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The Journal of Neuroscience, September 14, 2005, 25(37):8482-8497; doi:10.1523/JNEUROSCI.1848-05.2005
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Cellular/Molecular
Glutamate Transporter Studies Reveal the Pruning of Metabotropic Glutamate Receptors and Absence of AMPA Receptor Desensitization at Mature Calyx of Held Synapses
Robert Renden,1
Holger Taschenberger,2
Nagore Puente,3,4
Dmitri A. Rusakov,5
Robert Duvoisin,6
Lu-Yang Wang,7
Knut P. Lehre,3 and
Henrique von Gersdorff1
1The Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239, 2Abteilung Membranbiophysik, Max-Planck-Institut für Biophysikalische Chemie, D-37077 Göttingen, Germany, 3Centre for Molecular Biology and Neuroscience, Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway, 4Department of Neurosciences, Faculty of Medicine and Dentistry, Basque Country University, 699-48080 Bilbao, Spain, 5Institute of Neurology, University College London, London WC1N 3BG, United Kingdom, 6Neurological Sciences Institute, Oregon Health and Science University, Beaverton, Oregon 97006, and 7The Program for Brain and Behavioral Research and Division of Neurology, The Hospital for Sick Children and Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5G 1X8
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Abstract
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We examined the effect of glutamate transporter blockade at the calyx of Held synapse. In immature synapses [defined as postnatal day 8 (P8) to P10 rats], transporter blockade causes tonic activation of NMDA receptors and strong inhibition of the AMPA receptor-mediated EPSC amplitude. EPSC inhibition was blocked with a metabotropic glutamate receptor (mGluR) antagonist [1µM LY341495 (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid)], suggesting that elevated resting glutamate concentration specifically activates group II and group III mGluRs. Using mGluR subtype-specific agonists and antagonists, we determined that increased glutamate activates presynaptic mGluR2/3 and mGluR8 receptors but not mGluR4, although this receptor is present. Surprisingly, in older animals (P16–P18), transporter blockade had no effect on EPSC amplitude because of a developmental downregulation of group II/III mGluR activation in rats and mice. In contrast to other CNS synapses, we observed no effect of transporter blockade on EPSC decay kinetics, although expression of glutamate transporters was strong in nearby glial processes at both P9 and P17. Finally, using a low-affinity AMPA receptor antagonist ( -D-glutamylglycine), we show that desensitization occurs at P8–P10 but is absent at P16–P18, even during trains of high-frequency (100–300 Hz) stimulation. We suggest that diffusion and transporter activation are insufficient to clear synaptically released glutamate at immature calyces, resulting in significant desensitization. Thus, mGluRs may be expressed in the immature calyx to help limit glutamate release. In the more mature calyx, there is a far smaller diffusional barrier attributable to the highly fenestrated synaptic terminal morphology, so AMPA receptor desensitization is avoided and mGluR-mediated inhibition is not necessary.
Key words: auditory brainstem; MNTB; development; glutamate transporters; desensitization ; AMPA and NMDA receptors; -DGG ; diffusion modeling; group II mGluR; mGluR8; TBOA
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Introduction
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Rapid clearance of glutamate from the synaptic cleft after exocytosis is a prerequisite for fine temporal and spatial discrimination of signaling among neighboring synapses during high-frequency firing. The kinetics of transmitter clearance are controlled by the interplay of passive diffusion, receptor affinity, and active translocation. Diffusion depends on the morphology of the synapse, which may change during development. Transmitter translocation depends on the subtypes and density of glutamate transporters (Diamond, 2005 ) and their proximity to release sites and, thus, also depends on glial and synaptic geometry.
The mechanisms and speed of glutamate clearance are synapse specific and may depend on stimulation frequency (Rusakov and Kullmann, 1998; Bergles et al., 1999). Bouton-like terminals, such as those found in the hippocampus, allow rapid diffusion of transmitter. Thus, the synaptic glutamate transient is terminated by escape of neurotransmitter out of the synaptic cleft (Hestrin et al., 1990; Isaacson and Nicoll, 1993; Sarantis et al., 1993). At the large climbing fiber–Purkinje cell synapse of the cerebellum, formed by multiple bouton-like terminals, postsynaptic and glial transporters are a major mechanism for glutamate clearance (Huang and Bergles, 2004), and, at parallel fiber synapses, glutamate transporters limit the activation of metabotropic glutamate receptors (mGluRs) (Brasnjo and Otis, 2001). In contrast, at the morphologically tortuous mossy fiber–unipolar brush cell synapse of the cerebellum, diffusional exits are limited, and extracellular glutamate clears very slowly. Here, EPSC termination is also mediated by AMPA receptor (AMPAR) desensitization (Rossi et al., 1995; Kinney et al., 1997), and there is an additional effect of transporters on rapid glutamate clearance (Overstreet et al., 1999). In some calyx-type synapses, in which there are both multivesicular release and limited diffusion out of the synaptic cleft, AMPA receptor desensitization is a major factor shaping fast synaptic transmission (Trussell et al., 1993; Isaacson and Walmsley, 1996; Otis et al., 1996b), with an additional role of transporters in terminating synaptic transmission (Otis et al., 1996a; Turecek and Trussell, 2000).
The calyx of Held is a large glutamatergic nerve terminal that can fire action potentials at high frequencies (up to 1 kHz), which result in precisely timed postsynaptic responses (Guinan and Li, 1990; Wu and Kelly, 1993). Presumably, mechanisms for terminating the glutamate transients must be exquisitely tuned and robust. In immature animals, the presynaptic terminal envelops 40–50% of the postsynaptic soma in a contiguous manner [postnatal day 9 (P9 rats)] (Satzler et al., 2002). However, by postnatal day 14, the terminal becomes highly fenestrated, with many diffusional exits (Kandler and Friauf, 1993; Taschenberger et al., 2002). What is the functional significance of these dramatic morphological changes? Does the rate of glutamate clearance change during development, and does this lead to differing degrees of AMPA receptor desensitization? What role do glutamate transporters play during high-frequency firing? Previous reports showed they are not present in either presynaptic or postsynaptic compartments (Palmer et al., 2003). We study here the mechanisms underlying termination of the synaptic glutamate transient in immature and morphologically mature calyces of Held and discern the subtypes of mGluRs activated by transporter blockade.
