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Volume 17, Number 19,
Issue of October 1, 1997
pp. 7503-7522
Copyright ©1997 Society for Neuroscience
Differential Presynaptic Localization of Metabotropic Glutamate
Receptor Subtypes in the Rat Hippocampus
Ryuichi Shigemoto1,
Ayae Kinoshita1,
Eiki Wada1,
Sakashi Nomura3,
Hitoshi Ohishi1,
Masahiko Takada1,
Peter J. Flor4,
Akio Neki2,
Takaaki Abe2,
Shigetada Nakanishi2, and
Noboru Mizuno1
Departments of 1 Morphological Brain Science and
2 Biological Sciences, Faculty of Medicine, and
3 College of Medical Technology, Kyoto University, Kyoto
606, Japan, and 4 Novartis Pharma Inc., Nervous System
Research, CH-4002 Basel, Switzerland
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Neurotransmission in the hippocampus is modulated variously through
presynaptic metabotropic glutamate receptors (mGluRs). To establish the
precise localization of presynaptic mGluRs in the rat hippocampus, we
used subtype-specific antibodies for eight mGluRs (mGluR1-mGluR8) for
immunohistochemistry combined with lesioning of the three major
hippocampal pathways: the perforant path, mossy fiber, and Schaffer
collateral. Immunoreactivity for group II (mGluR2) and group III
(mGluR4a, mGluR7a, mGluR7b, and mGluR8) mGluRs was predominantly
localized to presynaptic elements, whereas that for group I mGluRs
(mGluR1 and mGluR5) was localized to postsynaptic elements. The medial
perforant path was strongly immunoreactive for mGluR2 and mGluR7a
throughout the hippocampus, and the lateral perforant path was
prominently immunoreactive for mGluR8 in the dentate gyrus and CA3
area. The mossy fiber was labeled for mGluR2, mGluR7a, and mGluR7b,
whereas the Schaffer collateral was labeled only for mGluR7a. Electron
microscopy further revealed the spatial segregation of group II and
group III mGluRs within presynaptic elements. Immunolabeling for the
group III receptors was predominantly observed in presynaptic active
zones of asymmetrical and symmetrical synapses, whereas that for the group II receptor (mGluR2) was found in preterminal rather than terminal portions of axons. Target cell-specific segregation of receptors, first reported for mGluR7a (Shigemoto et al., 1996
), was
also apparent for the other group III mGluRs, suggesting that transmitter release is differentially regulated by
2-amino-4-phosphonobutyrate-sensitive mGluRs in individual synapses on
single axons according to the identity of postsynaptic neurons.
Key words:
metabotropic glutamate receptor;
hippocampus;
perforant
path;
mossy fiber;
Schaffer collateral;
axon terminal;
preterminal;
immunohistochemistry;
lesion
INTRODUCTION
Metabotropic glutamate receptors
(mGluRs) have various modulatory functions on neuronal excitability,
transmitter release, and synaptic plasticity in the CNS (Pin and
Duvoisin, 1995). These functions have been studied most extensively in
the hippocampus because of its roles in learning and memory and of its
architecture, which is compartmentalized well with the three major
excitatory pathways: the perforant path, mossy fiber, and Schaffer
collateral. The mGluRs consist of at least eight subtypes that are
classified into three groups (Nakanishi and Masu, 1994; Pin and
Duvoisin, 1995). Group I mGluRs (mGluR1/mGluR5) are selectively
activated by 3,5-dihydroxyphenylglycine (DHPG) (Schoepp et al., 1994)
and coupled to inositol phospholipid hydrolysis. On the other hand, group II mGluRs (mGluR2/mGluR3) and group III mGluRs
(mGluR4/mGluR6/mGluR7/mGluR8), which are linked to inhibition of the
cAMP cascade in receptor-transfected cell lines, are selectively
activated by 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (Hayashi et
al., 1993) and 2-amino-4-phosphonobutyrate (L-AP4), respectively.
Excitability of hippocampal neurons is modulated directly through group
I mGluRs (Davies et al., 1995; Gereau and Conn, 1995b) coupled to
various calcium, potassium, and nonselective cationic channels (Swartz
and Bean, 1992; Crépel et al., 1994; Guérineau et al.,
1994, 1995). This is consistent with the postsynaptic location of
mGluR1/mGluR5 immunoreactivity in hippocampal neurons (Luján et
al., 1996). On the other hand, presynaptic mGluRs are thought to
suppress transmitter release in various regions (Pin and Duvoisin,
1995) by inhibiting voltage-dependent calcium channels (Takahashi et
al., 1996) and/or interfering directly with the release machinery
(Scanziani et al., 1995). In the hippocampus, group I and group III
mGluRs mediate the inhibition of excitatory transmission in the
Schaffer collateral-CA1 cell synapses (Gereau and Conn, 1995a),
whereas the group II and group III mGluRs are involved in the
presynaptic inhibition in the perforant path-granule cell synapses
(Bushell et al., 1996a; Macek et al., 1996). Furthermore, recent
studies with mGluR-deficient mutant mice have indicated that not only
postsynaptic but also presynaptic mGluRs have roles in hippocampal
synaptic plasticity (Bushell et al., 1996b; Yokoi et al., 1996).
The mRNAs for all mGluRs except mGluR6 are expressed in the hippocampus
and entorhinal cortex (Shigemoto et al., 1992; Ohishi et al., 1993a,b,
1995a; Fotuhi et al., 1994; Saugstad et al., 1994). Immunolabeling for
mGluR2/3 and mGluR7a was found in presynaptic elements in the
hippocampus using subtype-specific antibodies (Shigemoto et al., 1995,
1996; Bradley et al., 1996; Petralia et al., 1996; Yokoi et al., 1996).
We also developed antibodies specific to mGluR4a and mGluR8 and found
these immunoreactivities in axon terminals in other brain regions
(Kinoshita et al., 1996a,b). In the present study, we systematically
used 10 subtype-specific mGluR antibodies, including a newly developed
mGluR7b-specific antibody, to examine the precise localization of
presynaptic mGluRs in the rat hippocampus by light and electron
microscopy, in combination with lesion experiments to establish the
origins of presynaptic mGluR-bearing pathways.
MATERIALS AND METHODS
Preparation of affinity-purified antibodies against
mGluRs. Antibodies for mGluR1
, mGluR1, mGluR2/3, mGluR2,
mGluR5, and mGluR7a were raised against bacterial fusion proteins
containing mGluR sequences (amino acid residues 859-1199 of mGluR1,
104-154 of mGluR1, 87-134 and 813-872 of mGluR2, 863-1171 of
mGluR5a, and 874-915 of mGluR7a) as described previously (Shigemoto et al., 1993, 1994, 1996; Ohishi et al., 1994, 1995b; Neki et al., 1996).
One mGluR1 antibody (G18) raised against extracellular amino acid
residues 104-154 is specific to all spliced forms of mGluR1 (pan
mGluR1 antibody) (Shigemoto et al., 1994), whereas another (A52) raised
against intracellular C-terminal residues 859-1199 of mGluR1 is
specific to mGluR1 (see Results). The antibody (H12) raised against
intracellular C-terminal residues 813-872 of mGluR2 is reactive to
both mGluR2 and mGluR3 (mGluR2/3 antibody) (Ohishi et al., 1994),
whereas another mGluR2 monoclonal antibody (mG2Na-5) raised against
putative extracellular amino acid residues 87-134 is mGluR2-specific
(Neki et al., 1996). The mGluR5 antibody is reactive to the C-terminal
domain common for mGluR5a and mGluR5b (Minakami et al., 1993).
Antibodies for mGluR4a (K44), mGluR6, and mGluR8 (K88) were raised
against synthetic peptides corresponding to C-terminal sequences (amino
acid residues 890-912 of mGluR4a, 853-871 of mGluR6, and 886-908 of
mGluR8) as described previously (Nomura et al., 1994; Kinoshita et al., 1996a,b). The antibodies for mGluR1, mGluR1, mGluR2/3, mGluR5, mGluR6, and mGluR7a are polyclonal antibodies raised in rabbits, whereas those for mGluR4a and mGluR8 are polyclonal antibodies raised
in guinea pigs. An antibody for mGluR7b (K74) was newly raised against
a synthetic peptide corresponding to the human mGluR7b-specific
C-terminal sequence (NCIPPVRKSVQKSVTWYTIPPTV) (Flor et al., 1997) as
described previously (Kinoshita et al., 1996a). The amino acid sequence
used for the antibody production is identical between the human and rat
mGluR7b (F. Ferraguti, personal communication). After conjugation with
maleimide-activated bovine serum albumin (Pierce, Rockford, IL), the
peptide was injected subcutaneously in rabbits and guinea pigs
(0.5-1.0 mg of peptide/animal). The immunized animals were boosted
every 4 weeks and bled 1-2 weeks after each boost. The collected
antisera were purified by sodium sulfate fractionation, followed by
affinity chromatography on a SulfoLink (Pierce) coupled to the
C-terminus peptide. The purified mGluR7b antibodies derived from
rabbits and guinea pigs gave identical results.
