HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 49, 47796-47803, December 6, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/49/47796    most recent M207531200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Millán, C. Articles by Sánchez-Prieto, J. Search for Related Content PubMed PubMed Citation Articles by Millán, C. Articles by Sánchez-Prieto, J. Social Bookmarking                      What's this?

Subtype-specific Expression of Group III Metabotropic Glutamate Receptors and Ca²⁺ Channels in Single Nerve Terminals^*

Carmelo Millán, Rafael Luján^§, Ryuichi Shigemoto^¶, and José Sánchez-Prieto

From the  Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain, the ^§ Centro Regional de Investigaciones Biomédicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Campus de Albacete, 02071 Albacete, Spain, and the ^¶ National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan

Received for publication, July 26, 2002, and in revised form, September 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca²⁺ channel activity. By imaging Ca²⁺ in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca²⁺ channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca²⁺ channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for L(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca²⁺ response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca²⁺ channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca²⁺ channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca²⁺ channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The release properties of presynaptic terminals are determined by factors such as the presence of auto- or heteroreceptors, the subtype of voltage-dependent Ca²⁺ channel coupled to neurotransmitter release at the active zone, and the machinery of synaptic vesicle fusion. Thus, it is possible to observe different release properties among presynaptic terminals even when considering terminals on the same axon as a consequence of a target cell-specific distribution of presynaptic metabotropic glutamate receptors (mGluRs)¹ (1, 2). Group III mGluRs expressed in the brain consist of three different subtypes (mGluR4, mGluR7, and mGluR8) (for review, see Refs. 3 and 4) that are localized within presynaptic active zones (1, 5, 6), where they act as autoreceptors mediating the inhibition of glutamate release (7-10). Although these receptors are also responsible for reducing cAMP levels (11-15), the inhibition of induced glutamate release is primarily mediated by the inhibition of Ca²⁺ channel activity (8, 10, 16-19).

Voltage-dependent Ca²⁺ channels are fundamental components of the presynaptic release machinery whereby glutamate release and hence synaptic strength can be modulated (20). Indeed, a number of electrophysiological studies have shown that the release of glutamate depends primarily on the activity of P/Q- and N-type Ca²⁺ channels (21-25). These same electrophysiological studies have suggested that Ca²⁺ channels exhibit a heterogeneous distribution in glutamatergic nerve terminals, which may express N- or P/Q-type calcium channels or a combination of both. Because the Ca²⁺ channel profile may confer specific release properties to glutamatergic nerve terminals, it is possible that the distinct group III mGluRs are also restricted to subpopulations of nerve terminals depending upon the type or combination of Ca²⁺ channels present. However, due to the small size of the synaptic boutons and because synaptic transmission studies provide average measures of the release from a large number of nerve terminals, the precise distribution of Ca²⁺ channels in single nerve terminals remains largely unknown. It also remains to be established whether presynaptic mGluRs are expressed in terminals endowed with a particular subtype of Ca²⁺ channels.

In this study, by imaging Ca²⁺ in single nerve terminals, we have established the distribution of Ca²⁺ channels and group III mGluRs. In the cerebrocortical preparation of nerve terminals from young rats, two mGluRs exhibiting high and low affinity for L(+)-2-amino-4-phosphonobutyrate (L-AP4) and immunocytochemically identified as mGluR4 and mGluR7, respectively, reduced the entry of Ca²⁺ and the release of glutamate in a different subpopulation of terminals. Interestingly, we found that mGluR4 was primarily located in terminals that coexpress N- and P/Q-type Ca²⁺ channels, whereas mGluR7 was preferentially expressed with N-type Ca²⁺ channels. This distinct distribution of Ca²⁺ channel types in mGluR4- and mGluR7-expressing nerve terminals may account for the terminal-specific modulation of glutamate release through group III mGluRs. These data demonstrate for the first time the selective distribution of two group III mGluRs in a different population of Ca²⁺ channel-containing nerve terminals.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synaptosomal Preparation-- Synaptosomes were purified on discontinuous Percoll gradients (Amersham Biosciences, Uppsala, Sweden) as previously described (26, 27). Briefly, cerebral cortices were isolated from young male Wistar rats (3 weeks old) and homogenized in medium containing 0.32 M sucrose (pH 7.4). The homogenate was centrifuged at 2000 × g for 2 min at 4 °C, and the supernatant was spun again at 9500 × g for 12 min. From the pellets formed, the white loosely compacted layer containing the majority of the synaptosomes was gently resuspended in 8 ml of 0.32 sucrose (pH 7.4). Of this synaptosomal suspension, 2 ml was placed onto a 3-ml Percoll discontinuous gradient containing 0.32 M sucrose; 1 mM EDTA; 0.25 mM DL-dithiothreitol; and 3, 10, or 23% Percoll (pH 7.4). After centrifugation at 25,000 × g for 10 min at 4 °C, the synaptosomes were recovered from between the 10 and 23% Percoll bands and diluted in a final volume of 30 ml of HEPES buffer medium (HBM) containing 140 mM NaCl, 5 mM KCl, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.4). Following centrifugation at 22,000 × g for 10 min, the synaptosomes were resuspended in 8 ml of HBM, and the protein content was determined by the biuret method. Finally, 1 mg of the synaptosomal suspension was diluted in 8 ml of HBM and spun at 3000 × g for 10 min. The supernatant was discarded, and the pellets containing the synaptosomes were stored on ice. Under these conditions, the synaptosomes remained fully viable for at least 4-6 h as judged by the extent of KCl- and 4-aminopyridine-induced glutamate release.

