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 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 2001, 21(6):1838-1847
Differential Subcellular Localization of mGluR1a and mGluR5 in
the Rat and Monkey Substantia Nigra
George W.
Hubert,
Maryse
Paquet, and
Yoland
Smith
Yerkes Regional Primate Research Center, Division of Neuroscience
and Department of Neurology, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
Neurons in the rat substantia nigra (SN) are enriched in group I
metabotropic glutamate receptor (mGluR) subtypes and respond to group I
mGluR activation. To better understand the mechanisms by which mGluR1
and mGluR5 mediate these effects, the goal of this study was to
elucidate the subsynaptic localization of these two receptor subtypes
in the rat and monkey substantia nigra. At the light microscope level,
neurons of the SN pars reticulata (SNr) displayed moderate to strong
immunoreactivity for both mGluR1a and mGluR5 in rats and monkeys.
However, mGluR1a labeling was much stronger in monkey than in rat SN
pars compacta (SNc) neurons, whereas a moderate level of mGluR5
immunoreactivity was found in both species. At the electron microscope
level, the immunoreactivity for both group I mGluR subtypes was
primarily expressed postsynaptically, although light mGluR1a labeling
was occasionally seen in axon terminals in the rat SNr. Immunogold
studies revealed a striking difference in the subcellular distribution
of mGluR1a and mGluR5 immunoreactivity in SNr and SNc neurons. Although
the bulk of mGluR1a was attached to the plasma membrane, >80% of
mGluR5 immunoreactivity was intracellular. Plasma membrane-bound
immunoreactivity for group I mGluRs in both SNc and SNr neurons was
mostly extrasynaptic or in the main body of symmetric, putative
GABAergic synapses. On the other hand, asymmetric synapses
either were nonimmunoreactive or displayed perisynaptic labeling. These
data raise important questions about the trafficking, internalization,
and potential functions of group I mGluRs at extrasynaptic sites or
symmetric synapses in the substantia nigra.
Key words:
metabotropic; glutamate; receptor internalization; receptor trafficking; dopamine neurons; Parkinson's disease; immunogold method
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INTRODUCTION |
The substantia nigra pars reticulata
(SNr) and the internal segment of the globus pallidus are the
main output nuclei of the basal ganglia, whereas the SN pars compacta
(SNc) is a group of dopaminergic neurons that project to the striatum.
Both the SNr and SNc receive glutamatergic innervation from the
subthalamic nucleus), cerebral cortex, and pedunculopontine tegmental
nucleus (Wichmann and DeLong, 1998 ).
The effects of glutamate in the CNS are mediated by activation of
ionotropic and metabotropic receptors. Ionotropic receptors are
ligand-gated cation channels that mediate fast excitatory neurotransmission, whereas metabotropic glutamate receptors (mGluRs) belong to a family of G-protein-coupled receptors that mediate modulatory effects of synaptic transmission by activation of a number
of intracellular metabolic pathways. The family of mGluRs is subdivided
into three groups (Conn and Pin, 1997). The group I mGluRs consist of
mGluR1, mGluR5, and all their splice variants. These receptors are
coupled to phosphoinositol hydrolysis and usually induce slow
depolarization. On the other hand, group II (mGluR2,3) and group III
(mGluR4,6,7,8) mGluRs are negatively coupled to adenylate cyclase and
often induce presynaptic inhibition of transmitter release. Electron
microscopic studies revealed that group I mGluRs are located
perisynaptically to the postsynaptic specializations of asymmetric
glutamatergic synapses in the hippocampus and cerebellum in the rat
(Baude et al., 1993; Nusser et al., 1994; Lujan et al., 1996; Ottersen
and Landsend, 1997) and various basal ganglia structures in monkeys
(Hanson and Smith, 1999; Smith et al., 2000). However, we recently
demonstrated that group I mGluRs are also associated with the active
zones of GABAergic striatopallidal synapses in the monkey (Hanson and
Smith, 1999), which raises interesting questions about the functions
and mechanisms of activation of group I mGluRs in the primate basal
ganglia. Obviously, the localization of a receptor relative to its
source of activation might have a significant effect on the
transduction of the glutamatergic signal by a number of means. For
instance, the proximity of mGluRs to other receptor subtypes may affect the response of these receptors to synaptic activation via various mechanisms, including activation of common second-messenger systems (Greengard et al., 1999), modulation of membrane excitability (Conn and
Pin, 1997), or direct protein-protein interactions (Liu et al.,
2000).