Immunoblot analysis. Immunoblot analysis was performed as
described previously (Shigemoto et al., 1994). The crude membrane preparations from rat hippocampus and mGluR4-, mGluR6-, mGluR7a- and
mGluR7b-expressing Chinese Hamster Ovary (CHO) cells (Tanabe et al.,
1992; Nakajima et al., 1993; Okamoto et al., 1994; Flor et al., 1997)
were separated by 7% SDS-PAGE and transferred to polyvinylidene
difluoride (PVDF) membranes. The membranes were blocked with Block-Ace
(Dainippon Pharmaceutical) and then reacted with the affinity-purified
mGluR antibodies (0.2-1.0 µg/ml) in the absence or presence of
respective fusion proteins. Alkaline phosphatase-labeled secondary
antibodies (Chemicon, Temecula, CA) were used to visualize the reacted
bands.
Surgery. At least three adult male Wistar rats
(250-300 gm) were used for each series of experiments. All surgical
manipulations were performed using a David Kopf stereotaxic apparatus
under anesthesia with sodium pentobarbital (50 mg/kg, i.p.). A lesion generator (Radionics Inc.) was used at the setting of 60°C for 2 min
to make lesions in the entorhinal cortex and subiculum. For destruction
of dentate granule cells, colchicine (Sigma, St. Louis, MO) (3 µg in
0.9 µl of 25 mM PBS, pH 7.3) was injected through a 1 µl Hamilton microsyringe into the dorsal and ventral hilus of the
left hippocampus as described previously (McGinty et al., 1983). For
destruction of CA3 pyramidal cells, kainic acid (Sigma) was injected
into the CA areas of the left hippocampus or bilateral ventricles at
the dose of 0.2 µg in 0.2 µl PBS or 0.6 µg in 1.2 µl PBS,
respectively. In some experiments, PBS alone was injected into the
corresponding sites of the right hippocampus or bilateral ventricles to
serve as controls. Seven days after the lesioning or injections of
colchicine or kainic acid, the rats were killed and processed for
immunohistochemistry.
Immunohistochemistry. Immunohistochemical staining was
performed by the ABC and double-immunofluorescence methods as described previously (Shigemoto et al., 1993, 1996). For light microscopy, adult
male Wistar rats as well as wild-type and mGluR-deficient mice were
deeply anesthetized and perfused transcardially with 3.5%
paraformaldehyde, 1% picric acid, and 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.3. For immunolabeling with the mGluR2 antibody (mG2Na-5), brains were further fixed for 3 d
at 4°C in a mixture containing 2% formaldehyde and 1% picric acid
in 0.1 M PB, pH 7.3 (Neki et al., 1996). Tissue blocks
containing the hippocampus were cryoprotected in 20% sucrose in 0.1 M PB overnight at 4°C and cut on a freezing microtome
into 40-µm-thick sections. The sections were incubated with 0.5-1.0
µg/ml antibodies for mGluRs in PBS containing 2% normal goat serum
and 0.1% Triton X-100 overnight at 15°C. After washes in PBS, the
sections were incubated with biotinylated goat anti-rabbit, goat
anti-guinea pig, or goat anti-mouse IgG (Vector Laboratories,
Burlingame, CA). The sections were then washed again, reacted with the
ABC kit (Vector), and finally incubated with 0.05% diaminobenzidine and 0.0006% hydrogen peroxide. For double-immunofluorescence
histochemistry, 20-µm-thick sections were reacted with antibodies to
mGluRs (0.5-1.0 µg/ml) and then with fluorescein
isothiocyanate-conjugated anti-[rabbit IgG] antibody and biotinylated
anti-[guinea pig IgG] antibody combined with Texas Red-conjugated
avidin. When either of the primary antibodies was omitted, no
fluorescence signal for the omitted primary antibody was observed (not
shown). For electron microscopy, rats were perfused with 4%
paraformaldehyde, 0.2-1.0% picric acid, and 0.05-0.1%
glutaraldehyde in 0.1 M PB, pH 7.3. Tissue blocks were then
processed by two separate methods. In the first method, 50-µm-thick
sections were cut on a vibratome and processed as described above for
light microscopy, except that 0.2% photoflo (Kodak) was used instead
of Triton X-100. In the second method, tissue blocks were freeze-thawed
in liquid nitrogen and PBS at room temperature, and 50-µm-thick
sections were cut on a vibratome. The sections from freeze-thawed
tissues were incubated with mGluR antibodies in PBS containing 2%
normal goat serum without Triton X-100 or photoflo. After washes in
PBS, sections were incubated with biotinylated or 1.4 nm gold-labeled (Nanoprobes, Stony Brook, NY) secondary antibodies and then reacted with the ABC or HQ Silver kit (Nanoprobes), respectively. After osmification, the immunostained sections were block-stained with uranyl
acetate, dehydrated, and flat-embedded in Epon. Ultrathin sections were
then prepared and examined with a Hitachi H-7100 electron microscope.
Some sections with immunogold labeling were counterstained with lead
citrate.
RESULTS
Immunoblot analysis of mGluRs in the hippocampus
Specificity of the antibodies used in the present study was
verified with immunoblot analysis of a crude membrane fraction prepared
from the rat hippocampus (Fig.
1A). Pan mGluR1
antibody (G18) gave rise to two immunoreactive products with estimated molecular masses of 145 and 97 kDa. The former corresponds to mGluR1
(Masu et al., 1991) and the latter to mGluR1
(Tanabe et al., 1992),
mGluR1c (Pin et al., 1992), and mGluR1d (Laurie et al., 1996). Only the
former was detected with another mGluR1 antibody (A52) raised against
C-terminal residues of mGluR1, indicating that A52 is specific to
mGluR1. Two immunoreactive products of 98 kDa and ~200 kDa were
observed with the mGluR2/3 (H12) and mGluR2 (mG2Na-5) antibodies. The
former product corresponds to mGluR2, and the latter probably
represents dimeric forms of mGluR2/3 proteins (Hayashi et al., 1993;
Ohishi et al., 1994). Presumed dimeric forms were found for most mGluRs
(Baude et al., 1993; Shigemoto et al., 1993; Nomura et al., 1994;
Petralia et al., 1996; Romano et al., 1996) (also see the present
results for mGluR4a, mGluR7a, mGluR7b, and mGluR8). The extracellular mGluR2-specific antibody (mG2Na-5) gave a similar immunoreactive product of 98 kDa but only weakly reacted with the product of ~200
kDa. This result may be attributed to low accessibility of the
extracellular epitope in the dimeric form (Romano et al., 1996). In the
present study, the mGluR3-immunoreactive product of 106 kDa observed in
the cerebral cortex (Hayashi et al., 1993) and whole brain (Ohishi et
al., 1994) was scarcely detected in the hippocampus. Immunoreactive
bands of molecular masses around 100 kDa were observed for mGluR4a,
mGluR7a, and mGluR8, and that of 145 kDa was found for mGluR5, as
reported previously (Shigemoto et al., 1993, 1996; Kinoshita et al.,
1996a,b). Immunoreactivity for mGluR5 and mGluR7a was strong in the
hippocampus, whereas that for mGluR4a or mGluR8 was much weaker in the
hippocampus than in the cerebellum (Kinoshita et al., 1996b) or in the
piriform cortex (Kinoshita et al., 1996a), respectively. The newly
developed mGluR7b antibody gave rise to an immunoreactive doublet with
estimated molecular masses of 102 and 96 kDa. These molecular masses
agree well with that of the cloned human mGluR7b (103,082 Da) (Flor et
al., 1997). Two such immunoreactive bands with slightly different molecular masses were also observed for mGluR6 in the retina (Nomura et
al., 1994) and may result from different post-translational modification, such as glycosylation and proteolytic cleavage. All
immunoreactivities described above disappeared after preadsorption of
the antibodies with the corresponding fusion proteins or synthetic peptides (Fig. 1A; shown only for mGluR7b). The
homology between the amino acid sequences used for the production of
the mGluR antibodies was highest among group III mGluRs (52% for
mGluR4a and mGluR8, 35% for mGluR4a and mGluR7a, and 30% for mGluR7a
and mGluR8). To exclude the possibility of cross-reactivity to other group III mGluRs, the mGluR4a, mGluR7a, and mGluR8 antibodies were
incubated with excess amounts (10 times more antigens than those used
for the homologous preadsorption controls) of heterologous antigens for
other group III mGluRs. The preincubated antibodies gave results (data
not shown) identical to those shown in Figure 1A. The
mGluR7a antibody was purified with a column coupled to a glutathione
S-transferase-fusion protein containing 20 amino acid
residues from the C-terminal (Ohishi et al., 1995b) that have four
amino acid residues (896-899) in common with mGluR7b (Flor et al.,
1997). To exclude possible cross-reactivity of the mGluR7a and mGluR7b
antibodies to each other and to other group III mGluRs, membrane
fractions prepared from cDNA-transfected cell lines expressing mGluR4a,
mGluR7a, mGluR7b, and mGluR8 were subjected to immunoblot analysis.