Glutamate Release-- Glutamate release was assayed by on-line fluorometry as described previously (28). Synaptosomal pellets were resuspended in HBM (0.67 mg/ml) and preincubated at 37 °C for 1 h in the presence of 16 µM bovine serum albumin to bind any free fatty acids released from synaptosomes during the preincubation. A 1-ml aliquot was transferred to a stirred cuvette containing 1 mM NADP⁺, 50 units of glutamate dehydrogenase (Sigma), and 1.33 mM CaCl₂ or 200 nM free Ca²⁺, and the fluorescence of NADPH was followed in a PerkinElmer Life Sciences LS-50 luminescence spectrometer at excitation and emission wavelengths of 340 and 460 nm, respectively. Traces were calibrated by addition of 2 nmol of glutamate at the end of each assay. Data points were obtained at 2-s intervals and corrected for Ca²⁺-independent release. Thus, the Ca²⁺-dependent release was calculated by subtracting the release obtained during a 5-min period of depolarization at 200 nM free Ca²⁺ from the release at 1.33 mM CaCl₂.

Ca²⁺ Imaging: Ca²⁺ Responses in Single Synaptosomes-- Synaptosomes in HBM (2 mg/ml) with 16 µM bovine serum albumin were preincubated with 5 µM fura-2/AM and 1.33 mM CaCl₂ for 40 min. The synaptosomal suspension was attached to a polylysine-coated coverslip for 1 h. A superfusion chamber was carved in small Petri dish, which was mounted on a Nikon inverted stage microscope. The medium was fed into the chamber by gravity at 1-1.5 ml/min from a prewarmed (37 °C) reservoir and continuously removed by aspiration. Synaptosomes were illuminated alternately at 340 and 380 nm for 0.8 s through a ×100 objective with the aid of a monochromator (Kinetic Imaging, Ltd.), and the fluorescence emitted from the nerve terminals was collected through a band-pass filter centered at 510 nm. The video images were obtained using a slow-scan CCD camera (Hamamatsu C4880) operating at 12-bit digitalization (4096 levels), and the output from the camera was stored by a computerized imaging system (Kinetic Imaging, Ltd.). Individual synaptosomes were visualized as bright round spots in fluorescence images. Background at F_340 nm and F_380 nm was substracted from each series of F_340 nm and F_380 nm images. A binary mask constructed by local equalization and thresholding of row images was applied to isolate round measuring areas, zeroing pixels outside of the bright spots. Using this, some synaptosomes were not analyzed, but never >5-10%. Ratio images were stored as 32-bit floating point number data. The cytosolic free [Ca²⁺] was derived from the F_340 nm/F_380 nm ratio with the equation derived by Grynkiewicz et al. (29) using Lucida Version 3.0 (Kinetic Imaging, Ltd.). R_max, R_min, and  parameters were determined from in vitro calibration by recording fluorescence from small droplets of fura-2 free acid (Molecular Probes, Inc., Eugene, OR) dissolved in intracellular solution containing 100 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10 mM MOPS, and 10 µM fura-2 (pH 7.0) plus 2 mM CaCl₂ (saturated Ca²⁺) or 2 mM EGTA (0 Ca²⁺). For cytosolic free [Ca²⁺] measurements, synaptosomes were stimulated by 10-s pulses (indicated in each graph by a bar) of 30 mM KCl in the absence and presence of pharmacological agonists or antagonists. Drugs were applied to the nerve terminals by switching the perfusion solution.

Antibody Controls-- The affinity-purified rabbit polyclonal antibody against mGluR7a and the affinity-purified guinea pig polyclonal antibodies against mGluR4a and mGluR8a used here have been described elsewhere (6). The monoclonal anti-synaptophysin antibody was obtained from Sigma. The rabbit polyclonal antibodies against the vesicular glutamate transporters VGLUT1 and VGLUT2 were obtained from Synaptic Systems (Göttingen, Germany). The rabbit polyclonal anti-glutamate decarboxylase (GAD-65) antiserum were purchased from Chemicon International, Inc. (Temecula, CA). To control the immunochemical reaction, primary antibodies were omitted from the staining procedure, whereupon no immunoreactivity resembling that obtained using the specific antibodies could be detected.

