediated components of EPSCs during a developmental period that covers maturation of endbulb morphology (Neises et al. 1982) and spans the opening of the external ear canal and the onset of sensory input to bushy cells. Over this period of development, we have demonstrated that quantal AMPA EPSCs show an increase in conductance without major changes in their time course, while quantal NMDA EPSCs show a decrease in conductance with a major change in decay time course. Further to the increased quantal AMPA conductance, we also found that the quantal content of the AMPA component of single-fibre-evoked EPSCs increases.

Previous studies of developmental changes in AMPA receptor-mediated quantal EPSCs have been made in frog tectum (Wu et al. 1996) and in the medial nucleus of the trapezoid body at a somatic calyceal synapse similar to the endbulb synapse (Chuhma & Ohmori, 1998). While the former study found that quantal AMPA EPSCs increased in amplitude with increasing cell maturity, as determined by a rostro-caudal developmental gradient, it is possible that developmental changes in dendritic morphology or synaptic location could account for this correlation. In the latter study, which examined spontaneous EPSCs originating from a somatic location in animals aged up to 13 days, no change in quantal AMPA EPSC charge or decay time constant was found, while evoked AMPA EPSC amplitude showed a significant increase. In addition, developmental changes in the NMDA component of EPSCs were not studied.

Since the AMPA receptors which underlie synaptic transmission at this and other calyceal synapses show decay time constants which are consistently amongst the fastest recorded in central neurones (Forsythe & Barnes-Davies, 1993; Raman et al. 1994; Isaacson & Walmsley, 1996), our finding of a small absolute decrease in AMPA spontaneous EPSC τdecay suggests that AMPA receptor subunit composition is unlikely to undergo significant developmental changes in spherical bushy cells. Immunohistochemical studies of the endbulb-bushy cell contacts have demonstrated that the major glutamate receptors present in mature animals are AMPA receptor complexes containing mainly GluR3 and GluR4 (Wang et al. 1998), consistent with rapid EPSC kinetics (Raman et al. 1994). Increased AMPA quantal amplitude may therefore be due to a developmental increase in AMPA receptor number or density at individual postsynaptic densities.

Several studies have examined developmental changes in NMDA receptor-mediated EPSCs (Hestrin, 1992; Carmignoto & Vicini, 1992; Burgard & Hablitz, 1993; Wu et al. 1996; Shi et al. 1997). In all of these studies, the synaptic locations of the EPSCs have been unknown, making any quantification of amplitude changes difficult to interpret. However, all have reported that NMDA EPSCs show no developmental change in amplitude. This contrasts markedly with our finding of a major decrease in quantal NMDA EPSC amplitude at somatically located synaptic sites. One possible explanation is that NMDA receptors may not be sufficiently co-activated by ambient levels of glycine in our slice preparation. However, since addition of saturating levels of glycine to bathing solutions does not change NMDA EPSC amplitude in slices from animals aged 10 days and older (Isaacson & Walmsley, 1995b), we think it unlikely that this affects our results. It is also unlikely that morphological changes in endbulb-bushy cell synaptic contacts underlie changes in AMPA and NMDA receptor-mediated EPSCs. While initial contact between developing endbulbs and bushy cells is via somatic appendages which retract into the soma as the endbulb matures (Neises et al. 1982), the length of these appendages is quite short and thus is unlikely to cause any significant electrotonic distortion of synaptic currents recorded at the bushy cell soma. The change in NMDA-mediated EPSC kinetics we observed may be due to a developmental change in the subunit composition of the NMDA receptors (Hestrin, 1992; Carmignoto & Vicini, 1992; Burgard & Hablitz, 1993; Wu et al. 1996; Shi et al. 1997). NMDA receptors incorporating the NR2B subunit exhibit slow deactivation kinetics, high agonist affinity, and are expressed in early development, while the incorporation of NR2A subunits later in development significantly speeds up deactivation kinetics (Farrant et al. 1994; Feldmeyer & Cull-Candy, 1996; Flint et al. 1997). Since the amplitude of the NMDA receptor-mediated component of spontaneous endbulb EPSCs is also markedly reduced, it is thus possible that a developmental loss of NMDA receptors, in addition to changes in NMDA receptor subunit composition, takes place at individual postsynaptic densities of endbulb synaptic contacts.