Both the mGluR7a and mGluR7b antibodies as well as mGluR4a and mGluR8
antibodies reacted specifically with the corresponding cell lines (Fig.
1B). These results indicate that the mGluR antibodies
react specifically with their respective mGluRs.
Fig. 1.
Immunoblot analysis of rat hippocampus
(A) and receptor-expressing cells
(B). Crude membrane preparations from rat
hippocampus and CHO cells expressing mGluR4a (4a),
mGluR7a (7a), or mGluR7b (7b), or COS
cells transfected with mGluR8 cDNA (8) were
subjected to 7% SDS-PAGE and transferred onto PVDF filters.
A, The filters with the hippocampus were reacted with
antibody to pan mGluR1 (G18), mGluR1 (A52), mGluR2/3 (H12), mGluR2
(mG2Na-5), mGluR4a (K44), mGluR5 (G53), mGluR7a (G71), mGluR7b (K74),
or mGluR8 (K88). Immunoreactive bands for mGluR7b were completely
abolished by preadsorption of the antibody with the corresponding
peptide (adsorbed). B, The filters with the
receptor-expressing cells were reacted with the mGluR7a (G71), mGluR7b
(K74), mGluR4a (K44), or mGluR8 (K88) antibody. Each of the
immunoreactive products was absent in nontransfected cells (not shown).
Positions of molecular mass markers (Bio-Rad) in kDa are indicated
on the left.
[View Larger Version of this Image (50K GIF file)]
Immunohistochemistry of mGluRs in normal hippocampus
All mGluR antibodies revealed specific and distinct patterns of
immunoreactivity in the normal rat hippocampus (Figs.
2, 3, 4, 5, 6). All of these immunostaining
patterns were abolished by preadsorption of the antibodies with the
homologous but not the heterologous antigens (data not shown). For
example, mGluR7a immunolabeling, which overlaps with immunolabeling for
mGluR4a/7b/8, was abolished after preadsorption of the antibody with
the mGluR7a fusion protein and an mGluR7a-specific synthetic peptide
(residues 900-915), but it was not affected by corresponding
C-terminal peptides for mGluR4a/7b/8. Specificity of the immunostaining
for mGluR1, mGluR1, mGluR2, mGluR5, mGluR7a, and mGluR7b was further verified in the mice lacking the respective mGluR genes (Aiba et al.,
1994; Bushell et al., 1996b; Yokoi et al., 1996; Lu et al., 1997). In
wild-type mice, immunolabeling patterns similar to those in rat were
observed for these receptors in the hippocampus, but they were totally
absent in the respective mGluR-deficient mice (data not shown).
Immunolabeling of mGluR4a in the hippocampus of wild-type mice had a
laminar distribution different from that in the rat (R. Shigemoto,
unpublished result), but it was also absent in mGluR4-deficient mice
(Pekhletski et al., 1996). Moreover, cross-reactivity of the antibodies
for mGluR7a/7b/8 to mGluR4 and of those for mGluR4a/8
to mGluR7 were excluded by unaltered immunostaining patterns observed
in mGluR4- and mGluR7-deficient mice, respectively (data not
shown).
Fig. 2.
Distribution of immunoreactivity for eight mGluRs
in rat hippocampus. Parasagittal sections through the hippocampus were
reacted with antibody to pan mGluR1 (A),
mGluR1 (B), mGluR2/3
(C), mGluR2 (D), mGluR4a
(E), mGluR5 (F),
mGluR6 (G), mGluR7a
(H), mGluR7b (I), or mGluR8
(J). Immunoreactivity for mGluR5
(F) and mGluR7a (H) is distributed in all dendritic layers
throughout the hippocampus, whereas immunoreactivity for the other
mGluRs is restricted to distinct regions. Immunoreactivity for mGluR1
(A) is strong in dendritic fields of the dentate
gyrus (DG) and CA3, as well as in the CA1 stratum
oriens/alveus border (arrows). Immunoreactivity for
mGluR1 (B) is strong only in the CA1 stratum oriens/alveus border (see
Results). Immunoreactivity for mGluR2/3
(C) and mGluR2 (D)
is strong in terminal zones of the perforant path and mossy fibers (see
Results), whereas that for mGluR7b (I) is
restricted to the mossy fiber terminal zone, and that for mGluR8
(J) to the lateral perforant path terminal
zone, i.e., the outer third (filled arrowheads)
of the dentate molecular layer and outer layer (double arrowhead) of CA3 stratum lacunosum moleculare.
Immunoreactivity for mGluR4a (E) is weak
but prominent in the inner third (open arrows) of the
molecular layer. Labeling for mGluR6 (G) is
hardly detected in the hippocampus. The rectangle in
C indicates a corresponding region shown with a higher
magnification in Figure 3. fi, Fimbria; S, subiculum. Scale bar, 500 µm.
[View Larger Version of this Image (121K GIF file)]
Fig. 3.
Distribution of immunoreactivity for mGluR1
(A), mGluR5 (B), mGluR2/3
(C), and mGluR7a (D) in the
hippocampal area corresponding to the rectangle in Figure
2C. Immunoreactivity for mGluR1 and mGluR5 is relatively
uniform throughout the dentate molecular layer (Mo),
whereas that for mGluR2/3 and mGluR7a is prominent in the middle
one-third of the layer (mid), which is the terminal zone
of the medial perforant path. In the hilus (Hi),
dendritic profiles are immunopositive for mGluR1, mGluR5, and mGluR7a,
whereas neuropil is immunopositive for mGluR2/3. Dendritic fields
(stars) of CA3 pyramidal cells (Py) are
immunopositive for mGluR1, mGluR5, and mGluR7a but immunonegative for
mGluR2/3. Intense mGluR2/3 labeling is seen in CA1 stratum lacunosum
moleculare (LM). The broken line
indicates the hippocampal fissure. Gr, Granule cell layer; in, inner one-third of the molecular layer;
out, outer one-third of the molecular layer. Scale bar,
100 µm.
[View Larger Version of this Image (127K GIF file)]
Fig. 4.
High-magnification micrographs showing
immunoreactivity for mGluR4a (A), mGluR7a
(B), and mGluR8 (C) in the
inner (A, B) and outer (C) third
of the dentate molecular layer. Note dendritic profiles decorated with
puncta immunopositive for mGluRs in the inner (Min) and
outer (Mout) third of the molecular layer.
Gr, Granule cell layer. Scale bar, 25 µm.
[View Larger Version of this Image (72K GIF file)]
Fig. 5.
Immunoreactivity for mGluR2/3
(A), mGluR7a (B), and
mGluR7b (C) in CA3 stratum lucidum. Axon
bundle-like profiles are immunopositive for mGluR2/3, whereas punctate
decorations of dendrites are immunopositive for mGluR7a and mGluR7b in
stratum lucidum (Lu). The pyramidal cell layer
(Py) is devoid of immunoreactivity. Scale bar, 25 µm.
[View Larger Version of this Image (79K GIF file)]
Fig. 6.
Double-immunofluorescence study for mGluR7b,
mGluR1, and mGluR7a in the hilus and CA3 stratum lucidum.
Fluorescence micrographs of sections double-immunolabeled for mGluR7b
and mGluR1 (A, A') or mGluR7b and mGluR7a
(B, B') were taken from identical fields of the
hilus (A, A') and CA3 (B,
B') under different filters. Punctate immunolabeling
for mGluR7b (A, visualized with Texas Red) decorates
mGluR1-immunopositive interneurons (A', visualized with fluorescein) in the hilus (Hi). In CA3, all
profiles decorated with mGluR7b (B) are
also decorated with mGluR7a immunoreactivity (B',
visualized with fluorescein) in stratum lucidum (Lu).