Group I mGluR mRNA and the receptor proteins are expressed in rat SNc
and SNr neurons (Berthele et al., 1998; Kosinski et al., 1998; Testa et
al., 1994, 1998). Furthermore, electrophysiological studies have
demonstrated that metabotropic glutamatergic stimulation has myriad
effects on the firing rate and firing pattern of neurons in both
portions of the SN (Meltzer et al., 1997; Fiorello and Williams, 1998;
Wigmore and Lacey, 1998; Marino et al., 1999). To better
understand the synaptic mechanisms by which mGluRs mediate these
effects, the goal of the present study was to characterize the
subsynaptic localization of Group I mGluRs in the rat and monkey SN.
Data presented in this study have been published previously in abstract
form (Hubert and Smith, 1999).
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MATERIALS AND METHODS |
Animals and tissue preparation
Twenty Sprague Dawley rats and five adult Rhesus monkeys were
deeply anesthetized with pentobarbital (100 mg/kg, i.v., for monkeys)
or a mixture of ketamine (100 mg/kg, i.p., for monkeys) and
dormitor (0.1 mg/kg, i.p., for rats) and transcardially perfused with
100-300 ml of cold, oxygenated Ringer's solution. This was followed
by 500-1000 ml of fixative containing 4.0% paraformaldehyde and
0.1-0.75% glutaraldehyde dissolved in phosphate buffer (PB; 0.1 M, pH 7.4). Free aldehydes were then washed from the brains by perfusion with 500 ml of cold PB. Brains were removed from the
skull, cut in 10-mm-thick slabs, and stored in PBS (0.01 M, pH 7.4) overnight at 4°C. The brains were then cut
into 60-µm-thick coronal sections on a vibrating microtome. Sections
were put in a 1.0% sodium borohydride solution, dissolved in PBS for
20 min, and rinsed with PBS before being processed for
immunocytochemistry. The anesthesia and euthanasia procedures were
performed according to the National Institutes of Health
Guidelines and have been accepted by the Institutional Animal
Care and Use Committee of Emory University. All efforts were made to
reduce the number of animals used and minimize animal suffering.
Immunoperoxidase localization of mGluR1a and mGluR5
Primary antisera. Two commercially available
affinity-purified rabbit polyclonal antibodies raised against synthetic
C-terminus peptides representing different amino acid sequences of
mGluR1a (PNVTYASVILRDYKQSSSTL; Chemicon, Temecula, CA) and mGluR5a/b
(KSSPKYDTLIIRDYTNSSSSL; Upstate Biotechnology, Lake Placid, NY) were
used in this study. In immunoblot analysis of rat brain microsomes or
rabbit brain extracts, both antibodies labeled a single band with an
estimated molecular weight of 145 kDA, which corresponds to that of
mGluR1a and mGluR5 proteins (Houamed et al., 1991; Abe et al.,
1992; Minakami et al., 1993; Testa et al., 1998).
Light microscope procedures. The sections were preincubated
in a solution containing 10% normal goat serum (NGS), 1.0% bovine serum albumin (BSA), and 0.3% Triton X-100 in PBS for 1 hr. They were
then incubated overnight with primary antisera diluted at 0.5-1.0 µg/ml in a solution containing 1.0% NGS, 1.0% BSA, and 0.3% Triton X-100 in PBS. Next, the sections were rinsed in PBS and
transferred for 1 hr to a secondary antibody solution containing biotinylated goat anti-rabbit IgGs (Vector Laboratories, Burlingame, CA) diluted 1:200 in the primary antibody diluent solution. After rinsing, sections were put in a solution containing 1:100
avidin-biotin-peroxidase complex (Vector). The tissue was then washed
in PBS and 0.05 M Tris buffer before being
transferred to a solution containing 0.01 M
imidazole, 0.0005% hydrogen peroxide, and 0.025%
3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in Tris
for 7-10 min. Sections were then mounted on gelatin-coated slides and
dried, and a coverslip was applied with Permount.
Electron microscope procedures. After vibratome cutting,
sections were transferred to a cryoprotectant solution (PB, 0.05 M, pH 7.4, containing 25% sucrose and 10%
glycerol) for 20 min. They were then frozen in a  80°C freezer for
20 min, returned to a decreasing gradient of cryoprotectant solutions,
and rinsed in PBS. Sections underwent immunocytochemical procedures for
the immunoperoxidase localization of mGluR1a and mGluR5 in a manner identical to that for the light microscope, except that the incubation in the primary antisera was performed at 4°C for 48 hr and Triton X-100 was omitted from all incubation solutions.