Immunohistochemical Methods-- The synaptosomes were allowed to attach to a polylysine-coated coverslip for 1 h and then fixed for 5 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Following several washes with 0.1 M phosphate buffer (pH 7.4), the synaptosomes were preincubated in 10% normal goat serum diluted in Tris-buffered saline (TBS; 50 mM Tris buffer (pH 7.4) containing 0.9% NaCl) with 0.2% Triton X-100 for 1 h. They were then incubated for 24 h with the appropriate primary antiserum diluted in TBS with 1% normal goat serum and 0.2% Triton X-100: mGluR7a (1 µg/ml), mGluR4a (1 µg/ml), mGluR8a (1 µg/ml), VGLUT1 (1:1000), VGLUT2 (1:1000), GAD-65 (1:1000), and synaptophysin (1:1000). After washing with TBS, the synaptosomes were incubated for 2 h with Cy2-coupled goat anti-rabbit or anti-mouse antibody or fluorescein-coupled donkey anti-guinea pig antibody. The secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were diluted 1:200 in TBS. After several washes with TBS, the coverslips were mounted with Fluoromount (Serva), and the synaptosomes were viewed with a Nikon Diaphot microscope equipped with a ×100 objective, a mercury lamp light source, and fluorescein-rhodamine Nikon filter sets.

Chemicals-- -Conotoxin GVIA (-CgTx) and -agatoxin IVA (-AgaTx) were supplied by Peptide Institute, Inc. (Osaka, Japan). L-AP4 was from Tocris Cookson (Bristol, UK). Fura-2/AM was from Molecular Probes, Inc. NADP⁺ and all other reagents were from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Responses to L-AP4 in Cerebrocortical Nerve Terminals from Young Rats Are Mediated by mGluR4 and mGluR7-- The depolarization elicited with 30 mM KCl in cerebrocortical nerve terminals from young rats (3 weeks old) induced a Ca²⁺-dependent release of 1.58 ± 0.02 nmol of glutamate/mg ± S.E. (n = 10). This release was reduced by 22.2 ± 1.4% (n = 8) by the group III mGluR agonist L-AP4 at 20 µM and by 46.3 ± 1.3% (n = 8) when the agonist concentration was increased to 1 mM (Fig. 1A). The inhibition of glutamate release by L-AP4 followed a biphasic dose-response curve, with EC₅₀ values of 1.37 and 371 µM, respectively. These data suggest that at least two mGluRs are involved in the inhibition of glutamate release in this preparation, one with a high and the second with a low affinity for L-AP4 (Fig. 1B).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   mGluRs with high and low affinity for L-AP4 inhibit glutamate release in cerebrocortical nerve terminals. A, inhibition of glutamate release by low and high concentrations of L-AP4. The Ca2+-dependent release of glutamate was induced by 30 mM KCl in the absence (Control; n = 10) and presence (n = 8) of 20 µM and 1 mM L-AP4, added 30 s before depolarization of the nerve terminals. B, concentration-response curve of L-AP4-induced inhibition of glutamate release (n = 3). The mGluR agonist was added 30 s prior to depolarization of nerve terminals with 30 mM KCl. Data are means ± S.E. n indicates the number of data averaged and the number of nerve terminal preparations. div, division.

Because group III mGluR-mediated inhibition of induced glutamate release is the consequence of a reduction in Ca²⁺ channel activity (8, 10, 17, 19), Ca²⁺ imaging experiments were performed to determine which subpopulation of nerve terminals expresses these receptors. Immobilized fura-2-loaded nerve terminals were used to determine the impact of activating group III mGluRs with respect to calcium influx induced by depolarization at the level of a single nerve terminal. A total of 2180 fura-2-loaded nerve terminals that responded to KCl were analyzed from five separate fields similar to that shown in Fig. 2A. Although the basal cytosolic free [Ca²⁺] was 78.1 ± 3.0 nM (n = 60), the responses to 10 s of depolarization with 30 mM KCl were transient, ranging from 500 to 800 nM. Nerve terminals expressing mGluRs with high and low affinity for L-AP4 were distinguished by their responses to applications of 20 µM and then 1 mM L-AP4 in the presence of 30 mM KCl. A subpopulation of nerve terminals (24.4 ± 1.2%) responded to 20 µM L-AP4 with a strong reduction of the Ca²⁺ response (Fig. 2B), whereas another subpopulation (30.3 ± 1.7%) responded only to the application of 1 mM L-AP4 (Fig. 2C). The viability of the preparation was assessed by further addition of 30 mM KCl, which elicited a Ca²⁺ response comparable to that produced by the first depolarization in the absence of L-AP4. In addition, in 45.3 ± 1.0% of the fura-2-loaded particles, L-AP4 did not alter the Ca²⁺ responses (Fig. 2D), indicating that this subpopulation of nerve terminals does not express functional L-AP4-sensitive mGluRs.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Low and high concentrations of L-AP4 reduce the Ca2+ responses of individual nerve terminals in two different subpopulations. Synaptosomes were fixed on polylysine-coated coverslips and loaded with fura-2 as indicated under "Experimental Procedures." A, representative field of fura-2-loaded synaptosomes under basal conditions at 380 nm. Ca2+ responses were induced by a 10-s application of 30 mM KCl in the absence and presence of 20 µM L-AP4 or 1 mM L-AP4. B and C, individual responses of the subpopulation of terminals that responded to 20 µM (n = 6) and 1 mM (n = 8) L-AP4, respectively. D, individual responses of the subpopulation of terminals that did not respond to any concentration of L-AP4 (n = 7). Data are means ± S.E. n indicates the number of individual responses averaged. The disc diagrams indicate the percent of nerve terminals showing a given response. Scale bar = 10 µm in A. [Ca2+]cyt, cytosolic free calcium concentration; div, division.