Stratum radiatum (Ra) is immunopositive only for mGluR7a
(B'). Arrows indicate cell bodies
decorated with mGluR7a/b and labeled for mGluR1 immunoreactivity.
Gr, Granule cell layer; Py, pyramidal cell layer. Scale bar, 30 µm.
[View Larger Version of this Image (142K GIF file)]
Immunoreactivity for the group I mGluRs was observed in neuropil of the
dendritic fields of the hippocampal principal cells as described
previously (Luján et al., 1996), whereas immunoreactivity for the
group II and group III mGluRs was observed more or less in a laminated
fashion corresponding to the terminal zones of the major hippocampal
pathways (Fig. 2). All hippocampal fields were immunopositive for
mGluR5 and mGluR7a, whereas immunoreactivity for the other mGluRs was
restricted to some areas or layers in the hippocampus. Immunoreactivity
for mGluR6 was hardly detected in the hippocampus (Fig.
2G).
For the group I mGluRs, we used the antibodies reactive to all splice
variants of mGluR1 (pan mGluR1) and mGluR5, as well as the
mGluR1-specific antibody. In CA1 dendritic fields, only the mGluR5
antibody showed intense immunolabeling of neuropil, whereas the pan
mGluR1 and mGluR5 antibodies revealed similar patterns of
immunolabeling in CA3 dendritic fields and dentate molecular layer
(Figs. 2A,F, 3A,B). Cell bodies of the
granule and CA3 pyramidal cells showed moderate to strong labeling for mGluR1 but only very weak labeling for mGluR5 (Fig.
3A,B). These labelings with
the pan mGluR1 antibody were not detected at all with the
mGluR1-specific antibody (Fig. 2B), indicating
that the mGluR1 immunoreactivity in CA3 pyramidal cells is ascribable to mGluR1, mGluR1c, and/or mGluR1d. Strong labeling for pan mGluR1 was also observed in cell bodies and dendrites of interneurons scattered in the hilus (Fig. 3A) and CA areas, being densest
in the border zone between CA1 stratum oriens and the alveus (Fig. 2A). These interneurons were the only cells that were
also immunopositive for mGluR1 (Fig. 2B). A
double-immunofluorescence labeling confirmed that all of the
interneurons labeled with the pan mGluR1 antibody were also
immunopositive for mGluR1 throughout the hippocampus (data not
shown). In the hilus, mGluR5 immunolabeling was seen in many dendritic
processes (Fig. 3B). The somatodendritic profiles labeled
for mGluR1 and mGluR5 were observed less frequently in CA3 stratum
lucidum.
For the group II mGluRs, we used the antibody reactive to both mGluR2
and mGluR3, as well as the mGluR2-specific antibody. The immunostaining
patterns with the mGluR2/3 and mGluR2 antibodies were similar (Fig.
2C,D), but some differences were seen in the strata oriens
and radiatum of the CA areas, where diffuse and weak immunostaining was
detected only with mGluR2/3 antibody (Fig. 2C). Such
mGluR2/3 immunoreactivity was also detected in mice lacking the mGluR2
gene (Yokoi et al., 1996) and ascribed to mGluR3 immunoreactivity of
glial cells (see the results of electron microscopy). The density of
the mGluR2/3 and mGluR2 immunolabeling was highest in neuropil of CA1
stratum lacunosum moleculare (Figs. 2C,D, 3C). In
the CA3 area, labeling was stronger in the inner than in the outer
layer of the stratum lacunosum moleculare, and in the dentate gyrus, it
was stronger in the middle than in the outer one-third of the molecular
layer (Figs. 2C,D, 3C). The strongly labeled layers correspond to the medial perforant path (Steward, 1976). A clear
boundary of the strong labeling was observed between the inner and
middle thirds of the molecular layer, whereas the other boundary
between the outer and middle thirds of the layer was less clear (Fig.
3C). This observation again agrees well with the reported
pattern of the medial perforant path projection (Steward, 1976).
Immunoreactivity for mGluR2/3 and mGluR2 was also observed in neuropil
of the mossy fiber terminal zone (Fig. 2C,D). Labeling of
axon bundle-like profiles was apparent in CA3 stratum lucidum (see Fig.
5A) as well as in the fimbria (Fig. 2C,D). In
mice lacking the mGluR2 gene, these mGluR2/3 immunolabelings were
almost absent (Yokoi et al., 1996), indicating that most of the
immunoreactivity in the perforant path and mossy fiber terminal zones
is ascribable to mGluR2.
Among the group III mGluRs, mGluR4a, mGluR7a, mGluR7b, and mGluR8 were
expressed in the hippocampus. In the CA1 area, strong neuropil labeling
was observed only for mGluR7a, being strongest in the strata oriens and
radiatum, followed by the stratum lacunosum moleculare (Figs.
2H, 3D). Labeling in the pyramidal cell
layer was very weak. In CA3 dendritic fields, very strong mGluR7a
labeling was observed in CA3 stratum lacunosum moleculare, especially
in the inner layer adjacent to the dentate molecular layer (Fig. 2H). The medial perforant path terminal zone in the
dentate gyrus, namely the middle third of the molecular layer, was also
prominently immunopositive for mGluR7a (Fig. 3D). The
distribution of immunoreactivity for other group III mGluRs was much
more restricted in the hippocampus. Labelings for mGluR4a and mGluR8
were weak and diffuse compared with those for mGluR2/3 and mGluR7a;
however, prominent staining for mGluR4a and mGluR8 was still observed
in neuropil of the inner and outer thirds of the dentate molecular
layer, respectively (Figs. 2E,J,
4A,C). The former
corresponds to the terminal zone of the associational/commissural
fibers (Swanson et al., 1978) and the latter to that of the lateral
perforant path (Steward, 1976). The mGluR8 immunolabeling was stronger
in the inner than in the outer blade of the dentate gyrus (Figs.
2J, 7H); this pattern is in good
accordance with that of preferential projections of the lateral
perforant path (Wyss, 1981). The lateral perforant path terminal zone
in CA3 stratum lacunosum moleculare (superficial portion of the layer)
was also prominently immunopositive for mGluR8, making the staining
pattern apparently complementary to that for mGluR2/3 (Fig.
2C). In these neuropil stainings, some dendritic profiles
showed up being decorated with many axon terminals that were intensely
labeled for the group III mGluRs (Fig. 4) (also see the results of
electron microscopy). They were most frequently observed for mGluR7a
followed by mGluR8, and only occasionally observed for mGluR4a in the
molecular layer. Most of these dendrites decorated with
labeled axon terminals seemed to originate from interneurons in the
hilus or granule cell layer. For mGluR7a and mGluR7b, the decoration of
many dendritic processes and some cell bodies of interneurons were
observed in the hilus (Figs. 2H,I, 3D,
6A). In CA3 stratum lucidum, the somatodendritic
decoration was observed more strongly for mGluR7b than for mGluR7a
(Fig. 5B,C). The strong
terminal labeling for mGluR7a on the somatodendritic profiles of
interneurons was distributed most densely in the stratum oriens/alveus
border zone in the CA1 area and less densely in other CA areas, as
described previously (Shigemoto et al., 1996). Because these decorated
interneurons were mGluR1-immunopositive (Shigemoto et al., 1996), we
also examined, by a double-immunofluorescence labeling, whether the
decoration with mGluR7b correlates with the mGluR1-immunopositive
interneurons in the mossy fiber terminal zone (Fig.
6A,A'). As in mGluR7a
labeling, mGluR7b-labeled puncta were mostly found on mGluR1-labeled
interneurons. Colocalization of mGluR7a and mGluR7b was also examined
(Fig. 6B,B'). Virtually all mGluR7b-labeled
structures were labeled for mGluR7a; however, the mGluR7a-labeled axon
terminals decorating interneurons in strata oriens and radiatum of the
hippocampus could be labeled only very slightly for mGluR7b.
Fig. 7.
Distribution of immunoreactivity for mGluRs in the
ipsilateral (A-C, E, G) and contralateral (D, F,
H) hippocampus on the seventh day after placing a
massive unilateral lesion in the perforant path. Adjacent horizontal
sections were reacted with antibodies to mGluR1
(A), mGluR5 (B), mGluR2/3
(C, D), mGluR7a (E, F), and mGluR8
(G, H). The lesion (asterisks)
involves the entorhinal cortex and subiculum. Marked reduction of
immunoreactivity for mGluR2/3 (C), mGluR7a
(E), and mGluR8 (G) is
observed in the ipsilateral perforant path terminal zones of the CA
areas, i.e., stratum lacunosum moleculare (arrowheads in
E) and the molecular layer of the dentate gyrus
(DG). No apparent changes are found in immunoreactivity for mGluR1 or mGluR5 (A, B). An mGluR2/3 immunoreactive
area remains (arrow in C) in CA1 stratum
lacunosum moleculare near CA1-CA2 transition. ipsi,
Ipsilateral to the lesion; contra, contralateral to the
lesion. Scale bar, 500 µm.