The sections were transferred to PB (0.1 M, pH 7.4) for 10 min and exposed to 1% osmium tetroxide for 20 min. They were then rinsed with PB and dehydrated in an increasing gradient of ethanol. Uranyl acetate (1%) was added to the 70% alcohol to increase contrast at the electron microscope. The sections were then treated with propylene oxide before being embedded in epoxy resin (Durcupan, ACM; Fluka, Buchs, Switzerland) for 12 hr, mounted on microscope slides, and placed in a 60°C oven for 48 hr.
Pieces of SNc and SNr tissue were taken out from the slides and glued
on the top of resin blocks with cyanoacrylate glue. They were cut into
60 nm ultrathin sections with an ultramicrotome (Ultracut T2; Leica,
Nussloch, Germany) and serially collected on single-slot
Pioloform-coated copper grids. The sections were stained with lead
citrate for 5 min (Reynolds, 1963) and examined with a Zeiss
EM-10C electron microscope (Thornwood, NY).
Immunogold localization of mGluR1a and mGluR5
Sections processed for pre-embedding immunogold were transferred
to the cryoprotectant solution and frozen at 80°C in the same way
as those processed for immunoperoxidase. They were then preincubated
for 1 hr in a solution containing 10% NGS in PBS-BSA (0.005% BSA,
0.05% Tween 20, and 0.001% gelatin in PBS) before being transferred
to a 1% NGS in PBS-BSA solution containing the primary
antibodies for either mGluR1a (0.5 µg/ml) or mGluR5 (1.0 µg/ml) for
48 hr at 4°C. Next, they were rinsed in PBS-BSA and incubated for 2 hr in the secondary 1.4 nm gold-conjugated goat anti-rabbit IgGs
(Nanogold; Nanoprobes, Stonybrook, NY) at a concentration of 1:100 in
1% NGS in PBS-BSA. The sections were then fixed overnight in 1-2%
glutaraldehyde and rinsed with PB before the silver intensification of
gold particles for 5-10 min using the HQ silver kit (Nanoprobes). Next, they were rinsed with PB, treated with osmium tetroxide (1.0% in
PB, 0.1 M, pH 7.4) for 20 min, and dehydrated in a graded series of alcohol and propylene oxide. The remainder of the procedure was the same as that described above for the immunoperoxidase material.
Control experiments
For both immunoperoxidase and immunogold reactions, controls
included sections of SN incubated in solutions in which the primary antisera were replaced by 1% nonimmune rabbit serum; the rest of the
procedure remained the same as described above. Sections processed in this way were totally devoid of immunoperoxidase deposit
or gold particles at the electron microscope level.
Analysis of material
Immunoperoxidase material. A total of 14 blocks
from rat and monkey SNr [4 (2 for mGluR1a, 2 for mGluR5) from rat, 3 (2 for mGluR1a, 1 for mGluR5) from monkey] and SNc [7 (6 for mGluR1a, 1 for mGluR5) from monkey] were cut out from the slides, mounted on
resin blocks, and glued into place using cyanoacrylate glue. In both
rats and monkeys, all blocks were collected from the mid rostrocaudal
level of the substantia nigra (bregma 5.6 mm in rat) where the SNc
and SNr are clearly separate. The SNc blocks were taken from the dense
cluster of dopaminergic neurons in the dorsomedial part of the SN,
whereas SNr blocks were collected from the ventral part of the SN along
the cerebral peduncle. Ultrathin sections were scanned for the presence
of immunoperoxidase-labeled structures that were easily distinguishable
from unlabeled elements by their electron-dense reaction product.
Low-power (10,000×) and high-power (<20,000×) electron micrographs
of randomly selected immunoreactive elements were taken for analysis.
Immunoreactive elements were categorized as presynaptic (axons,
terminals) or postsynaptic (perikarya, dendrites, spines) neuronal
structures or glial cells on the basis of ultrastructural features.