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Materials and Methods
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Slice preparation. Sprague Dawley rat pups (Charles River Laboratories, Wilmington, MA) aged P5–P18 or CD1/CD57 mice aged P6–P18 were used in this study. After rapid decapitation, the brainstem was quickly removed from the skull and immersed in ice-cold (for P5–P14) or warm (for P16–P18) saline containing the following (in mM): 125 NaCl, 2.5 KCl, 3 MgCl2, 0.2 CaCl2, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, and 2 Na-pyruvate, pH 7.3–7.5 when bubbled with carbogen (95% O2, 5% CO2; osmolality of 310–320 mOsm). Cyanoacrylic glue (World Precision Instruments, Sarasota, FL) was used to glue the brainstem to the stage of a vibratome slicer (VT1000; Leica, Bannockburn, IL), and 180- to 200-µm-thick transverse slices were made of the region containing the medial nucleus of the trapezoidal body (MNTB). Slices were then transferred to an incubation chamber containing normal saline (P5–P14) or a low-sodium, high-magnesium incubation saline (P16–P18) bubbled with carbogen (95% O2,5%CO2), maintained for 30–45 min at 35°C and thereafter at room temperature (RT) (22–25°C). Normal saline was the same as slicing saline but with 1 mM MgCl2 and 2mM CaCl2. Incubation saline for P16–P18 contained 85 mMNa, 7 mM MgCl2, and 0.5 mM CaCl2 to inhibit endogenous synaptic activity; sucrose (75 mM) was added to maintain osmolarity (310–320 mOsm) (Hefft et al., 2002).
Electrophysiology. Whole-cell patch-clamp recordings were performed in normal saline at room temperature (22–24°C) unless otherwise specified. Slices were perfused at 1–3 ml/min and visualized using infrared-differential interference contrast microscopy (Leica) and a 40x water-immersion objective. The pipette internal solution for postsynaptic recordings contained the following (in mM): 130 Cs-gluconate, 10 CsCl, 5 Na2-phosphocreatine, 10 HEPES, 5 EGTA, 10 tetraethylammonium (TEA)-Cl, 4 Mg-ATP, and 0.3 GTP, pH adjusted to 7.3 with CsOH. Presynaptic recordings of capacitance and Ca2+ currents used a pipette solution containing the following (in mM): 130 Cs-gluconate, 15 CsCl, 5 Na2-phosphocreatine, 10 HEPES, 0.2 EGTA, 20 TEA-Cl, 4 Mg-ATP, and 0.3 GTP, pH adjusted to 7.3 with CsOH. D-APV (50 µM) and MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] (5µM) were present in most experiments to isolate AMPA receptor currents, and strychnine (0.5 µM) was added to eliminate inhibitory transmission present especially in older MNTB (Awatramani et al., 2004), unless specified otherwise. For presynaptic recordings, TEA (5 mM) and tetrodotoxin (1 µM) were added to the bath to block voltage-activated potassium and sodium channel currents, respectively.
Pipettes were pulled from borosilicate glass (World Precision Instruments) with a Sutter P-97 electrode puller (Sutter Instruments, Novato, CA) to open tip resistances of 1.6–2.5 M for postsynaptic recordings and 3.5–4.5 M for presynaptic recordings. Access resistance (Rs) was  6 M for postsynaptic recordings and 20 M for presynaptic recordings. Rs was compensated >85% for postsynaptic recordings and  50% for presynaptic recordings. Principal cells were voltage clamped at a holding potential of –70 mV, and presynaptic terminals were held at –80 mV, unless otherwise noted. Occasionally, the EPSC amplitude in older (P16–P18) principal cells exceeded –20 nA, saturating the recording amplifier. In these instances, the holding potential was changed to –30 mV to reduce driving force, or the cells were rejected from analysis.
EPSCs were stimulated with a bipolar platinum/iridium electrode (Frederick Haer Company, Bowdoinham, ME) placed near the midline spanning the afferent fiber tract of the MNTB. An Iso-Flex stimulator driven by a Master 8 pulse generator (A.M.P.I., Jerusalem, Israel) delivered pulses <15 V (direct current at constant voltage) triggered by computer. Data was acquired at 10–25 µs sampling rate, using an EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) controlled by Pulse 8.4 software (Instrutech, Port Washington, NY) and filtered on-line at 2.9 kHz. Software was controlled by a Power Macintosh G3 computer (Apple Computers, Cupertino, CA). Data were analyzed off-line and presented using Igor Pro (Wavemetrics, Lake Oswego, OR). All traces for kinetic analysis and display were corrected off-line for series resistance errors (Schneggenburger et al., 1999). Data presented were the average of several (usually three to five) sweeps under the conditions described.
Capacitance recordings were calculated from a 1 kHz, 20 mV amplitude sine wave on the holding potential of –80 mV using the software lock-in capability of the EPC-9 amplifier (sine + DC method, as per Gillis, 1995). The reversal potential was assumed to be 0 mV. Membrane capacitance (Cm) was not measured during step depolarizations (Taschenberger et al., 2002; Sun and Wu, 2001), and 30–60 s was allowed between depolarizations to allow complete recovery of exocytosis. Baseline was measured for 500–1000 ms before step depolarization and corrected for baseline drift in Cm.  Cm was calculated 500–700 ms after depolarization to avoid depolarization-induced changes in membrane conductance (Yamashita et al., 2005). Presynaptic calcium currents were leak subtracted using the P/n method.
Immunohistochemistry. Slices of MNTB were made as for electrophysiology but fixed 30 min in 4% paraformaldehyde in 0.1 M PBS immediately after cutting, washed repeatedly, and incubated 20 min at RT in antibody blocking solution consisting of 3% goat serum, 0.025% sodium azide, and 0.5% Triton X-100 in 0.1 M PBS. Slices were incubated in blocking solution with primary antibodies raised against Rab3a (mouse anti-Rab3a, 1:1000 dilution; PharMingen-Transduction Labs, San Diego, CA) and mGluR8 (guinea pig anti-mGluR8, 1:1000 dilution; Chemicon, Temecula, CA) for 48 h at 4°C. Primary antibody was detected with appropriate fluorescent Alexa dye-labeled secondary antibodies (Molecular Probes, Eugene, OR), and slices were coverslipped on glass slides under Gel/Mount (Biomeda, Foster City, CA) for microscopy. Confocal fluorescent images were taken on Zeiss (Oberkochen, Germany) LSM510 microscope. Images were analyzed with Zeiss LSM Browser and prepared for presentation with Adobe Photoshop (Adobe Systems, San Jose, CA).
Immunoblotting, perfusion fixation of Wistar and Sprague Dawley rats, and preembedding light and electron microscopic immunocytochemistry for glutamate transporters [glutamate transporter 1 (GLT1) and glutamate–aspartate transporter (GLAST)] were performed as described previously (Lehre et al., 1995), with the following modifications. The GLAST labeling shown in Figure 11 was done with 1.5% normal goat serum in PBS. For immunoblots, 10–20% gradient gels and a chemiluminescent detection system were used (Supersignal West Pico; Pierce, Rockford, IL).
Drugs and reagents. All salts and serum were purchased from Sigma (St. Louis, MO). All pharmacological reagents were purchased from Tocris Cookson (Ellisville, MO). Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel). A lexicon of the mGluRs examined in this study and specific pharmacological agents used to target them has been provided for clarity, as supplemental Table 1 (available at www.jneurosci.org as supplemental material).