[View Larger Version of this Image (155K GIF file)]
Effects of unilateral and bilateral lesions in the entorhinal
cortex on immunoreactivity for mGluRs in the hippocampus
The light microscopic findings on the distribution of mGluRs in
the hippocampus support the idea that the group I and group II/III
mGluRs are localized in postsynaptic and presynaptic elements, respectively. It is not always possible, however, to distinguish between the pre- and postsynaptic immunolabelings by light microscopy, especially if receptors are highly targeted to postsynaptic sites receiving particular presynaptic elements (Nusser et al., 1996) or to
presynaptic sites terminating on particular postsynaptic elements
(Shigemoto et al., 1996). Furthermore, in addition to the major
excitatory pathways, the hippocampus contains many types of extrinsic
and intrinsic afferent fiber groups that have selective termination
areas (Frotscher and Leranth, 1985; Bakst et al., 1986; Wouterlood et
al., 1990; Han et al., 1993; Maglóczky et al., 1994). To clarify
the origins of axon terminals showing mGluR immunoreactivity in the
hippocampus, we next generated various lesions in the sites of origin
of the major hippocampal excitatory pathways.
First, massive unilateral lesions were placed in the entorhinal cortex
to damage the perforant path, a major excitatory afferent path to the
hippocampus (Amaral and Witter, 1995). In the rats with lesion
involving most of the unilateral entorhinal cortex as well as a part of
the adjacent perirhinal area and subicular complex (Fig.
7A-C,E,G, asterisks),
immunoreactivity for mGluR1 and mGluR5 in the hippocampus ipsilateral
to the lesion (Fig. 7A,B) was not different from that on the
contralateral side (not shown). On the other hand, immunoreactivity for
mGluR2/3, mGluR7a, and mGluR8 was markedly reduced ipsilaterally in the
terminal zones of the perforant path (Fig. 7C-H). In
the CA1 stratum lacunosum moleculare, the inner layer of CA3 stratum
lacunosum moleculare, and the middle third of the dentate molecular
layer, mGluR2/3 and mGluR7a immunoreactivity was greatly reduced (Fig.
7C,E). In the outer layer of CA3 stratum lacunosum
moleculare and the outer third of the dentate molecular layer, mGluR7a
and mGluR8 immunoreactivity was reduced (Fig. 7E,G). In a
sector of CA1 near the CA1-CA2 transition, moderate mGluR2/3
immunoreactivity remained in stratum lacunosum moleculare (Fig.
7C, arrow). The mGluR2/3 labeling in this region, however,
disappeared in the rats with massive lesions in the bilateral
entorhinal cortices (not shown), indicating that mGluR2/3
immunoreactivity in this region originates not only from the
ipsilateral but also from the contralateral entorhinal cortex. This
observation is consistent with that of the previous tracer study
showing the contralateral projection of the perforant path to the CA1
sector (Steward, 1976). Labeling for mGluR7a in the inner molecular
layer remained intact (Fig. 7E). In accordance with the
reduction of the mGluR7a immunoreactivity in neuropil, the decoration
of interneuron dendrites in the molecular layer disappeared in the
outer two-thirds of the layer but remained in the inner third of the
layer (not shown). Moderate immunoreactivity for mGluR7a also remained
in a superficial layer of CA1 stratum lacunosum moleculare in the
ipsilateral hippocampus (Fig. 7E), which may suggest
additional sources, such as the nucleus reuniens thalami (Wouterlood et
al., 1990), for mGluR7a-immunoreactive afferent fibers in this layer.
Thus, immunoreactivity for mGluR2/3, mGluR7a, and mGluR8 in the
perforant path terminal zone originates mostly from the ipsilateral and
partly from the contralateral entorhinal cortex.
Effects of unilateral colchicine injection in the hilus on
immunoreactivity for mGluRs in the hippocampus
To examine the contribution of the dentate granule cells to the
mGluR immunoreactivity in the hippocampus, we next used colchicine, which is known to preferentially degenerate the dentate granule cells
(Goldschmidt and Steward, 1980). At the seventh day after unilateral
colchicine injection (3 µg in 0.9 µl of 25 mM
PBS, pH 7.3) into the hilus of the dorsal and ventral hippocampus, a
massive degeneration of the granule cells and mod-erate atrophy of the
whole hippocampus were observed in the dentate gyrus ipsilateral to the
injection (Fig. 8A,B).
Immunoreactivity for mGluR1 and mGluR5 almost disappeared in the
ipsilateral molecular layer, but was affected only slightly in the
hilus and CA3 area (Fig. 8C,D,G,H). On the other
hand, immunoreactivity for mGluR2/3, mGluR7a (Fig.
8I, arrows), and mGluR7b was markedly
reduced in the ipsilateral mossy fiber terminal zone, namely, the hilus
and CA3 stratum lucidum (Fig. 8E,F,I-L), whereas
mGluR2/3 labeling in the molecular layer was reduced only slightly
(Fig. 8E,F). Although immunoreactivity for
mGluR7a was moderately reduced in the molecular layer (Fig. 8I,J), the prominent labeling in the middle
third of the layer was preserved. The reduction of mGluR7a
immunolabeling was observed in the decoration of the dendritic profiles
as well as in neuropil.
Fig. 8.
Effects of unilateral colchicine injection into
the hilus on immunoreactivity for mGluRs in the ipsilateral (A,
C, E, G, I, K) and contralateral (B, D, F, H, J,
L) hippocampus. Adjacent frontal sections were reacted with
antibody to mGluR1 (C, D), mGluR2/3 (E,
F), mGluR5 (G, H), mGluR7a
(I, J), or mGluR7b (K, L). Massive
degeneration of granule cells in the dentate gyrus (DG)
ipsilateral to the colchicine injection is seen in Nissl-stained sections (A, B). On the seventh day after the colchicine
injection, marked reduction of immunoreactivity for mGluR1
(C) and mGluR5 (G) is
observed in the dentate molecular layer ipsilateral to the lesion,
whereas marked reduction of immunoreactivity for mGluR2/3 (arrowhead in E), mGluR7a
(arrows in I), and mGluR7b
(K) is observed in the ipsilateral mossy
fiber terminal zone. Scale bar, 500 µm.
[View Larger Version of this Image (156K GIF file)]
Effects of local and intraventricular kainate injections on
immunoreactivity for mGluRs in the hippocampus
It has been known that local kainate injection degenerates only
postsynaptic elements in the injection site (Coyle and Schwarcz, 1976)
and that intraventricular kainate injection selectively degenerates CA3
pyramidal cells in the hippocampus (Nadler et al., 1980). At the
seventh day after the local kainate injections into the CA3 area (0.2 µg in 0.2 µl of PBS), pyramidal cells in the restricted regions
degenerated with gliosis (Fig.
9A). Immunoreactivity for
mGluR1 and mGluR5 was markedly reduced in the dendritic fields of the
degenerative pyramidal cells (Fig. 9B,D), whereas that for
mGluR2/3 and mGluR7a was unaffected (Fig. 9C,E). The local kainate injection directed to CA1 pyramidal cells also reduced mGluR5
immunoreactivity but not mGluR7a immunoreactivity in the dendritic
fields of the degenerative pyramidal cells (data not shown). After the
bilateral kainate injection (0.6 µg in 1.2 µl of PBS), severe
epileptic response was observed for several hours, as reported
previously (Nadler et al., 1980). At the seventh day after the
injection, massive degeneration of CA3 pyramidal cells and hilar
neurons was observed bilaterally, with most of the CA1 pyramidal cells
and granule cells being preserved (Fig. 9F). Some interneurons, including those in CA1 stratum oriens/alveus, also disappeared, as clearly shown by the lack of the dense
mGluR1-immunoreactive dendritic plexus (Fig. 9G, open
arrows; also see Fig. 2A). This might be
ascribable to susceptibility of these neurons to excitotoxicity after
prolonged stimulation (cf. Ouardouz and Lacaille, 1995). Immunoreactivity for mGluR1 and mGluR5 in the hilus and dendritic fields of CA3 pyramidal cells was also markedly reduced (Fig. 9G,I). In CA1 strata oriens and radiatum, which are
the dendritic fields receiving Schaffer collaterals from CA3 pyramidal
cells, mGluR5 immunoreactivity remained intact (Fig.