Immunocytochemical staining was performed to identify the mGluR that might mediate the responses to L-AP4. Among the L-AP4-sensitive receptors (mGluR4 and mGluR6-8), mGluR6 is not expressed in the brain (11). Thus, only the distribution of mGluR4, mGluR7, and mGluR8 and that of the vesicular protein synaptophysin was examined by immunocytochemistry. In these experiments, a lower concentration of nerve terminals was attached to the polylysine-coated coverslips to facilitate visualization. Among the synaptophysin-containing particles (589 particles from seven fields), mGluR7 was detected in 29.5 ± 3.4% (mean ± S.E.) of the terminals (Fig. 3, A-C). The mGluR4 protein was detected in 25.2 ± 2.8% of the synaptophysin-containing particles (734 particles from seven fields) (Fig. 3, D-F). In agreement with previous reports of the restricted expression of mGluR8 in the cerebral cortex (30), mGluR8 was found only in a small subpopulation (1.9 ± 0.6%) of synaptophysin-containing nerve terminals (1578 particles from 11 fields) (Fig. 3, G-I). The mGluR4 and mGluR7 subtypes have been shown to exhibit high (12) and low (13) affinity for L-AP4, respectively, in modulating G protein-coupled effectors. Thus, it is likely that these two mGluRs mediate the majority of the responses to L-AP4 in cerebrocortical nerve terminals from young rats.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   mGluR7, mGluR4, and mGluR8 are expressed in the preparation of cerebrocortical synaptosomes. Synaptosomes were fixed on polylysine-coated coverslips, and images of double-stained nerve terminals were obtained by performing immunocytochemistry with antisera against mGluR7, mGluR4, and mGluR8 and the vesicular marker synaptophysin. Antibody localization was visualized with Cy3 filters for synaptophysin (A, D, and G) and with fluorescein filters for mGluR7, mGluR4, and mGluR8 (B, E, and H, respectively). Merged panels are shown in C, F, and I. Scale bar = 10 µm.

The finding that 20 µM L-AP4 inhibited the release of glutamate by 22.2 ± 1.4% and the Ca²⁺ response in 24.4 ± 1.2% of the nerve terminal population strongly suggests that most of the nerve terminals that respond to low concentrations of L-AP4 are glutamatergic. A similar conclusion was reached when considering the mGluR with low affinity for L-AP4 because at 1 mM, this agonist reduced the release of glutamate by 24.1 ± 1.3% and the Ca²⁺ response in 30.3 ± 1.7% of the nerve terminals. To further study this possibility, synaptosomes were labeled with antiserum against the vesicular protein synaptophysin, and glutamatergic nerve terminals were identified with antisera against the vesicular glutamate transporters VGLUT1 (31, 32) and VGLUT2 (33, 34). To identify GABAergic nerve terminals, co-labeling was performed with synaptophysin and antiserum against the GABA-synthesizing enzyme glutamate decarboxylase, GAD-65 (35). VGLUT1 was detected in 62.0 ± 3.3% (mean ± S.E.) of the synaptophysin-containing particles (625 particles from 11 fields) (Fig. 4, A-C). VGLUT2 was present in 16.6 ± 2.2% of the synaptophysin-expressing particles analyzed (319 particles from seven fields) (Fig. 4, D-F). GAD-65 was detected in 23.0 ± 2.8% of a total of 304 particles from six fields (Fig. 4, G-I). Because nerve terminals coexpressing both VGLUT1 and VGLUT2 have not been found (36), we concluded that ~80% of the nerve terminals in the synaptosomal preparation were glutamatergic. These data are consistent with the quantitative distribution of glutamatergic and GABAergic synapses in the rodent cerebral cortex (37). Although our Ca²⁺ imaging and glutamate release data suggest that most of the nerve terminals in which L-AP4 alters the Ca²⁺ responses are glutamatergic, It also seems likely that a proportion of nerve terminals that respond to L-AP4 are GABAergic (38, 39).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Relative content of glutamatergic and GABAergic nerve terminals in the synaptosomal preparation. Synaptosomes were fixed on polylysine-coated coverslips, and images of double-stained nerve terminals were obtained using antisera against the vesicular glutamate transporters VGLUT1 and VGLUT2 and the GABA-synthesizing enzyme GAD-65. Antiserum against synaptophysin was used to label synaptic vesicles. Nerve terminals were visualized with Cy3 filters for synaptophysin (A, D, and G) and with Cy2 filters for VGLUT1, VGLUT2, and GAD-65 (B, E, and H, respectively). Merged panels are shown in C, F, and I. Scale bar = 10 µm.