Analysis. Curve fits of normalized EPSC decay kinetics were performed using a modified single- or double-exponential function in Igor (Wavemetrics). The single-exponential function was x(t) = y0 + A1exp(–x/ 1), and the double-exponential function was of the form x(t) = y0 + A1exp(–x/1) + A2exp(–x/2), where x was the time of the EPSC peak amplitude, 1 was the fast rate of decay, and 2 was the slow rate of decay. The fit was constrained such that A < 0. The fit was made from the EPSC peak to baseline when possible. All fits returned  2 0.10, and A1 + A2 1.10. Weighted mean time constant (m) is reported as A11 + A22.
Statistical tests and curve fits were calculated using Prism 4.0 software (GraphPad Software, San Diego, CA). Wilcoxon's nonparametric matched rank test was used for internally matched controls, and one-way ANOVA was used for multiple age comparisons, unless otherwise stated. Significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Data are shown as mean ± SEM. Curve fit for dose–response curves used the following equation: Y = Ymin + (Ymax–Ymin)/(1 + 10^(log10(EC50)–X)), where X is log10[drug], and Y is percentage inhibition. Curves were constrained as defined in Results.
Modeling. Computer simulations of glutamate diffusion were performed by adjusting a three-dimensional compartmental model of the calyx (Rusakov, 2001) to the structural data obtained from EM reconstructions in 9-d-old rats (Satzler et al., 2002). General parameters of the model are as follows: calyx radius, 10 µm; cleft width, 30 nm; 106 glutamate molecules (200 release sites, 5000 molecules each, D = 0.3 µm2/ms) released synchronously over the 90% cleft area (Fig. 12, red shadow), with an a-function time course ( = 39 ms–1). For additional details on modeling and transporter kinetics, see previous work by Rusakov (2001). In agreement with the present findings (see Figs. 10, 11), glutamate transporters were placed in the space compartments representing glial protrusions (an 200-nm-wide layer) that surround the calyx and principal cell, with an extracellular transporter concentration of 1mM. The latter corresponds to the total glutamate transporter level in glia of the adult hippocampus (Lehre and Danbolt, 1998).
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Results
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Transporter blockade inhibits transmission in the immature MNTB
To study the role of glutamate transporters on fast glutamatergic transmission at the calyx of Held synapse, we applied various concentrations of the high-affinity nontransportable glutamate transporter blocker DL-threo-h-benzyloxyaspartate (TBOA) to brainstem slices from P8–P10 or P16–P18 rats and recorded EPSCs in the principal cells of the MNTB. Concentrations of TBOA  100 µM inhibited AMPAR-mediated EPSCs significantly after drug application in immature MNTB (P8–P10) (Fig. 1). We also recorded NMDA receptor (NMDAR)-mediated EPSCs because NMDA receptors have a high affinity for glutamate and desensitize very slowly and are thus ideal for sensing glutamate concentration increases. Recording in 0 Mg2+ bath conditions, the EPSC was composed of both AMPAR and NMDAR components (Fig. 1A). When measured in the same cell, AMPAR and NMDAR EPSCs were inhibited by TBOA (300 µM) to a similar extent (75 ± 3% inhibition; n = 29), indicative of a presynaptic locus of inhibition (Fig. 1A,B). Application of TBOA likely increases basal glutamate concentration throughout the slice. If so, the increase in ambient glutamate might result in tonic NMDA receptor activation and could be observed as an increase in holding current (Ihold). When the principal cell was voltage clamped at Vhold of –70 mV, addition of 300 µM TBOA increased Ihold (54 ± 13 pA; n = 9 cells), and this increase could be completely reversed by addition of 50 µM APV (Fig. 1C). Thus, blockade of glutamate transporters by TBOA increases basal glutamate concentration in the MNTB and can be detected by NMDA receptor activation, as an increase in holding current.
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Figure 1. Glutamate transporter blockade by TBOA elevates extracellular glutamate concentration, detectable by NMDA receptors in Mg2+-free saline. A, Sample traces showing inhibition of EPSC by application of TBOA in 0 Mg2+ saline. B, Expanded timescale of sweeps in A, illustrating similar level of inhibition of NMDA and AMPA receptor-mediated EPSCs by TBOA. Note that addition of 50 µM APV abolished NMDA receptor current but had no effect on peak AMPA current. C, Bath application of TBOA resulted in a reliable increase in Ihold at low-frequency stimulation (0.05 Hz) and could be reversed by coapplication of 50 µM APV.
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In normal saline (1 mM Mg2+, 2 mM Ca2+), 200 µM TBOA strongly inhibited EPSCs in P8–P10 rats (49 ± 6% inhibition; n = 8; p < 0.0001) (Fig. 2A) but had little effect on transmission at P16–P18 (9 ± 7% inhibition; n = 5; p = 0.249) (Fig. 2B). At this concentration of TBOA, previous studies predict that 83–97% of all transporters should be blocked (Shimamoto et al., 1998). The inhibitory effect of TBOA in young animals could be reversed by washout (data not shown). Addition of 1 µM 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495), a selective antagonist for mGluR2/3 and mGluR8, also reversed the inhibitory effect of TBOA (Fig. 2A). This result indicates that inhibition was likely attributable to endogenous elevation of glutamate concentrations in the slice and activation of mGluR receptors at the calyx of Held. Application of LY341495 alone had a small but significant effect on EPSC amplitude in P8–P10 MNTB (107 ± 2%; n = 11; p = 0.011) and no effect by P16–P18 (104 ± 3%; n = 7; p = 0.303), indicating there may be a small endogenous activation of group II/III mGluRs in the young MNTB.
The inhibitory effect of TBOA could be attributable to direct activation of mGluRs because of the relatively high concentrations used. Previous studies showed that 100 µM TBOA did not directly activate mGluR1 or mGluR5 expressed in CHO cells (Shimamoto et al., 2004). Another study excluded 200 µM TBOA from direct mGluR activation in cerebellum Purkinje cells (Takayasu et al., 2004). However, there have been no studies directly assessing the possibility that TBOA can act as a ligand for group II/III mGluRs. We therefore used a structurally different glutamate transporter blocker to study mGluR activation at the calyx of Held synapse. Dihydrokainate (DHK) is a kainate derivative, as opposed to TBOA, which is an aspartate derivative. Application of 800 µM DHK significantly inhibited AMPAR EPSC amplitude in young (P8–P10) MNTB (27.1 ± 6.3% inhibition; n = 4; p = 0.0229) and was reversed by 1 µM LY341495 (10.7 ± 10.8% residual inhibition; n = 3; p = 0.3325). This result indicates that glial transporter blockade by TBOA or DHK likely acts to raise the extracellular glutamate concentration. A similar conclusion was also reached using L-trans-pyrrolidine-2,4-dicarboxylic acid (a transportable glutamate transporter blocker) on P8–P10 rats (Taschenberger and von Gersdorff, 2001). These results suggest that transporter block indirectly activates mGluRs in young (P8–P10) but not older (P16–P18) brainstem by increasing interstitial concentrations of glutamate throughout the slice.