Immunogold material. A total of 19 blocks (Table
1) from rat and monkey SNr and SNc were
cut out from the slides, mounted on resin blocks, and glued into place
using cyanoacrylate glue. Blocks were collected in the same regions of
SNc and SNr as those taken for immunoperoxidase observations (see
above). Ultrathin sections from the surface of the blocks were examined
under the electron microscope, and randomly selected elements
containing gold particles were photographed at low (10,000×) and high
( 20,000×) magnifications. Low-power micrographs were taken to
determine the overall distribution of immunogold particles, whereas
high-power micrographs were used to determine the relationship of
immunoreactivity to specific synaptic contacts. Immunogold particles
were designated as membrane-bound if they were in direct contact with
the plasma membrane. All other gold particles were categorized as
intracellular. On the basis of their localization relative to synapses,
membrane-bound immunogold particles were classified into three main
categories: (1) extrasynaptic, if they were attached to parts of
the plasma membrane not involved in synaptic contacts; (2)
perisynaptic, if they were located <20 nm away from the edges of
symmetric or asymmetric postsynaptic specializations; or (3) synaptic,
if they were located in the main body of postsynaptic specializations. Portions of dendrites and cell bodies that were poorly preserved or cut
in a plane of a section that was not suitable to distinguish the
presynaptic and postsynaptic membranes were not considered in this
analysis. To ascertain the specificity of labeling, some immunoreactive
synapses were examined in a series of three to five ultrathin
sections.
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Table 1.
Summary of material used for the immunogold localization of
mGluR1a and mGluR5 in the substantia nigra of rat and monkey
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To measure the surface of dendrites and the length of synaptic
junctions, micrographs of immunolabeled dendrites taken from the SNr
were scanned with a digital scanner (Powerlook II; Umax, Fremont,
CA) and analyzed for total dendritic membrane length and total
length of synaptic active zones using a Neurolucida setup and Morph
software (MicroBrightField, Colchester, VT).
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RESULTS |
Light microscope observations
At the light microscope level, the pattern of labeling for both
mGluR subtypes followed a mediolateral gradient, being heaviest ventrolaterally and progressively lighter dorsomedially (Fig. 1A,C,E).
Both antibodies stained neuronal processes more intensely than cell
bodies. The SNc displayed a light to moderate staining for mGluR1a and
mGluR5, respectively (Fig.
1A,C).
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Figure 1.
Light microscope examination of Group I mGluR
distribution in the right ventral midbrain of rat and monkey using the
immunoperoxidase method. A, Low-power micrograph of
mGluR1a in the rat. B, Low-power micrograph of mGluR1a
in the monkey. C, Low-power examination of mGluR5 in the
rat. D, Low-power micrograph of mGluR5 in the monkey. In
A-D, medial is on the left, and dorsal
is on the top of each micrograph. E,
High-power micrograph of mGluR5 in the ventrolateral part of SNr in the
rat. F, High-power micrograph of mGluR1a-immunoreactive
dendrites of SNc neurons descending into the SNr. CP,
Cerebral peduncle; Mk, monkey. Scale bars:
A, 500 µm (valid for C);
B, 500 µm (valid for D);
E, 50 µm (valid for F).
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In the monkey, the pattern of immunoreactivity for mGluR5 resembled
that in the rat (Fig. 1D). In contrast, the antibody
for mGluR1a strongly labeled both SNc and SNr neurons (Fig.
1B). As in the rat, neuronal processes were
preferentially labeled over cell bodies. High-power light microscope
examination of the SNc showed intensely labeled mGluR1a-containing
dendrites of SNc neurons descending into the SNr (Fig.
1F).
Electron microscope observations
Immunoperoxidase labeling
Blocks of SNc and SNr immunostained for mGluR1a or mGluR5 were
examined at the electron microscope level, and immunoreactive elements
were categorized according to their ultrastructural features. Although
blocks from the SNr were collected from the ventral part of the SN (see
Materials and Methods), they also contained dopaminergic dendrites from
neurons in the ventral tier of the SNc that travel dorsoventrally
throughout the SN (Fig. 1F). Because of technical limitations in combining mGluR immunostaining with tyrosine hydroxylase labeling, we could not use any marker to differentiate dopaminergic from nondopaminergic elements in this study. However, on the basis of
previous findings, we used ultrastructural criteria to categorize dendrites as belonging to SNr or SNc neurons. Indeed, it is well established that neurons in the SNr are tightly surrounded by striatal
GABAergic terminals that display a very particular rosette-like pattern
of dendritic innervation (Grofova and Rinvik, 1970; Hajdu et al., 1973;
Smith and Bolam, 1991; Smith et al., 1998) (Fig. 2B,D).
On the other hand, dendrites of SNc neurons are innervated much less by
striatal terminals and often are devoid of synaptic contacts (Bolam and
Smith, 1990; Smith et al., 1996) (Fig.
2A,C). In the following account,
the characterization of SNc and SNr dendrites was therefore based on
these ultrastructural features. Dendrites that could not be categorized
as SNc or SNr elements on the basis of these criteria were omitted from
the study.