9I), whereas mGluR7a immunoreactivity was markedly
reduced (Fig. 9J). Labeling for mGluR7a was also
reduced in CA1 stratum lacunosum moleculare (Fig. 9J,
arrows) but not in CA3 stratum lacunosum moleculare and the middle
third of the dentate molecular layer (Fig. 9J). The
same pattern of reduced immunostaining in the perforant path terminal zone was also apparent for mGluR2/3 immunoreactivity (Fig. 9H, arrows). This effect of the bilateral intraventricular kainate injection is probably attributable to preferential degeneration of the
entorhinal layer III neurons (Nadler et al., 1980) projecting to CA1
stratum lacunosum moleculare (Steward and Scoville, 1976). In fact,
Nissl staining showed massive degeneration of neurons in layer III but
not in layer II of the entorhinal cortex (data not shown). No changes
in immunoreactivity for mGluR2/3 and mGluR7a were detected in CA3
stratum lacunosum moleculare and the outer two-thirds of the dentate
molecular layer, which receive projection fibers from layer II neurons
(Steward and Scoville, 1976). In the inner third of the molecular
layer, labeling for mGluR7a appeared to be reduced, probably because of
loss of associational/commissural projections from the hilar neurons
(Amaral and Witter, 1995). Immunoreactivity for mGluR2/3 and mGluR7a in
the mossy fiber terminal zone remained (Fig. 9H,J),
despite the massive degeneration of postsynaptic elements in this
region.
Fig. 9.
Effects of small unilateral injection
(A-E) and bilateral intraventricular injection
(F-J) of kainate on immunoreactivity for
mGluRs in the hippocampus. A small amount of kainate (0.2 µg in 0.2 µl of PBS) was injected into two small areas in CA3, resulting in
degeneration of CA3 pyramidal cells in the restricted areas as shown
with Nissl staining (arrowheads in A). A
larger amount of kainate (0.6 µg in 1.2 µl of PBS) was injected
into bilateral ventricles to make massive bilateral degeneration of CA3
pyramidal cells, with most CA1 pyramidal cells being preserved (F). Adjacent frontal sections were
reacted with antibodies to mGluR1 (B, G), mGluR2/3
(C, H), mGluR5 (D, I), or
mGluR7a (E, J). On the seventh day after placing the lesions, immunoreactivity for mGluR1 and
mGluR5 is reduced in dendritic fields corresponding to the lesions
(arrowheads in B and D;
CA3 area in G and I), whereas that
for mGluR2/3 and mGluR7a shows no changes after the small injection
(C, E). After the bilateral intraventricular kainate injection, reduction of mGluR2/3 immunoreactivity is apparent in CA1
stratum lacunosum moleculare (arrows in
H), whereas that of mGluR7a immunoreactivity is
marked not only in the same layer (arrows in
J) but also in strata radiatum
(Ra) and oriens (Or) of both CA1 and CA3.
Interneurons immunoreactive to mGluR1 (see Results; compare Fig.
2A) also disappear completely in the CA1 stratum
oriens/alveus border (open arrows in G).
DG, Dentate gyrus; LM, stratum lacunosum
moleculare; Lu, stratum lucidum. Scale bars: 250 µm
for A-E; 500 µm for F-J.
[View Larger Version of this Image (168K GIF file)]
Localization of immunoreactivity for mGluRs as detected by
electron microscopy
As a final step, we performed immunoelectron microscopy in
the hippocampus with the antibodies for mGluR2/3, mGluR2, mGluR4a, mGluR7a, mGluR7b, and mGluR8 (Figs.
10, 11, 12, 13). The previous study revealed
that immunolabeling for mGluR1 and mGluR5 in neuropil of the CA areas
is attributable to staining of dendrites and dendritic spines of
pyramidal and granule cells (Luján et al., 1996).
Immunoreactivity for the group I mGluRs in presynaptic elements,
however, was not detected in the hippocampus. In the present study,
immunolabeling for all of the group II/III mGluRs was observed in
presynaptic elements, with differential patterns of location between
the group II and group III mGluRs.
Fig. 10.
Electron micrographs showing immunoreactivity for
mGluR2/3 (A, C, D, E) and mGluR2
(B) in CA1 stratum lacunosum moleculare (A-C), CA3 stratum lucidum
(D), and CA1 stratum radiatum
(E) as detected by preembedding immunoperoxidase
(A, B, D, E) and immunogold (C)
methods. A, Peroxidase reaction product for mGluR2/3 is
accumulated intracellularly in a preterminal axon
(arrows), which is continuous to an unlabeled axon
terminal making asymmetrical synapses (arrowheads). B, Peroxidase reaction product for mGluR2 is accumulated
extracellularly along axons and axon terminals (double
arrowheads), but not in the synaptic clefts of asymmetrical
synapses (arrowheads). C1, C2, and
C3 were taken from three serial sections. Silver-enhanced immunogold particles for mGluR2/3 are found along a preterminal axon
(arrows), which is continuous to an axon terminal making unlabeled asymmetrical synapses (arrowheads).
Open and closed stars indicate
corresponding regions in the serial sections. D, Peroxidase labeling for mGluR2/3 is observed in mossy fibers
(arrows), one of which (asterisk) is
continuous to a giant mossy fiber terminal (MT)
making unlabeled asymmetrical synapses (arrowhead) with
spines of CA3 pyramidal cells. E, Glial processes are
also labeled for mGluR2/3. Scale bar, 0.5 µm.
[View Larger Version of this Image (225K GIF file)]
Fig. 11.
Electron micrographs showing immunoreactivity for
mGluR4a in the inner third of the dentate molecular layer (A, C,
D) and in CA2 stratum oriens (B).
Peroxidase (A, C, D) and immunogold (B) labeling for mGluR4a is found in presynaptic
membrane specialization of asymmetrical synapses (A, B)
on spines (s) and dendrites (Den) or symmetrical synapses (C, D) on dendrites (Den,
D1). The labeled axon terminal (asterisk in
D) in symmetrical synaptic contact with D1
makes another unlabeled asymmetrical synapse on another dendrite
(D2). T, Axon terminal. Scale bar, 0.5 µm.
[View Larger Version of this Image (90K GIF file)]
Fig. 12.
Electron micrographs showing immunoreactivity for
mGluR7a (A, B) and mGluR7b (C-E)
in CA1 stratum radiatum (A), hilus
(B), and CA3 stratum lucidum
(C-E). Peroxidase reaction product for mGluR7a
is accumulated along presynaptic membrane specialization of
asymmetrical synapses on spines (s) of CA1
pyramidal cells (A) and symmetrical synapses on a
soma (So) in the hilus (B). Immunogold particles for mGluR7b are concentrated in presynaptic membrane specialization of asymmetrical synapses on dendrites and necks
of long spines (C). Symmetrical synapses on a
dendrite (Den) are also labeled for mGluR7b
(D). Note that the accumulation of the peroxidase
reaction product is restricted to active zones of presynaptic membrane
(D). E, A giant mossy fiber
terminal (MT) makes a labeled synapse on a
dendritic profile (asterisk) of a presumed interneuron
and also makes unlabeled synapses on spines (s)
of CA3 pyramidal cells. T, Axon terminal. Scale bars:
0.5 µm for A, B; 0.26 µm for C, D;
0.4 µm for E.
[View Larger Version of this Image (208K GIF file)]
Fig. 13.
Electron micrographs showing immunoreactivity for
mGluR8 in the dentate molecular layer (A, B, D) and CA2
stratum oriens (C). Peroxidase reaction product
is accumulated in axon terminals, which make asymmetrical synapses on
spines (s in A) or dendrites (Den in B; D1 in
D) or a symmetrical synapse on a soma (So
in C). Inset in B
indicates immunogold labeling for mGluR8 concentrated in presynaptic
membrane specialization. Most of the asymmetrical synapses on the
dendritic profile are labeled in B. The axon terminal in
D makes a labeled asymmetrical synapse on a dendrite
(D1) and an unlabeled asymmetrical synapse on another
dendrite (D2). Scale bar, 0.5 µm.
[View Larger Version of this Image (180K GIF file)]
Immunoreactivity for mGluR2/3 was frequently found in small
unmyelinated axons, especially in preterminal rather than terminal portions of axons, and most densely in CA1 lacunosum moleculare (Fig.
10A,C). Even in axon terminals, peroxidase and
immunogold labelings were often found along extrasynaptic membrane and
only rarely detected in the presynaptic membrane specialization.