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Figure 2. TBOA inhibited EPSC amplitude in immature (P8–P10) but not older (P16–P18) calyx of Held synapses. Ai, TBOA inhibited EPSCs in young (P10) calyx of Held synapses. Cells were stimulated at 0.1 Hz, and 200 µM TBOA was added to the bath solution when indicated. This effect was completely reversible by 1 µM LY341495, an antagonist of group II (mGluR2/3) and mGluR8 receptors. Aii, Sample EPSCs (average of 5 sweeps taken at the points indicated in Ai) show robust inhibition of EPSC peak amplitude. Bi, Bii, An example of a similar experiment as in A, performed at P16, showed no response to TBOA.
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Multiple trains of stimulation were delivered at 10 Hz with a 30 s interval between sweeps, to assess the effect of TBOA on repeated synaptic responses. EPSCs normally depress quickly during high-frequency stimulation in the P8–P10 MNTB (von Gersdorff et al., 1997). Stimulation at 10 Hz does not cause desensitization in control conditions of P8–P10 synapses in the MNTB (Wong et al., 2003) but does depress the EPSC by 70% before reaching a steady state of 31 ± 3% of the initial EPSC by 1 s (n = 30) (Fig. 3A,E). This depression recovers within 20 s and has a small contribution (10%) from presynaptic group III mGluRs (von Gersdorff et al., 1997). Application of 200 µM TBOA depressed the first EPSC and significantly reduced pairedpulse depression by 20% (EPSC2/EPSC1, 51 ± 6% in control; 79 ± 10% in TBOA; n = 8; p = 0.016) (Fig. 3E). This effect on paired-pulse depression is a common indicator of a presynaptic locus of inhibition (Zucker and Regehr, 2002). Furthermore, TBOA significantly relieved steady-state depression (44 ± 5% of initial EPSC amplitude; n = 8; p = 0.039, steady state vs control), as expected if enhanced presynaptic inhibitory mGluR activation in the presence of this drug reduced release probability (Fig. 3E). Presynaptic inhibition by 1 µM (S)-3,4-dicarboxyphenylglycine (DCPG), an agonist for mGluR8, also reduced the initial EPSC by 50% and also significantly relieved steady-state depression relative to the first stimulus (DCPG steady state, 50.91 ± 9% of initial EPSC amplitude; p = 0.0312 vs control; n = 6) (Fig. 3E). DCPG reduced the relative degree of synaptic depression from 70 to 50% probably by reducing synaptic vesicle pool depletion and ICa2+ inactivation (Xu and Wu, 2005). Additionally, these effects could be fully reversed to control values by 1 µM LY341495, supporting presynaptic activation of mGluRs by TBOA-induced glutamate accumulation in the brainstem slice. Note that glutamate increases within the synaptic cleft would presumably lead to AMPA receptor desensitization and would not be reversed by LY341495. LY341495 at 1–5 µM alone had no significant effect on the steady-state EPSC at 10 Hz (28 ± 7% of control; n = 5; p = 0.8125 vs control; data not shown). This result may indicate that mGluR4, but not mGluR2/3 and mGluR8, are activated by synaptically released glutamate (see below).
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Figure 3. Blockade of glutamate uptake relieved depression in P8–P10 synapses. A, Application of TBOA considerably reduced the peak EPSC amplitude throughout a 10 Hz train in the MNTB of a P10 rat. Samples shown for each treatment are averages of five sweeps taken 30 s apart. Inset shows first and last EPSC. Calibration:2 nA, 1ms. Calibration is the same for all insets in A and B. LY, LY341495. B, At P16, there was no effect of TBOA on EPSC size or kinetics of transmission at 10Hz. The sample shown is an average of five sweeps for each treatment. Ci, The first EPSC in P8–P10 MNTB showed negligible slowing of EPSC decay in the presence of 200µMTBOA. Decay in young rats was well fit by a double exponential, corresponding to fast and slow components of decay. Addition of 200 µM TBOA did not affect kinetics of fast or slow phases of AMPAR decay versus control. Cii, The 20th pulse in a 10 Hz train was also unaffected by 200µMTBOA. Di, Dii, EPSC decay in older MNTB was well fit by a single exponential, comparable with fast at P8–P10. Neither the first nor the 20th EPSC in P16–P18 (from B) showed any effect of TBOA on kinetics of fast transmission. Arrows point to noncalyceal inputs that were often present in older animals. E, Summary of cells at P8–P10, normalized to peak amplitude of the first EPSC in a train. Control synapses reached a steady state of 30% by 1 s (10 stimuli). Application of 200 µM TBOA significantly reduced depression of the second pulse and resulted in a striking relief from steady-state depression. DCPG, an mGluR8 agonist, similarly reduced depression and increased the steady state. The inhibitory effect of TBOA was fully reversible to control values by 1 µM LY341495, an antagonist for group II mGluRs and mGluR8. F, At P16–P18, there was no effect of TBOA or DCPG on synaptic depression, although the synapse showed less depression overall compared with younger synapses.
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By P16, there is little or no effect of the transporter blocker (TBOA) on EPSCs during a 10 Hz train on either initial or steady-state amplitude (Fig. 3B,F). This lack of effect suggests that the increase in extracellular glutamate concentration does not activate additional AMPA receptors in the synaptic cleft and is also insufficient to desensitize AMPA receptors in the MNTB. Thus, transporters do not appear to play a prominent role in glutamate clearance during 10 Hz trains. Passive diffusion may therefore suffice to terminate the synaptic glutamatergic transient at the functionally mature calyx of Held synapse.
Dissecting the identity of mGluRs activated by extrasynaptic glutamate
All three classes of mGluRs are present at the MNTB, and all have been shown to inhibit EPSCs (Barnes-Davies and Forsythe, 1995; Takahashi et al., 1996; von Gersdorff et al., 1997; Elezgarai et al., 1999, 2001; Kushmerick et al., 2004). Which mGluR subtype is activated after transporter block by TBOA?
It is unlikely that group I mGluRs are involved in the observed TBOA effect, because 1 µM LY341495 should not significantly block their activation. In non-neuronal cells expressing mGluRs, LY341495 has been shown effective at blocking group I mGluR activation by quisqualate, with IC50 of 7–8 µM (Kingston et al., 1998). Other studies have used 20–100 µM LY341495 as a broad-spectrum mGluR antagonist (Watabe et al., 2002; Howson and Jane, 2003; Palhalmi et al., 2004). However, in the MNTB, only 200 µM LY341495 was capable of blocking activation by 100 µM DHPG, a group I mGluR agonist (Kushmerick et al., 2004).
Group III mGluRs (mGluR4, mGluR7, and mGluR8) are present presynaptically at the young calyx of Held (Barnes-Davies and Forsythe, 1995; Takahashi et al., 1996; Leão and von Gersdorff, 2002). mGluR4 and mGluR8 are activated by 50 µM L-2-amino-4-phosphonobutyrate (L-AP-4), resulting in inhibition of presynaptic Ca2+ current (Takahashi et al., 1996) and EPSC (Barnes-Davies and Forsythe, 1995). Antibody staining for mGluR4a revealed that it is developmentally regulated at the rat MNTB, with levels peaking at 9 d after birth and then falling off dramatically by P14 (Elezgarai et al., 1999). No mGluR7 protein was detected in the MNTB during this time (Elezgarai et al., 1999), and it is doubtful whether the protein is expressed in the adult MNTB (Kinoshita et al., 1998). Expression of mGluR8 mRNA or protein has not been reported in the MNTB, but this nucleus has not been looked at specifically in previous studies (Duvoisin et al., 1995; Kinoshita et al., 1996; Saugstad et al., 1997; Wada et al., 1998).