Heterogeneity of Ca²⁺ Channels in Cerebrocortical Nerve Terminals from Young Rats-- In a number of studies of synaptic transmission in which Ca²⁺ channel blockers have been employed, the non-uniform distribution of the Ca²⁺ channels appears to be associated with glutamate release (21-25). However, only one study has addressed the heterogeneous distribution of Ca²⁺ channels at the level of single presynaptic terminals from rat hippocampal cells in culture (40). Because Ca²⁺ channels are key elements of the release machinery, and subpopulations of nerve terminals with different release properties express group III mGluRs (2), it is likely that different subtypes of group III mGluRs might be located in nerve terminals that contain different Ca²⁺ channels. To study this possibility, we first performed Ca²⁺ imaging experiments to determine which types of Ca²⁺ channels are expressed in the nerve terminals. Fixed fura-2-loaded nerve terminals were depolarized with 30 mM KCl in the absence and presence of -CgTx (41) and -AgaTx (22), selective inhibitors of N- and P/Q-type voltage-dependent Ca²⁺ channels, respectively. In total, 1644 fura-2-loaded particles from four fields were analyzed, from which four different types of responses were obtained. In a subpopulation of nerve terminals (47.5 ± 2.0%), the Ca²⁺ response was strongly reduced by -CgTx (2 µM), but not by -AgaTx (200 nM) (Fig. 5A), indicating the presence of N-type Ca²⁺ channels alone. In a second subpopulation (42.6 ± 1.1%), inhibition was observed following exposure to both toxins (Fig. 5B). In this population, although either toxin alone reduced the Ca²⁺ significantly, a stronger reduction was observed when the two toxins were applied together, clear evidence for the coexistence of N- and P/Q-type Ca²⁺ channels in these terminals. A small subpopulation of nerve terminals (3.9 ± 2.0%) expressed only P/Q-type Ca²⁺ channels as indicated by the ability of -AgaTx to reduce the Ca²⁺ response (Fig. 5C). Finally, in 6.1 ± 2.5% of the nerve terminals, the Ca²⁺ response was resistant to both toxins (Fig. 5D).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Inhibition of the KCl-induced Ca2+ responses by specific Ca2+ channel toxins. Ca2+ responses induced by 30 mM KCl were determined in the presence of 200 nM -AgaTx (-Aga-IVA), 2 µM -CgTx (-CgT-GVIA), and a combination of both toxins. Control Ca2+ responses were induced by 30 mM KCl in the absence of toxins at the end of the experiment. A, individual responses of the subpopulation of nerve terminals that responded only to -CgTx (n = 5); B, individual responses of the subpopulation of nerve terminals that responded to both -AgaTx and -CgTx (n = 8); C, individual responses of the subpopulation of nerve terminals that responded only to -AgaTx (n = 6); D, individual responses of the subpopulation of nerve terminals that did not respond to any of the Ca2+ channel toxins (n = 6). Data are means ± S.E. n indicates the number of individual responses averaged. The disc diagrams indicate the percent of nerve terminals showing a given response. [Ca2+]cyt, cytosolic free calcium concentration; div, division.

In another set of experiments in which 1526 fura-2-loaded particles from three fields were analyzed, we established that cerebrocortical nerve terminals were largely insensitive (95.5 ± 0.6%) to the L-type channel blocker nifedipine (10 µM) (Fig. 6A). In the remaining 4.5 ± 0.6% of the nerve terminals, the Ca²⁺ response was inhibited by blocking these channels (Fig. 6B). However, the KCl-induced Ca²⁺ response was completely abolished by the nonspecific Ca²⁺ channel blocker Cd²⁺ (30 µM) in all nerve terminals (Fig. 6, A and B).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   KCl-induced Ca2+ responses are largely insensitive to nifedipine, but are blocked by Cd2+. Ca2+ responses induced by 30 mM KCl were determined in the presence of 10 µM nifedipine (Nif.) and 30 µM CdCl2. Control Ca2+ responses were induced by 30 mM KCl in the absence of inhibitors at the end of the experiment. A, individual responses of the subpopulation of nerve terminals insensitive to nifedipine (n = 8); B, individual responses of the subpopulation of nerve terminals responding to nifedipine (n = 6). Data are means ± S.E. n indicates the number of individual responses averaged. The disc diagrams indicate the percent of nerve terminals showing a given response. [Ca2+]cyt, cytosolic free calcium concentration; div, division.