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Figure 2.
Electron microscope examination of group I mGluR
immunoperoxidase labeling in the ventral midbrain. A,
mGluR1a-immunoreactive (IR) dendrites (den) in the
monkey SNc. Note the apposition between the two immunoreactive
dendrites (arrows). B, mGluRla-IR
dendrite in the monkey SNr. Note the different pattern of innervation
of SNc and SNr dendrites (A, B).
C, An mGluR5-containing dendrite and spine
(sp) in the monkey SNc. Note the dense peroxidase
deposit at the axodendritic synapse (arrowhead).
D, An mGluR5-containing dendrite in the rat SNr.
Mk, Monkey. Scale bars: A, 0.25 µm
(valid for B, C); D, 0.25 µm.
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In keeping with light microscope observations, immunostaining for both
mGluR1a and mGluR5 was confined mainly to postsynaptic elements,
including dendritic processes and spine-like structures in the monkey
SNc (Fig. 2A,C) as well as in the
rat and monkey SNr (Fig. 2B,D). In
general, the peroxidase deposit was diffuse and did not display any
particular relationship with synaptic sites on the plasma membrane
(Fig. 2). Occasionally, light mGluR1a immunoreactivity was found in
axon terminals and glial processes in both rat and monkey SNr (Marino
et al., 1999). As expected on the basis of the light microscopic data,
putative dendrites and cell bodies of rat SNc neurons were mostly
devoid of mGluR1a immunoreactivity and displayed light mGluR5 labeling.
Immunogold labeling
The pre-embedding immunogold method was used to determine the
subsynaptic location of group I mGluRs. This method offers a higher
spatial resolution than the amorphous peroxidase reaction product and
allows better characterization of the localization of receptors in
relation to synaptic contacts. Because of the low level of labeling for
both receptor subtypes in rat SNc neurons, the immunogold data were
only gathered from SNr elements in rats, whereas both SNr and SNc
elements were analyzed in monkeys. The most striking finding that
characterized the pattern of group I mGluR immunogold labeling
in the SN was the difference in the relative proportion of mGluR1a and
mGluR5 labeling bound to the plasma membrane in rat and monkey SNr
neurons (Figs. 3,
4). Although up to two-thirds of mGluR1a
immunolabeling was attached to the plasma membrane, >80% of mGluR5
labeling was intracellular (Figs. 3A,B, 4). The bulk of intracellular
immunogold labeling in perikarya was associated with the endoplasmic
reticulum (Fig. 3C), but gold particles were also attached
to the nuclear membrane and endosome-like vesicles. In dendrites, gold
particles were often attached to microtubules or endosome-like
vesicles, but most were associated with unidentified intracellular
compartments (Fig. 3B,D).
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Figure 3.
Electron micrographs showing the subcellular
distribution of mGluR1a (A, C) and mGluR5
(B, D) immunogold labeling in the rat
SNr. A, Low-power electron micrograph of two mGluR1a-IR
dendrites (den). Note that most of the gold particles
are on the plasma membrane. B, Low-power electron
micrograph of mGluR5-positive dendrites. Note that most of the gold
particles are intracellular. C, High-power electron
micrograph of mGluR1a immunoreactivity associated with the endoplasmic
reticulum in a neuronal perikaryon. Small arrows
indicate immunoreactive gold particles at a symmetric synapse.
D, High-power electron micrograph of mGluR5
immunoreactivity in a dendrite. ER, Endoplasmic
reticulum. Scale bars: A, 0.5 µm (valid for
B); C, 0.25 µm (valid for
D).
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Figure 4.
Relative proportion of intracellular versus plasma
membrane-bound immunoreactive gold particles for Group I mGluRs in
dendrites of SNr neurons in the rat and monkey and SNc neurons in the
monkey. Number of gold particles (n) and
dendrites (den) examined: mGluR1a in rat SNr (n = 306, den = 20); mGluR1a in monkey SNr (n = 1811, den = 178); mGluR1a in monkey SNc (n = 1566, den = 230); mGluR5 in rat SNr (n = 820, den = 20); mGluR5 in monkey SNr (n = 5350, den = 200); mGluR5 in monkey SNc (n = 9699, den = 227).