Labeling detected with the mGluR2 antibody, which was raised against
amino acid residues 87-134, was found in the extracellular space (Fig. 10B), supporting the predicted transmembrane model
for mGluRs (Masu et al., 1991). This situation sometimes made it
difficult to decide which side of the space was the site of the
immunoreaction. In many cases, however, the peroxidase reaction end
product accumulated along the surface of small
unmyelinated axons and terminals in CA1 lacunosum moleculare (Fig.
10B). Around terminals, mGluR2 labeling was not
detected in the synaptic clefts, confirming the results obtained with
the mGluR2/3 antibody. In CA3 stratum lucidum, immunoreactivity for
mGluR2/3 was often observed in axon bundles of the mossy fibers (Fig.
10D) and occasionally in the giant mossy fiber
terminals, but again was not associated with the presynaptic junctional
sites. The mGluR2/3 labeling in the axons seemed to be concentrated on certain spots rather than distributed evenly along the axonal membrane.
Glial processes were also immunopositive for mGluR2/3 everywhere in the
hippocampus (Fig. 10E), probably reflecting a cross-reactivity of the antibody to mGluR3 (Ohishi et al., 1994). Consistently, mGluR3 mRNA expression was also observed in glial cells
in the hippocampus (Ohishi et al., 1993b)
Ultrastructural localization of immunoreactivity for the group III
mGluRs had quite a different pattern from that for mGluR2/3. Peroxidase
reaction end products for mGluR4a, mGluR7a, mGluR7b, and mGluR8 were
predominantly present in axon terminals, often attached to the
presynaptic sites of asymmetrical and symmetrical synapses in the
hippocampus (Figs. 11, 12, 13). Immunogold
particles for these mGluRs were concentrated in the presynaptic
membrane specialization (Figs. 11B, 12C,
and inset to Fig. 13B). Peroxidase reaction
products also accumulated less frequently in axonal profiles, and
single immunoparticles were found occasionally on axonal membranes.
In the dentate molecular layer, terminals making asymmetrical synapses
on spines were immunoreactive for mGluR4a (Fig. 11A), mGluR7a, and mGluR8 (Fig. 13A). Most of the asymmetrical
synapses on spines of granule cells are formed by perforant path
terminals in the outer molecular layer (Matthews et al., 1976) and by
associational/commissural terminals in the inner one-third of the layer
(Kishi et al., 1980). Less frequently, symmetrical synapses on
dendrites were also labeled for mGluR4a (Fig. 11C), mGluR7a,
and mGluR8. The decoration of dendritic profiles observed with light
microscopy (Fig. 4) originated from many immunopositive terminals
making asymmetrical synapses on dendrites of interneurons (Fig.
13B). Virtually all of the terminals making asymmetrical
synapses on the particular interneurons were immunopositive for
mGluR7a, whereas the proportion of immunopositive terminals was lower
for the decoration with mGluR4a and mGluR8. A few terminals with
labeled symmetrical synapses were also in contact with the decorated
dendrites. Occasionally, axon terminals with labeled asymmetrical
synapses on the decorated dendrites made other, unlabeled asymmetrical
synapses on spines. The segregation of receptors in single terminals
was also apparent between two synapses on dendrites (Figs.
11D, 13D). Although the identity of the
postsynaptic dendrites receiving labeled and unlabeled synapses remains
elusive, they seemed to originate from different interneurons.
In the dendritic fields of the CA areas, large numbers of asymmetrical
synapses on spines were immunoreactive for mGluR7a (Fig.
12A). The decoration
of somatodendritic profiles with many intensely labeled asymmetrical
synapses was scattered in the CA areas, as described previously
(Bradley et al., 1996; Shigemoto et al., 1996). For mGluR4a and mGluR8,
labeled synapses decorated dendrites (Fig. 11B) and
cell bodies (Fig. 13C) only
occasionally in the CA areas. In CA3 stratum lucidum and hilus,
immunoreactivity for mGluR7a and mGluR7b was observed in numerous small
axon terminals surrounding dendrites and long spines (Fig.
12C) of interneurons with asymmetrical synapses. Labeling
was found much less frequently in symmetrical synapses on dendrites
(Fig. 12D). On the other hand, cell bodies of the
interneurons were surrounded by many mGluR7a/b-labeled symmetrical
synapses (Fig. 12B). Occasionally, labeling was found in the giant mossy fiber terminals; however, reaction end products were
accumulated only in the presynaptic sites to presumed
mGluR1-positive interneuron dendrites but not in the presynaptic
sites to CA3 pyramidal cell spines (Fig. 12E).
DISCUSSION
The present study revealed the differential distribution of
presynaptic mGluRs in the major excitatory pathways in the hippocampus (Table 1). Type 2/7a/8, type 2/7a/7b, and
type 7a mGluRs are localized in the perforant path, mossy fibers, and
Schaffer collaterals, respectively. Electron microscopy further
revealed predominant localization of group II and group III mGluRs in
the extrasynaptic and synaptic sites, respectively. Finally, each group
III receptor in individual synapses of single presynaptic elements was
found to be segregated in correlation with postsynaptic target
neurons.
Presynaptic mGluRs in the hippocampal pathways
Massive degeneration of postsynaptic elements after lesions may
induce morphological changes in presynaptic elements in the same region
(Nadler et al., 1981), and vice versa (Matthews et al., 1976).
Nonetheless, in the present study, complementary results obtained with
lesions of pre- and postsynaptic elements unequivocally indicated the
major origins of mGluR immunoreactivity in the hippocampus. For
example, the kainate injection into CA1 depleted mGluR5
immunoreactivity in the dendritic fields of the degenerated pyramidal
cells but had no effect on mGluR7a immunoreactivity. Conversely,
extensive lesions made in the entorhinal cortex or CA3 reduced mGluR7a
immunoreactivity in CA1 terminal zones that receive fibers from the
degenerated regions, but exerted no effects on mGluR5 immunoreactivity.
These results clearly indicate that immunoreactivity for mGluR5 and mGluR7a in CA1 originates primarily from pyramidal cell dendrites and
perforant path/Schaffer collateral axons, respectively.
In the perforant path terminating in the CA3 area and the dentate
gyrus, we found complementary localization of mGluR2 and mGluR8 being
enriched in the medial and lateral perforant path, respectively.
Consistent with this finding, expression of mGluR2 and mGluR8 mRNAs was
prominent in layer II of the medial and lateral entorhinal cortex,
respectively (Ohishi et al., 1993a; A. Kinoshita, unpublished
observation). DCG-IV potently reduced field excitatory postsynaptic
potentials in the medial but not in the lateral perforant path, whereas
micromolar concentrations of L-AP4 reduced those in the lateral but not
in the medial perforant path (Macek et al., 1996). These
electrophysiological data are compatible with the complementary
localization of mGluR2 and mGluR8 in the dentate molecular layer.
Enrichment of mGluR7a in the medial perforant path also agrees with the
inhibitory effect of millimolar concentrations of L-AP4 in this path
(Koerner and Cotman, 1981; Macek et al., 1996) because mGluR7a has an
exceptionally high EC50 value (1 mM) for
glutamate (Okamoto et al., 1994). Although mRNA for mGluR4 is strongly
expressed in layers II and III of the entorhinal cortex (Saugstad et
al., 1994; Ohishi et al., 1995a), mGluR4a immunoreactivity was not
clearly detected in the perforant path terminal zones in the present
study. This may be attributable to the preferential expression of
another splice variant mGluR4b (Thomsen et al., 1997) in this pathway;
however, mGluR4 may not be involved in the synaptic depressant effects
in the lateral perforant path, because different pharmacology was found
between cloned mGluR4a and L-AP4-sensitive receptors mediating such
effects (Johansen et al., 1995). In fact, no changes were observed in
the L-AP4 effects on this pathway in mGluR4-deficient mice (Baskys et
al., 1996).
Labeling for mGluR4a was found in asymmetrical synapses on spines and
dendrites and in symmetrical synapses on dendrites in the inner
one-third of the molecular layer, which receives projections from
neurons in the hilus (Amaral and Witter, 1995), septal cholinergic neurons (Frotscher and Leranth, 1985), and supramammillary neurons (Maglóczky et al., 1994). The mossy cells in the hilus and
supramammillary neurons make asymmetrical synapses with granule cells
(Maglóczky et al., 1994; Buckmaster et al., 1996), but mGluR4
mRNA is strongly expressed only in the hilus (Ohishi et al., 1995a),
supporting the mossy cells as the origin of the mGluR4a-labeled axon
terminals. Axon terminals making both mGluR4a-labeled symmetrical
synapses and unlabeled asymmetrical synapses may originate from septal cholinergic neurons, because they form both types of synapses in the
dentate gyrus (Frotscher and Leranth, 1985).