Until recently, there were no pharmacological tools capable of distinguishing between group III mGluR subtypes, specifically mGluR4 and mGluR8 (Schoepp et al., 1999). The development of DCPG, an agonist specific for mGluR8 over mGluR4 or mGluR7 (Thomas et al., 2001), allows a pharmacological discrimination of these receptors. In addition, the potent group II antagonist LY341495 has unique properties that result in selective potency for mGluR8 over mGluR4 or mGluR7 and thus can aid in further elucidating the physiological role of these group III mGluR receptors (Kingston et al., 1998). We applied these drugs to slices containing MNTB to dissect the presence and contribution of group III mGluR subtypes to EPSC inhibition (Fig. 4).
Application of DCPG resulted in up to 80% inhibition of AMPAR EPSCs in P8–P10 MNTB (Fig. 4A). We began to see significant inhibition at 100 nM DCPG (Fig. 4A–C), suggesting mGluR8 activation. The dose–response curve described by DCPG inhibition was fitted well by a sigmoid curve (R2 = 0.939) (Fig. 4C, black line); however, the fit returned an EC50 of 393 nM, a much higher value than that reported in transfected cell lines expressing only mGluR8 or from spinal cord (EC50 of 31 nM) (Thomas et al., 2001). This may be attributable to mGluR4 expression in MNTB at this age, supported by immunohistochemistry (Elezgarai et al., 1999), which would be activated by higher concentrations of DCPG (EC50 of 8.8µM for mGluR4) (Thomas et al., 2001). Thus, the affinity data from transfected cell culture experiments together with the data from the present work suggest independent contributions from both mGluR4 and mGluR8 at this synapse.
Using the maximal inhibition attributable to mGluR4 alone (60.6% in 50 µM L-AP-4 and 1 µM LY341495) (Fig. 5) and the EC50 of mGluR4 for DCPG reported by Thomas et al. (2001), we calculated a hypothetical dose–response curve to DCPG in the MNTB by mGluR4 alone (Fig. 4C, gray trace) (see Materials and Methods). This curve shows a negligible contribution to EPSC inhibition by mGluR4 at concentrations 1 µM. We then calculated a similar hypothetical curve for mGluR8, constraining the equation with the reported EC50 from transfected cells, and used the difference in DCPG inhibition at 1 µM and the hypothetical mGluR4 curve to determine maximum inhibition from mGluR8 alone (Fig. 4C, hatched trace). At high concentrations of DCPG, the effects of these two receptors do not linearly sum. This is likely attributable to saturation of the presynaptic inhibitory machinery (Leão and von Gersdorff, 2002). However, this result suggests that DCPG can effectively be used at 1 µM to discriminate between mGluR4 and mGluR8 at a mixed receptor synapse.
Similarly, we investigated the ability of low doses of LY341495 to inhibit mGluR8 activation by DCPG in the MNTB (Fig. 4D,E). Nanomolar concentrations of LY341495 strongly antagonized 1 µM DCPG-mediated inhibition, and LY341495 fully blocked inhibition at concentrations 1 µM (Fig. 4D). Figure 4E shows a dose–response curve for LY341495 antagonism of 1 µM DCPG. Curve fit returned an IC50 of 16.1 nM (R2 = 0.990; see Materials and Methods), consistent with the reported affinity of LY341495 for mGluR8 in spinal cord (14–57 nM) (Howson and Jane, 2003) and much less than that reported for mGluR4 (22 µM) (Kingston et al., 1998). Thus, low micromolar concentrations of LY341495 can be used to completely and specifically block mGluR8-mediated inhibition of transmission in the MNTB.
In addition, we used antibodies specific for mGluR8 to localize the receptor at the calyx of Held in P8–P10 rats (Fig. 4F). Signal for mGluR8 was restricted to the calyx terminal and was not present in the axon at this age. By comparison, the synaptic vesicle-associated protein Rab3a signal is strong in the terminal and is also present in the axon for some distance (20 µm). Minimal cross-reactivity or nonspecific staining of secondary antibodies was observed at the MNTB in control experiments (data not shown).
Group II mGluRs (mGluR2 and mGluR3) have also been localized in the MNTB via immunohistochemistry and are reported to be located in the presynaptic calyx membrane, apposed to glial cells and also occasionally facing the postsynaptic principal cell (Elezgarai et al., 2001). Previous reports on the physiology of these receptors have used 50 µM (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) or 5 µM (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) as agonists for group II mGluRs at the MNTB and reported EPSC inhibition by 40–50% (von Gersdorff et al., 1997). However, neither of these agonists is clearly specific for group II mGluRs; ACPD at 50 µM can also activate group I and III mGluRs (Cartmell and Schoepp, 2000), and DCG-IV could also activate NMDA receptors (Breakwell et al., 1997). We used instead the agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC), which shows specificity for group II mGluRs over all other mGluRs by almost two orders of magnitude (Cartmell and Schoepp, 2000). APDC (50 µM) reversibly inhibited the AMPAR EPSC peak amplitude by 55% (Figs. 5, 6, Table 1). The level of inhibition by group II mGluRs is similar to that seen by 200 µM TBOA. Activation of the high-affinity group II mGluRs may thus account for the majority of the effect on EPSCs of transporter blockade by TBOA.
The effect of group II activation by APDC (50 µM) did not occlude the effect of group III mGluRs, because DCPG (10 µM) further inhibited the EPSC amplitude (Fig. 5A). Likewise, DCPG (1 µM) did not occlude the effect of transporter block by TBOA (200 µM) (Fig. 5B). However, APDC (50 µM) did occlude a majority of the inhibitory effect of TBOA, indicating that transporter block may preferentially activate group II mGluRs (n = 4 cells) (Fig. 5C). There was some additional inhibition of the EPSC by TBOA, likely attributable to mGluR8 activation. In summary, these results suggest that increased glutamate concentrations attributable to transporter blockade predominately activate group II (subtype 2/3) mGluRs and also may activate mGluR8 at the calyx of Held but has no detectable effect on group I mGluR-mediated modulation.