Both mGluR4 and mGluR7 Are Coexpressed with Different Ca²⁺ Channels-- We next set out to determine whether mGluRs with different affinities for L-AP4 are preferentially distributed in any of these subpopulations of nerve terminals or whether they are randomly distributed. To this end, nerve terminals were stimulated with 30 mM KCl in the presence of 20 µM L-AP4, 1 mM L-AP4, 2 µM -CgTx, or 200 nM -AgaTx. In these experiments, 3074 fura-2-loaded particles from six fields were analyzed. mGluRs with low affinity for L-AP4 were found largely (69.9 ± 1.5%) in N-type Ca²⁺ channel-expressing nerve terminals (Fig. 7A) and to a lesser extent (30.1 ± 1.5%) in nerve terminals endowed with N- and P/Q-type Ca²⁺ channels (Fig. 7B). Co-localization of mGluRs with low affinity for L-AP4 and P/Q-type Ca²⁺ channels was very rarely observed (2 out of 3074 particles) (data not shown). In contrast, mGluRs sensitive to 20 µM L-AP4 were primarily expressed (73.7 ± 1.4%) in nerve terminals exhibiting both N- and P/Q-type calcium channels (Fig. 8A). In these terminals, L-AP4 reduced the Ca²⁺ response further than exposure to either of the toxins alone, indicating that the mGluR inhibited both types of Ca²⁺ channels. In the remaining terminals sensitive to 20 µM L-AP4 (26.3 ± 1.4%), only N-type Ca²⁺ channels were found (Fig. 8B). We did not detect any response to 20 µM L-AP4 in P/Q-type Ca²⁺ channel-containing nerve terminals.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Inhibition of the KCl-induced Ca2+ responses by high concentrations of L-AP4 and Ca2+ channel toxins. Ca2+ responses were determined in the presence of 20 µM or 1 mM L-AP4, 200 nM -AgaTx (-Aga-IVA), or 2 µM -CgTx (-CgT-GVIA). Control Ca2+ responses were induced by 30 mM KCl in the absence of toxins at the end of the experiment. A, individual responses of the subpopulation of nerve terminals that responded to 1 mM L-AP4 and 2 µM -CgTx (n = 8); B, individual responses of the subpopulation of nerve terminals that responded to 1 mM L-AP4, 200 nM -AgaTx, and 2 µM -CgTx (n = 6). Data are means ± S.E. n indicates the number of individual responses averaged. The disc diagrams indicate the percent of nerve terminals showing a given response. [Ca2+]cyt, cytosolic free calcium concentration; div, division.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Inhibition of the KCl-induced Ca2+ responses by low concentrations of L-AP4 and Ca2+ channel toxins. Ca2+ responses were determined in the presence of 20 µM or 1 mM L-AP4, 200 nM -AgaTx (-Aga-IVA), or 2 µM -CgTx (-CgT-GVIA). Control Ca2+ responses were induced by 30 mM KCl in the absence of toxins at the end of the experiment. A, individual responses of the subpopulation of nerve terminals that responded to 20 µM L-AP4, 2 µM -CgTx, and 200 nM -AgaTx (n = 6); B, individual responses of the subpopulation of nerve terminals that responded to 20 µM L-AP4 and 2 µM -CgTx (n = 6). Data are means ± S.E. n indicates the number of individual responses averaged. The disc diagrams indicate the percent of nerve terminals showing a given response. [Ca2+]cyt, cytosolic free calcium concentration; div, division.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By combining Ca²⁺ imaging and immunocytochemistry from single nerve terminals with techniques to determine the release of glutamate from a population of cerebrocortical nerve terminals, we have identified the mGluRs that mediate L-AP4 responses in this preparation and the types of Ca²⁺ channels with which these receptors might interact. Inhibition of glutamate release was largely mediated by mGluR4 and mGluR7 as a result of reduction in the activity of N- and P/Q-type Ca²⁺ channels, which were heterogeneously distributed in the nerve terminals. Interestingly, mGluR4 was predominantly located in terminals containing both N- and P/Q-type Ca²⁺ channels, whereas mGluR7 was primarily expressed in terminals containing only N-type Ca²⁺ channels.

mGluR4 and mGluR7 Mediate Release Inhibition in Cerebrocortical Glutamatergic Terminals from Young Rats-- Because mGluR6 is absent in the brain (11), we studied the immunocytochemical distribution of mGluR4, mGluR7, and mGluR8 to identify the receptors mediating the responses to L-AP4. The presence of mGluR4 and mGluR7 in cerebrocortical nerve terminals is consistent with the expression of these two proteins in rat cerebral cortex (39, 42-44). Moreover, mGluR4 and mGluR7 have been shown to exhibit high and low affinity, respectively, for L-AP4 and glutamate in modulating G protein-coupled effectors (12, 13). Thus, we conclude that mGluR4 mediates the responses to low concentrations of L-AP4, whereas mGluR7 will respond to only high concentrations of this mGluR agonist. It is also possible that some responses to low L-AP4 concentrations are mediated by mGluR8. However, this receptor was detected only in a minor subpopulation of nerve terminals (1.9 ± 0.6%), consistent with the restricted expression of mGluR8 that has been described in the piriform cortex (30).