AMPA and NMDA receptors are co-localized at a variety of central synapses (Bekkers & Stevens, 1989; Silver et al. 1992; Forsythe & Barnes-Davies, 1993; Isaacson & Walmsley, 1995b). NMDA receptor activation is thought to be important in the establishment and modification of appropriate glutamatergic connections (Kandel & O'Dell, 1992; Fox & Daw, 1993; Goodman & Schatz, 1995; Constantine-Paton et al. 1996), such as are seen during the developmental formation of an auditory space map in the central auditory pathways (Schnupp et al. 1995). It is thought that developmental modification of synaptic strength may be due to Hebbian mechanisms similar to those underlying stimulus-induced long-term potentiation (LTP) in the hippocampus (Kandel & O'Dell, 1992; Bliss & Collingridge, 1993; Kullmann, 1994; Isaac et al. 1995; Goodman & Schatz, 1995; Oliet et al. 1996). In stimulus-induced LTP, NMDA receptor activation and Ca2+ entry through NMDA channels is necessary to alter co-localized AMPA receptors, either increasing AMPA receptor-mediated synaptic currents (Bliss & Collingridge, 1993) or inducing their appearance at previously ‘silent’ synapses (Kullmann, 1994; Isaac et al. 1995; Oliet et al. 1996; Wu et al. 1996). It remains to be determined whether a process analogous to stimulus-induced LTP, involving NMDA receptor activation and postsynaptic Ca2+ entry, is necessary for normal developmental changes in amplitude in AMPA receptor-mediated synaptic transmission. Certainly, if such a process did underlie the developmental increase in AMPA receptor-mediated quantal EPSC amplitude demonstrated here, the large amplitude and long duration of NMDA receptor-mediated synaptic current seen at early postnatal ages in this study would maximize the effects of NMDA receptor activation.

The physiological activation of postsynaptic NMDA receptors depends on presynaptic nerve activity and glutamate release. In rats, the ear canal opens and acoustic inputs to bushy cells commence at postnatal day 12 (Blatchley et al. 1987). Our results comparing the periods before and after ear canal opening have demonstrated major development changes in synaptic transmission at the endbulb synapse in rats. However, it is unlikely that acoustic inputs are the primary determinants of developmental changes in synaptic transmission, since basic tonotopic map formation can form in the brainstem auditory pathways by postnatal day 5 (Kandler & Friauf, 1993) and auditory nerve fibres are spontaneously active in the absence of acoustic stimulation, even before ear canal opening (Liberman, 1991). It is possible that this spontaneous activity is important in developmental changes in synaptic transmission, as endbulbs of high spontaneous activity fibres are larger and contain more synaptic specializations than endbulbs of low spontaneous activity fibres (Ryugo et al. 1996). Such an increase in the number of specializations may be due to NMDA receptor activation and the splitting of existing synapses (Dyson & Jones, 1984; Edwards, 1995), and may underlie the increased AMPA quantal content seen in our study.

In conclusion, we have shown for the first time that a developmental increase in synaptic strength at a mammalian glutamatergic synapse is due to an increase in both quantal size and quantal content mediated by AMPA receptors. We suggest that this increase in synaptic efficacy occurs via both an increase in the total number of synaptic specializations in a single endbulb and an increase in AMPA receptor number or density in individual postsynaptic densities. These changes are temporally correlated with, and potentially due to, the transient presence of NMDA receptors co-localized at the same sites during early development.

Acknowledgments

We thank Dr J. Dempster for supplying the WCP software, and Drs G. Stuart and J. Bekkers for their helpful comments on earlier versions of this manuscript. Supported by National Health and Medical Research Council (B. W.), Australian Research Council (M. C. B.), and Clive and Vera Ramaciotti Foundation (B. W. and M. C. B.) grants.

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