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Figure 4. DCPG-mediated inhibition is attributable to both mGluR8 and mGluR4 at the rat MNTB in vitro. Peak EPSC amplitude attributable to a nondepressing (0.1 Hz) stimulation protocol was challenged by various concentrations of DCPG. A, Example of data collected showing incremental and reversible inhibition of EPSC by DCPG. Drug was applied as indicated by bars, and EPSC amplitude was monitored at 0.1 Hz. Submicromolar concentrations of DCPG resulted in inhibition and was maximal by 1 µM. B, Sample EPSCs taken at the times (A–E) shown in A. Increasing DCPG concentration resulted in greater inhibition of EPSC amplitude, without affecting kinetics. C, The dose–response curve described by DCPG was well fit by a sigmoidal curve. n = 3–8 at each concentration. Using the EC50 values reported from expression systems for mGluR4 or mGluR8, we overlaid the predicted EPSC inhibition attributable to DCPG activation of mGluR4 (gray trace) and mGluR8 (hatched trace) (see Results). At concentrations <1 µM, there is contribution to DCPG-mediated inhibition exclusively by mGluR8. D, Example trace showing concentration dependence of LY341495 (LY) relief of DCPG inhibition. EPSCs were stimulated at 0.1 Hz, and peak amplitude was measured. Addition of 1 µM DCPG inhibited EPSCs by 50% in P9 rat MNTB slice. This effect was fully reversible by 100 nM or 1 µM LY341495. LY341495 blocked mGluR8 activation at low micromolar concentration. E, Summary data were well fit by a sigmoid curve. n = 3–4 at each concentration. The open symbol indicates inhibition by 1 µM DCPG alone. F, Immunoreactivity for mGluR8 was located presynaptically at the developing calyx of Held. The calyx from a P10 rat was labeled with antibodies against Rab3a (left panel, green), a synaptic vesicle associated protein. Arrows show calyceal structures in the MNTB. Note that Rab3a is also present in the axon. Immunoreactivity to mGluR8 is shown in the middle panel (red) and colocalized entirely with Rab3a immunoreactivity in the calyx (Merge, especially arrows).
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Figure 5. Occlusion experiments between mGluRs and TBOA (glutamate transporter antagonist): inhibitory mechanisms in the MNTB of P8–P10 rats are not saturated by APDC (group II mGluR agonist) or DCPG (mGluR8 agonist). A, The group II mGluR agonist APDC (50µM) inhibits EPSC peak amplitude but does not occlude activation of mGluR8 by DCPG (10 µM) and is fully blocked by 200 nM LY341495 (LY). B, Application of 1 µM DCPG, an mGluR8 agonist, does not occlude inhibition attributable to transporter blockade by 200 µM TBOA. The effect of both DCPG and TBOA can be fully reversed by 1µM LY341495. C, Group II activation via 50 µM APDC occludes most of the inhibitory effect of 200 µM TBOA.
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Developmental loss of mGluRs at the calyx of Held
We determined the presence of group II and group III mGluRs in the MNTB during postnatal maturation of the calyx of Held synapse between P5 and P18 to see whether the loss of inhibition attributable to transporter block by TBOA could be correlated with a loss of mGluR activity (Fig. 6). There are multiple inhibitory G-protein-coupled receptors (GPCRs) present presynaptically at the calyx of Held, linked to Gi/o activation (Barnes-Davies and Forsythe, 1995; Takahashi et al., 1996, 1998; Isaacson, 1998; Leão and von Gersdorff, 2002; Kimura et al., 2003). Previous studies have shown strong inhibition of the EPSC by group II and group III mGluRs at the MNTB, up to age P14 (Barnes-Davies and Forsythe, 1995; von Gersdorff et al., 1997; Leão and von Gersdorff, 2002). Activation of group II mGluRs with 50 µM APDC resulted in >50% inhibition but disappeared by P14 (Fig. 6, Table 1). Activation of either mGluR4 or mGluR8 (50 µML-AP-4 plus 1 µM LY341495, or 1 µM DCPG, respectively) also resulted in >50% inhibition in P5–P7 and P8–P10. Simultaneous activation of both receptors by 50 µM L-AP-4 inhibited transmission by >80%, less than that expected by additive inhibition. Because mGluR4 and mGluR8 likely share the same second-messenger pathway (for review, see Cartmell and Schoepp, 2000), it is possible that we may be saturating the presynaptic inhibitory mechanisms present at the calyx with 50 µML-AP-4. After the onset of hearing (P12–P14) (Blatchley et al., 1987), the effect of mGluR4 or mGluR8 activation was greatly reduced to 25% for both receptors (Fig. 6C). At this age, activation of both mGluRs (by 50 µM L-AP-4) results in 50% inhibition, equivalent to summation of an independent effect of the two receptors. Application of 1 mM L-AP-4, which activates mGluR7 in addition to mGluR4 and mGluR8, did not have any additional inhibitory effect on transmission, indicating that mGluR7 is unlikely to be expressed in the MNTB at this age. By P16–P18, only activation of mGluR4 and mGluR8 in combination (50 µM L-AP-4) resulted in significant inhibition (Fig. 6B), and this was considerably less than at earlier ages (Fig. 6C, Table 1). However, similar to the result observed in younger animals, mGluR7 did not contribute significantly to synaptic inhibition at P16–P18. The developmental loss of functional group III mGluRs was also seen in the mouse MNTB, with a slightly accelerated time course (supplemental data, available at www.jneurosci.org as supplemental material). Even a large dose, L-AP-4 (100 µM) had no inhibitory effect at a P18 mouse calyx of Held EPSC, suggesting a complete absence of group III mGluRs at this age.
GABAB receptors, also Go-coupled metabotropic receptors, are present in the mouse and rat MNTB throughout the first 2 weeks of development (Takahashi et al., 1998). In contrast to mGluRs, activation of these receptors with baclofen (20 µM) resulted in strong inhibition of excitatory transmission in rats and mice throughout the developmental time period studied here (Fig. 6, Table 1) (supplemental data, available at www.jneurosci.org as supplemental material). However, note that the effect of baclofen was reduced by one-half at P18 compared with P9–P10 mouse slices (supplemental data, available at www.jneurosci.org as supplemental material). This indicates that the transduction cascade, which is shared between these two classes of metabotropic receptors, has not been disrupted during the course of postnatal development.
mGluR8 directly inhibits Ca2+ influx and exocytosis
The occlusion experiments in Figure 5 indicated that mGluR8 was only mildly activated by TBOA. We used voltage-clamp recordings from the calyx of Held to determine the magnitude and precise mechanism of inhibition by mGluR8. Recordings were made in P8–P10 calyces, in which DCPG exerts a very strong effect, and presynaptic recordings are more feasible (Borst and Sakmann, 1996). To independently confirm that mGluR8 activation inhibits synaptic transmission, we recorded directly from the presynaptic terminal to examine the effect of 1 µM DCPG on synaptic vesicle exocytosis (Fig. 7A). Brief depolarizations (to 0 mV, 2 ms) resulted in fast, reliable increases (jumps) in Cm relative to baseline, representing the addition of synaptic vesicle membrane to the terminal via exocytosis (see Materials and Methods). Application of 1 µM DCPG caused a significant 38 ± 0.1% decrease in capacitance jump (control, 88 ± 16 fF; DCPG, 57 ± 16 fF; p = 0.0312 vs control; n = 6) and was reversed by 1µM LY341495 (DCPG plus LY341495, 126 ± 33 fF). Capacitance jumps were measured 500–700 ms after the end of the depolarization (Fig. 7A, gray bar) to avoid contamination by Ca2+-dependent conductances (Yamashita et al., 2005). Summarized results from serial application of DCPG and LY341495 are shown in Figure 7B and show that capacitance is significantly decreased by activation of mGluR8. The capacitance jump amplitude after a 2 ms depolarization to 0 mV is consistent with previous experiments (Sun and Wu, 2001; Taschenberger et al., 2002). Inhibition of capacitance by DCPG was similar at 2 or 5 ms depolarization to 0 mV (data not shown). This result shows that presynaptic activation of mGluR8 acts to decrease exocytosis and glutamate release and explains the effect of DCPG on EPSCs.