In the mossy fiber synapses on CA3 pyramidal cells, DCG-IV but not
L-AP4 suppressed excitatory transmission in the rat (Lanthorn et al.,
1984; Yokoi et al., 1996). This effect was reduced to ~50% of the
control level in mice lacking mGluR2 (Yokoi et al., 1996), indicating
the involvement of mGluR2 as well as other DCG-IV-sensitive receptors.
In the present study, mGluR7a and mGluR7b were found in the mossy fiber
but not in synapses on CA3 pyramidal cells. They are present almost
exclusively in synapses on mGluR1-positive interneurons, which
correspond to somatostatin/GABA-immunoreactive interneurons (Baude et
al., 1993). These interneurons are heavily innervated by mossy fiber
collaterals (Leranth et al., 1990), and virtually all asymmetrical
synapses made on the interneurons had high densities of mGluR7a/b,
showing up as almost continuous profiles of dendrites in light
microscopy (Fig. 6). Cell bodies also showed up with many symmetrical
synapses labeled for mGluR7a/b. The somatostatin-immunoreactive
interneurons constitute symmetrical synapses on their cell bodies and
dendrites with septal cholinergic (Leranth and Frotscher, 1987) and
GABAergic (Milner and Bacon, 1989) axon terminals, and mGluR7 mRNA is
strongly expressed in the medial septal and hilar neurons (Ohishi et
al., 1995a). Smaller numbers of symmetrical synapses on interneurons
were also labeled for mGluR4a and mGluR8 in the present study. Although
depression of inhibitory synaptic transmission is mediated by group II
mGluRs in the hippocampal pyramidal cells (Poncer et al., 1995), no
such effects mediated by group III mGluRs have been reported so
far.
Several lines of evidence have suggested that group I and III mGluRs
are involved in presynaptic inhibition of excitatory transmission from
Schaffer collaterals to CA1 pyramidal cells in the adult rat (Gereau
and Conn, 1995a; Manzoni and Bockaert, 1995). In the Schaffer
collateral terminal zones in CA1, mGluR5 and mGluR7a immunoreactivity
was strongly detected in the present study. Although the presynaptic
location of mGluR7a is consistent with the reported effects of
millimolar concentrations of L-AP4, we found no evidence for the
presence of presynaptic mGluR5 (also see Luján et al., 1996). It
should be noted, however, that the inhibitory effect of DHPG depends on
NMDA receptor activation in these synapses (Harvey et al., 1996). This
finding raises the possibility of a synergistical mechanism involving
postsynaptic mGluR5 and NMDA receptors to depress presynaptic glutamate
release through another messenger, such as adenosine (Manzoni et al., 1994; Di Iorio et al., 1996) or nitric oxide (Boulton et al., 1994).
Extrasynaptic versus synaptic localization of group II and group
III mGluRs
One of the most striking observations in the present study is the
segregation of group II and group III mGluRs in presynaptic elements.
The difference in mGluR localization in relation to glutamate release
sites suggests that effector mechanisms as well as the sources of
glutamate activating these receptors may be distinct from each other.
It is conceivable that the group III mGluRs in the presynaptic active
zone act as autoreceptors activated with glutamate released from the
very synapse to which the receptors are localized. Patch-clamp
recordings from the presynaptic terminals directly indicated that
L-AP4-sensitive mGluRs are coupled to P/Q-type voltage-sensitive
calcium channels to suppress excitatory postsynaptic currents in the
trapezoid body nucleus (Takahashi et al., 1996). In the present study,
the group III mGluRs were found also in symmetrical synapses, which
have much lower glutamate concentrations than asymmetrical synapses in
the hippocampus (Bramham et al., 1990). For such nonglutamatergic
synapses, a major question would be the source of glutamate that
activates the presynaptic mGluRs. GABA release may be inhibited by
glutamate spilled over from nearby synapses (Hayashi et al., 1993;
Ohishi et al., 1994) or released from dendrosomatic regions (Glitsch et
al., 1996). The functional significance of presynaptic mGluR7a/b in
nonglutamatergic synapses is unclear, however, because it is unlikely
that receptors with such a low affinity to glutamate are activated by
diluted signals from remote synapses.
In contrast to group III mGluRs enriched at synaptic sites, mGluR2 is
mainly localized apart from synaptic sites. Localization of mGluR3 in
neuronal cells is inconclusive in the present study; only a small
portion of mGluR2/3 immunoreactivity is ascribable to mGluR3 because of
weak cross-reactivity of this antibody to mGluR3 (Hayashi et al.,
1993). Extrasynaptic mGluR2 may be activated not only with glutamate
released from homologous presynaptic elements but also with glutamate
from heterologous presynaptic elements that make asymmetrical synapses
near the mGluR2-bearing axons. Recent studies showed that glutamate
accumulated by repeated stimulations with short intervals, but not by
single stimulations, activates presynaptic group II mGluRs in mossy
fibers to induce long-term depression (Yokoi et al., 1996) and to
suppress excitatory transmission (Scanziani et al., 1997). Although
these studies are suggestive of the autoreceptor function for mGluR2,
it is also possible that mGluR2 is activated by prolonged glutamate
release from nearby synapses to cause heterosynaptic interaction. For
the effectors of group II mGluRs, N- and L-type calcium channels were
reported in cultured hippocampal neurons (Sahara and Westbrook, 1993)
and in heterologous expression systems (Ikeda et al., 1995). Each type
of neuron, however, expresses a distinct set of calcium channel subtypes in certain subcellular compartments (Sakurai et al., 1996),
and it is not clear whether mGluR2 is coupled to these channels in the
preterminal axons. An alternative possible effector mechanism for the
axonal mGluR is reducing efficacy of axonal signal transmission by
facilitating potassium channels. In the perforant path, which was most
strongly labeled for mGluR2 in the present study, voltage-dependent
potassium channel subunits Kv1.2/1.4 (Sheng et al., 1993) and
G-protein-coupled inwardly rectifying potassium channels (Ponce et al.,
1996) are abundant, and the latter is efficiently coupled to mGluR2 in
a Xenopus oocyte expression system (Saugstad et al., 1996).
High-resolution colocalization of mGluR2 and these channel/effector
molecules in axons would help to clarify the physiological implication
of this receptor.
Target-specific segregation of group III mGluRs in
single axons
A target cell-specific concentration of receptor proteins in the
presynaptic active zone was first reported for mGluR7a in the
hippocampus (Shigemoto et al., 1996). This type of receptor segregation
seems to be a general feature of the group III mGluRs. In the present
study, mGluR7b was concentrated, even more exclusively than mGluR7a, in
synapses making contacts with presumed mGluR1-positive interneurons
in the mossy fiber terminal zone. Synapses on CA3 pyramidal cells made
by the same mossy fiber had very little labeling for mGluR7b. Not only
glutamatergic synapses but also nonglutamatergic synapses on cell
bodies of the mGluR1-positive interneurons were labeled for
mGluR7a/b. Although it is not known whether nonglutamatergic neurons
also locate these receptors according to distinct targets along single
axons, this finding is suggestive of some postsynaptic influence common
to axon terminals of different types. A similar segregation according
to postsynaptic targets was found for mGluR4a and mGluR8, but not all
of the synapses on the interneurons were labeled. This may be
attributable to the lack of expression of these mGluRs in some neurons
providing afferents to those interneurons, or to unknown local third
factor(s) governing the selective distribution of these mGluRs in
individual synapses. In any case, such a segregation should be
regulated in a spatially restricted way, probably through nondiffusable
membrane-bound molecules. Different regulation of transmitter release
mediated by L-AP4-sensitive mGluRs may result in differential
transmission of time-coded signals to principal neurons and to
interneurons (Shigemoto et al., 1996). In mGluR4- and mGluR7-deficient
mice, smaller EPSCs than in wild-type mice were detected in the second
or third response to high-frequency stimulation (Bushell et al., 1996b;
Pekhletski et al., 1996). This implies that synapses equipped with
these mGluRs transmit high-frequency signals with higher efficiencies
than do synapses without them. It is interesting to note that all cells
decorated with synapses heavily labeled for group III mGluRs seem to be interneurons. This situation may allow effective feedback for high-frequency signals through interneuron subtypes that have selective
targets (Buhl et al., 1994). Thus, the physiological significance of
the differential synaptic regulation should be understood in light of
identified neuronal connections in the complex circuitry.
FOOTNOTES
Received March 28, 1997; revised July 14, 1997; accepted July 16, 1997.
This work was supported by research grants from the Ina