It is important to note that the immunocytochemical data nicely correlate with data obtained in Ca²⁺ imaging experiments. Thus, for example, mGluR7 was detected in 29.5 ± 3.4% of the synaptophysin-positive particles, whereas this same receptor reduced the Ca²⁺ responses in 30.3 ± 1.7% of the nerve terminals. This would indicate, first, that most of the mGluR7-containing nerve terminals are functional and, second, that mGluR7 reduces the activity of Ca²⁺ channels in the majority of such terminals. However, immunocytochemical and Ca²⁺ imaging data do not provide a direct estimate of the fraction of glutamatergic nerve terminals that contain mGluR7 because both techniques detect the receptor both in glutamatergic and GABAergic nerve terminals. Nevertheless, when we consider that 1 mM L-AP4 reduced glutamate release by 24.1%, and taking into account the relative abundance of glutamatergic over GABAergic terminals in this preparation, it would appear that mGluR7 is predominantly, but not exclusively, expressed in glutamatergic terminals (43). Indeed, both mGluR4 and mGluR7 have also been found in GABAergic nerve terminals (38, 39). However, mGluR8 does not co-localize with GAD-65,² although it is expressed in glutamatergic terminals from the piriform cortex (30).

Ca²⁺ Channel Heterogeneity in Cerebrocortical Nerve Terminals-- Based on the supra-additive inhibition of synaptic transmission caused by the different Ca²⁺ channel toxins, a non-uniform distribution of Ca²⁺ channels in nerve terminals has been anticipated (21-25). Directly quantifying this heterogeneity has been limited by the fact that electrophysiological recordings normally represent the averaged responses of many synapses, particularly if the field excitatory postsynaptic potential is measured. However, it is necessary to establish the precise distribution of Ca²⁺ channels within nerve terminals to understand synaptic function. In this study, by directly measuring the Ca²⁺ responses from single cerebrocortical nerve terminals, we have described a heterogeneous distribution of Ca²⁺ channels. Two major subpopulations of nerve terminals containing either N-type (47.5%) or a combination of N- and P/Q-type (42.6%) Ca²⁺ channels were identified, as well as two minor subpopulations of terminals containing either P/Q-type calcium channels (3.9%) or calcium channels insensitive to -CgTx and -AgaTx (6.1%). It is unlikely that this non-uniform distribution of Ca²⁺ channels observed in the cerebrocortical preparation is the result of differential expression of Ca²⁺ channel subtypes in neuronal populations because a heterogeneous distribution of Ca²⁺ channels has also been observed in single hippocampal neurons (24, 40). It therefore seems that the heterogeneous pattern for Ca²⁺ channel distribution is a widespread phenomenon. It is important to note that our results are remarkably similar to those found by Reuter (40), despite the presumably greater anatomical heterogeneity of the cerebrocortical nerve terminal preparation compared with hippocampal cell cultures. In that study, the exocytosis (measured with the fluorescent dye FM1-43) was completely inhibited by -CgTx in 45% of the boutons, whereas in the remaining boutons, exocytosis was sensitive to a combination of -CgTx, -AgaTx, and the L-type Ca²⁺ channel blocker isradipine. In contrast, Reid et al. (24) found that 45% of the nerve terminals expressed only -AgaTx-sensitive Ca²⁺ channels in cultured hippocampal neurons. It is important to note the age differences between the cultures used in these two studies (24, 40); and thus, the different results could well be due to developmental changes in the distribution of Ca²⁺ channels (45). In the calix-type rat medial nucleus of the trapezoid body, P/Q-, N-, and R-type Ca²⁺ channels coexist at individual release sites (46).

Although the functional implications of a non-uniform distribution of Ca²⁺ channels are unknown, it is likely that this heterogeneous distribution of Ca²⁺ channels could contribute to different release properties of the terminals. In this respect, a number of factors related to Ca²⁺ channel function can influence release, including the biophysical properties of individual Ca²⁺ channels, Ca²⁺ channel density at the release site, and the efficiency with which the Ca²⁺ channels trigger exocytosis (25). In addition, an obvious advantage of having more than one Ca²⁺ channel type in a nerve terminal is the differential regulation of these channels by neuromodulators, including those acting on G protein-coupled receptors, that can generate a terminal-specific modulation of synaptic transmission.