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Figure 6. Group II and group III mGluR-mediated inhibition was strikingly reduced in morphologically mature MNTB synapses (P16–P18). Addition of group II agonists and combinations of group III mGluR agonists and subtype-specific antagonists showed robust and reversible inhibitory effects on peak EPSC amplitude in young (P8–P10) but not older (P16–P18) MNTB synapses. APDC, APDC at 50 µM, specific for group II mGluRs. L-AP-4,L-AP-4 at 50 µM, specific form GluR4 and mGluR8. LY, LY341495 at 5 µM, a group II- and mGluR8-specific antagonist at this concentration; thus, L-AP-4 plus LY341495 is specific for mGluR4 activation. DCPG, DCPG at 10µM, an mGluR8-specific agonist. Baclofen, Baclofen at 20µM, an agonist for GABAB, is shown here to compare differentially coupled Gi/o-dependent inhibitory mechanisms and illustrate that the GPCR mechanism has not rundown over the course of the recording. A, An example recording from the MNTB of a P7 rat. Many Gi/o-coupled GPCRs are present: group II, mGluR4, mGluR8, and GABAB all cause dramatic inhibition. This inhibition was, in all cases, reversible. There was no apparent endogenous activation of mGluR8 because 5µM LY341495 did not significantly increase the EPSC over that of control amplitude; however, some run-up of the size of the response was present on occasion. Timescale is shared between A and B. B, By P16, the MNTB was essentially resistant to group II and group III mGluR agonists. Inhibition attributable to GABAB activation was still very strong and reversible, indicating that the GPCRGi/opath way is still in tact at this age. C, Inhibition of synaptic transmission by group II and group III mGluRs is developmentally regulated. Combinations of specific agonists and antagonists were used to pharmacologically dissect the presence and contribution of group III mGluRs to synaptic inhibition at the rat MNTB throughout postnatal development (see also Table 1). Amplitudes of EPSCs were recorded at 0.1 Hz and are shown relative to control amplitude.
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Brief depolarizations of the presynaptic terminal (2 or 5 ms from –80 to 0 mV, 30 s apart) result in influx of Ca2+; we used the integral of the current to measure charge transfer across the membrane (2 ms; control, 1.46 ± 0.11 pC; n = 8) (Fig. 7C,D). Ca2+ current responses were very stable between sweeps, indicating that the interstimulus interval was long enough to buffer intraterminal [Ca2+]i to baseline levels (Borst and Sakmann, 1998). Takahashi et al. (1996) showed that activation of group III mGluRs (by 50 µM L-AP-4) in the MNTB inhibits presynaptic Ca2+ currents. As shown in Figure 7, C and D, the Ca2+ current was also significantly inhibited by 1 µM DCPG (1.07 ± 0.08 pC; p < 0.01 vs control), indicating that mGluR8 is acting presynaptically to inhibit Ca2+ influx. Similar to capacitance results, this inhibition could be fully recovered by the addition of 1 µM LY341495 (1.34 ± 0.1 pC). The Ca2+ flux attributable to 2 ms depolarization was inhibited by 26 ± 5% (Fig. 7D), similar to that seen for application of 50 µM L-AP-4 (25 ± 4%) (Takahashi et al., 1996). Consistent with reports using L-AP-4, the inhibitory effect of DCPG was similar for 2 or 5 ms depolarization and did not alter the Ca2+ channel current–voltage relationship (data not shown).
A double-pulse depolarization protocol showed that the mechanism of inhibition by DCPG is attributable to direct interaction of G-protein  subunits with Ca2+ channels (Fig. 7C). Prepulse depolarization to +100 mV has been shown to alleviate inhibition of Ca2+ channels by subunits (Bean, 1989; Kajikawa et al., 2001; Leão and von Gersdorff, 2002). A test depolarization (0 mV, 2 ms; first pulse) was followed 50 ms later by "prepulse" depolarization (+100 mV, 10 ms) and then a second test depolarization (0 mV, 2 ms; second pulse) 10 ms later. Consistent with a -dependent mechanism of action, strong depolarization relieved the inhibition of ICa2+ by DCPG (Fig. 7C,D). This prepulse depolarization did not change the relative current in control cells or after addition of 1 µM LY341495 (p = 0.65 vs control first pulse), indicating that mGluR8 is likely not endogenously activated. These results suggest that mGluR8 is an inhibitory mGluR receptor present in the calyx of Held, and mGluR8 activation acts directly through subunits to inhibit Ca2+ influx.
TBOA indirectly inhibits presynaptic Ca2+ current
We have shown that the transporter blocker TBOA (200 µM) inhibits excitatory transmission in the immature MNTB, which can be reversed by an mGluR antagonist (Figs. 1, 2, 3). The reversal by 1 µM LY341495 is indicative of presynaptic group II and mGluR8 receptors, which disappear with a similar time course as the loss of TBOA effect (Figs. 4, 5, 6), and mGluR activation can fully explain the inhibitory effect of TBOA on transmission. To test this hypothesis further, we recorded presynaptic Ca2+ currents directly from the presynaptic terminal in the presence of 200 µM TBOA (Fig. 7E). Blockade of transporters resulted in a small but significant decrease in Ca2+ current, inhibiting Ca2+ influx by 12% (control, 844 ± 55 fC; TBOA, 746 ± 60 fC; p = 0.039; n = 8) (Fig. 7F), which could then be reversed by 1 µM LY341495 to control amplitude (851 ± 13 fC). The effect of TBOA on Ca2+ current is relatively small. However, because of the power relationship of 3–4 on exocytosis, this inhibition is sufficient to explain EPSC inhibition (Schneggenburger et al., 1999). A 12% decrease in Ca2+ influx thus results in 32–40% inhibition of exocytosis, roughly similar to the 50% inhibition of EPSC by TBOA. Therefore, this result can account for the inhibitory effect of TBOA on EPSCs, via activation of presynaptic mGluRs.
TBOA does not affect the EPSC kinetics
The rate of glutamate clearance from CNS synapses is often a critical determinant of the EPSC. In addition, glutamate transporters help limit the amount of mGluR activation and provide a buffer between adjacent neurons to allow independent processing by nearby synapses (for review, see Bergles et al., 1999; Huang and Bergles, 2004). We thus wanted to know whether transporters aid in the termination of fast EPSCs at the calyx of Held, making it well suited for high-frequency transmission. We first examined the effect of transporter blockade on the decay kinetics of EPSCs when mGluRs were blocked by 1 µM LY341495 and also when AMPA receptor desensitization was additionally blocked by a low-affinity antagonist.
Previous reports showed that the decay kinetics of fast AMPAR EPSCs at RT in the MNTB are well fit by a double exponential in young animals [fast 1 ms, slow  2–4 ms (Barnes-Davies and Forsythe, 1995; Chuhma and Ohmori, 1998; Taschenberger and von Gersdorff, 2000)] but better fit by a single fast exponential in older animals (P12–P14) (Taschenberger and von Gersdorff, 2000; Iwasaki and Takahashi, 2001). In our hands, EPSCs from P8–P10 animals showed similar fast and slow components (fast = 0.64 ± 0.09 ms, carried 77 ± 4%% of decay; slow = 3.07 ± 0.46; n = 11). By P16–P18, >98% of a double-exponential fit in control was attributable to the fast decay constant, so only single-exponential values are reported ( = 0.29 ± 0.02 ms; n = 12). There was no effect of 1 µM LY341495 on EPSC kinetics at either age (n = 3). Surprisingly, we saw no effect of nearly complete transporter blockade (200 µM TBOA) on the decay kinetics of fast glutamatergic EPSCs, in young or older synapses (Fig. 3C,D). Neither fast nor slow components of EPSC decay were affected (n = 10 cells at P8–P10; n = 5 at P16–P18).
At physiological temperature (35–36°C), we still did not see any effect of the transporter blocker (TBOA) on decay kinetics or EPSC amplitude in the presence of 1 µM LY341495 (data not shown; n = 3 at P8–P10; n = 4 at P16–P18). These results indicate that glutamate transporters have little or no role in shaping the synaptic waveform and that AMPA receptor kinetics plus diffusion and/or receptor desensitization likely sculpt the EPSC waveform, in both young and older calyceal synapses of the MNTB.
AMPA receptor desensitization in the MNTB
At some auditory synapses, postsynaptic receptor desensitization aids in determining the shape and amplitude of EPSCs (Trussell et al., 1993; Otis et al., 1996b). Several groups have used the low-affinity competitive AMPA antagonist -D-glutamylglycine (-DGG) to effectively block receptor desensitization and saturation (Wadiche and Jahr, 2001; Wong et al., 2003; Takayasu et al., 2004). In particular, modeling studies at the calyx of Held have confirmed that 4 mM -DGG is effective at blocking AMPA receptor desensitization (Wong et al., 2003). The EPSC rise time is slowed because of displacement of -DGG by synaptically released glutamate (Wadiche and Jahr, 2001), but it is unclear how the antagonist affects decay rates. We used 4 mM -DGG to block AMPA receptor desensitization and examined the effect of transporter blockade on the resulting EPSCs in young and older animals.
Addition of -DGG reversibly reduced the EPSC amplitude to 10% of control, in both young and old animals (P8–P10, 87 ± 3% inhibition, n = 11; P16–P18, 90 ± 2% inhibition, n = 9). Scaling the EPSCs to peak amplitude reveals a small but significant effect of -DGG on the fast component of EPSC decay kinetics relative to control in both immature (fast = 0.96 ± 0.17 ms; n = 11; p = 0.0137) and older (fast = 0.39 ± 0.04 ms; n = 10; p = 0.0391) synapses (Fig. 8Aiii,Biii). This effect could be attributable to endogenous desensitization of AMPA receptors, especially in younger animals (Joshi et al., 2004). More likely, however, the effect on decay kinetics could be an artifact of the drug used. Competitive binding of AMPA receptors by -DGG and glutamate results in delayed rise time (Wadiche and Jahr, 2001), which would then amplify any inherent asynchronicity of synaptic vesicle release in an EPSC, resulting in prolonged decay. Decreasing the EPSC amplitude with -DGG did not occlude the inhibitory effect of transporter block (by 200 µM TBOA) in P8–P10 animals (Fig. 8A). Addition of TBOA still significantly reduced the EPSC amplitude in P8–P10 (51 ± 6% relative to -DGG; n = 10; p < 0.0001). The inhibitory effect of TBOA could be reversed by blocking mGluR activation (1 µM LY341495; 106 ± 52% of -DGG peak amplitude; n = 9), reinforcing the hypothesis that activation of the mGluRs mGluR2/3/8 are attributable to tonic increases in glutamate concentration. In older animals, transporter block had no effect on EPSC peak amplitude (89 ± 9% relative to -DGG; n = 6; p = 0.2840) (Fig. 8Bi,Bii), consistent with a loss of mGluR expression.
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Figure 7. Presynaptic recordings show that the inhibitory effect of DCPG and TBOA is attributable to reduced presynaptic Ca2+ currents.A,Activation of presynaptic mGluR8 inhibited exocytosis at the calyx of Held. Brief depolarization decreased synaptic exocytosis in the presence of DCPG. Capacitance of the calyceal terminal was monitored using the sine + DC method (see Materials and Methods), and the terminal was depolarized for 2 ms to 0 mV. The resulting capacitance change was measured 500–700 ms after depolarization (gray bar). The depolarizing pulse produced a capacitance jump (black circles). Application of 1 µM DCPG (gray circles) decreased the capacitance jump,and the control response could be fully recovered by the addition of 1 µMLY341495 (open circles). All traces were taken serially from the same cell over the course of 20 min. B, DCPG significantly decreased exocytosis at the calyx of Held. Pooled data from multiple experiments show a significant decrease in exocytosis by 1µM DCPG (*p<0.05 vs control). C, Ca2+ currents were inhibited by mGluR8 via subunits. DCPG at 1 µM (gray trace) reduced Ca2+ influx in the calyx terminal by 25%. This effect was reversible by 1 µM LY341495. Inhibition of Ca2+ channels by mGluR8 was attributable to subunit interaction with voltage-dependent Ca2+ channels,as revealed by double-pulse protocol. Double-pulse protocol and sample trace are shown from P9 rat and are averages of three to five sweeps 20–30 s apart for each treatment. Depolarization prepulse (+100 mV, 10 ms) removed -mediated inhibition and reversed DCPG-induced inhibition completely. DCPG-induced inhibition was recovered to control values by adding 1µM LY341495. D, Summary of double-pulse protocol results in P8–P10MNTB. DCPG(1µM)very significantly inhibited Ca2+ charge transfer (current area during depolarization) during 2 ms depolarization (**p < 0.01). Adding 1µM LY341495 completely reversed inhibition (DCPG + LY). Second pulse resulted in Ca2+ charge transfer that was similar to control in all cases. Transporter blockade inhibited presynaptic Ca2+ current. Ca2+ currents attributable to 2 ms depolarization were recorded from the calyx terminal, as in A. Perfusion of 200 µM TBOA inhibited Ca2+ current and could be relieved by serial addition of 1 µM LY341495, a group I | |
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Abstract
Introduction