mGluR7 and mGluR4 Are Selectively Located in Different Populations of Ca²⁺ Channel-containing Nerve Terminals-- By measuring the Ca²⁺ responses of single nerve terminals when exposed to Ca²⁺ channel toxins and the agonist L-AP4, we found that the low affinity L-AP4-sensitive mGluR7 is largely (70%) located in N-type Ca²⁺ channel-expressing terminals, whereas the high affinity L-AP4-sensitive mGluR4 is largely (74%) located in N- and P/Q-type Ca²⁺ channel-expressing terminals. These results reveal the existence of a high level of heterogeneity among the L-AP4-sensitive mGluR-containing terminals. Thus, by simply considering the distribution of mGluR4 and mGluR7 in N-type and N- and P/Q-type Ca²⁺ channel-expressing terminals, six different subpopulations of nerve terminals can be described: N-type Ca²⁺ channel-expressing terminals with or without mGluR4 or mGluR7 and N- and P/Q-type Ca²⁺ channel-expressing terminals with or without mGluR4 or mGluR7. Although it is likely that the preparation of cerebrocortical synaptosomes might contain terminals from different anatomical locations, heterogeneity of group III mGluRs has previously been shown even among the nerve terminals from the same axon (2). An obvious functional advantage of this presynaptic heterogeneity can be seen in the different release properties exhibited by the terminal dependent on the target cell. We do not have an explanation for the preferential location of mGluR7 in N-type Ca²⁺ channel-expressing terminals and of mGluR4 in N- and P/Q-type Ca²⁺ channel-expressing terminals because the functional properties of these different cortical synapses are not known. However, the role of these two receptors should be influenced, at least in part, by their different affinities for glutamate (12, 13). Thus, although both receptors function in reducing glutamate release (10, 19), the remarkable differences in their affinities for glutamate should determine their activation properties. The low affinity of mGluR7 for glutamate may restrict its activation to the arrival of action potentials at high frequency. However, two different situations have to be considered for the mGluRs with high affinity for L-AP4. If they are not activated under basal conditions, then the release of glutamate induced by the arrival of an action potential should depress the release induced by a subsequent action potential. In contrast, if these receptors are tonically activated under basal conditions (47), then the arrival of consecutive action potentials would overcome the depression of Ca²⁺ channels with the subsequent facilitation of glutamate release. In this regard, it is important to note that N- and P/Q-type Ca²⁺ channels also exhibit differences in voltage-dependent facilitation (48). Further work is needed to understand the release properties of the many group III mGluR-containing synapses.

	ACKNOWLEDGEMENT

We thank M. Sefton for editorial assistance.

	FOOTNOTES

^* This work was supported by Ministerio de Ciencia y Tecnologia Grant BFI2001-1436 and Dirección General de Investigación de la Comunidad de Madrid Grant 08.5/0075.1/2000 (to J. S.-P.) and European Community Grant QLG3-CT-1999-00192 (to R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-91-394-3891; Fax: 34-91-394-3909; E-mail: jsprieto@vet.ucm.es.

Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M207531200

² C. Millán, R. Luján, R. Shigemoto, and J. Sánchez-Prieto, unpublished data.

	ABBREVIATIONS

The abbreviations used are: mGluRs, metabotropic glutamate receptors, L-AP4, L(+)-2-amino-4-phosphonobutyrate; HBM, HEPES buffer medium; MOPS, 4-morpholinepropanesulfonic acid; TBS, Tris-buffered saline; -CgTx, -conotoxin GVIA; -AgaTx, -agatoxin IVA; GABA, -aminobutyric acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				F. Ferraguti, L. Crepaldi, and F. Nicoletti Metabotropic Glutamate 1 Receptor: Current Concepts and Perspectives Pharmacol. Rev., December 1, 2008; 60(4): 536 - 581. [Abstract] [Full Text] [PDF]

				C. M. Niswender, K. A. Johnson, Q. Luo, J. E. Ayala, C. Kim, P. J. Conn, and C. D. Weaver A Novel Assay of Gi/o-Linked G Protein-Coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors Mol. Pharmacol., April 1, 2008; 73(4): 1213 - 1224. [Abstract] [Full Text] [PDF]

				D. A. Rusakov, A. Wuerz, and D. M. Kullmann Heterogeneity and Specificity of Presynaptic Ca2+ Current Modulation by mGluRs at Individual Hippocampal Synapses Cereb Cortex, July 1, 2004; 14(7): 748 - 758. [Abstract] [Full Text] [PDF]

				C. Millan, E. Castro, M. Torres, R. Shigemoto, and J. Sanchez-Prieto Co-expression of Metabotropic Glutamate Receptor 7 and N-type Ca2+ Channels in Single Cerebrocortical Nerve Terminals of Adult Rats J. Biol. Chem., June 20, 2003; 278(26): 23955 - 23962. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/49/47796    most recent M207531200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Millán, C. Articles by Sánchez-Prieto, J. Search for Related Content PubMed PubMed Citation Articles by Millán, C. Articles by Sánchez-Prieto, J. Social Bookmarking                      What's this?