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Although the density of gold particles was much lower than in the SNr,
significant immunostaining for both mGluR subtypes was found in
putative SNc neurons in monkeys (see Fig. 6). Overall, the pattern of
labeling in the monkey SNc was the same as in the SNr, i.e., a large
proportion of mGluR1a labeling was bound to the plasma membrane,
whereas the bulk of mGluR5 immunostaining was localized intracellularly
(Fig. 4). However, even the intracellular mGluR1a labeling was slightly
higher than the plasma membrane-bound immunoreactivity in the monkey
SNc (Fig. 4).
High-power photomicrographs were taken to determine the location of
plasma membrane-bound mGluRs in relation to symmetric or asymmetric
synaptic junctions in rat and monkey SNr and in the monkey SNc (Figs.
5, 6). As
described above, gold particles attached to the plasma membrane were
categorized as extrasynaptic, perisynaptic, or synaptic on the basis of
their localization relative to synaptic junctions. A common finding for
both compartments of the SN was that most plasma membrane-bound gold
particles were extrasynaptic (Fig.
7A,B).
Furthermore, as was the case in the monkey pallidum (Hanson and Smith,
1999), a substantial proportion of mGluR1a- and mGluR5-immunoreactive
gold particles were expressed in the main body of symmetric synapses
established by putative GABAergic striatal-like terminals in the rat
and monkey SN (Figs. 5A-D,F,
6B,D, 7). To ensure that this
labeling was not an artifact of single sections, we followed individual
synapses serially and found that the synaptic labeling at symmetric
synapses was maintained in at least three to five serial sections (Fig.
5B,D,F). Because gold particles range from 20 to 40 nm in diameter (Hanson and Smith,
1999), a single particle cannot be found in more than two sections.
Therefore, the fact that the labeling at some synapses was found in at
least three sections indicates that multiple gold particles contributed
to the immunostaining. Because SNr neurons are tightly surrounded by
striatal terminals, one may speculate that the expression of mGluRs at
striatonigral synapses merely reflects a random distribution of
labeling caused by the high frequency of these synapses. To rule out
this possibility, we measured the length of the active zones of
synapses on a series of 20 randomly chosen immunoreactive dendrites in
the rat SNr and compared the proportion of dendritic surface occupied
by the synaptic contacts with the proportion of plasma membrane-bound gold particles at symmetric synapses. These measurements revealed that
~70% of SNr dendritic membrane does not contribute to synaptic contacts, 26% contributes to symmetric synapses, and <3% contributes to asymmetric synapses. Therefore, the proportion of labeling at
symmetric synapses (35-45%) is significantly higher than what would
be expected on the basis of a random distribution, which further
indicates the specificity of labeling. The remaining plasma membrane-bound gold particles were located perisynaptically to asymmetric synaptic junctions (Figs. 5E,
6A,C, 7). None of the asymmetric
synapses examined displayed synaptic labeling, which is consistent with
previous data on group I mGluRs localization in other brain regions
(Ottersen and Landsend, 1997; Hanson and Smith, 1999; Smith et al.,
2000)
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Figure 5.
Subsynaptic distribution of Group I
mGluR-immunoreactive gold particles in dendrites (den)
of SNr neurons in rat and monkey. A, mGluR5
immunoreactivity at symmetric synapses (small arrows)
and at an asymmetric synapse (large arrow) in the monkey
SNr. The arrowhead indicates the postsynaptic
specialization of the asymmetric synaptic contact. B,
D, F, Serial sections of an mGluR1a-IR
dendrite in the rat SNr. Note that the immunoreactivity at symmetric
synapses established by three striatal-like terminals
(t1, t2, t3) is found in
serial sections. C, mGluR5 immunoreactivity at a
symmetric synapse in the rat SNr (small arrows).
E, mGluR1a immunoreactivity (arrow) at
the edge of an asymmetric axodendritic synapse
(arrowhead) in the rat SNr. Scale bars:
A, 0.25 µm; B, 0.25 µm (valid for
D, F); C, 0.25 µm
(valid for E).
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Figure 6.
Subsynaptic localization of Group I mGluRs in the
monkey SNc. A, mGluR1a-immunoreactive gold particles
(large arrows) perisynaptic to three asymmetric
postsynaptic specializations (arrowheads).
B, A mGluR1a-immunoreactive gold particle (small
arrow) in the main body of a symmetric synapse.
C, mGluR5 immunoreactivity (large arrows)
perisynaptic to an asymmetric axodendritic synapse
(arrowhead). D, mGluR5 immunoreactivity
(small arrow) in the main body of a symmetric synapse.
Scale bar, 0.25 µm (valid for B-D).
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Figure 7.
Relative subsynaptic distribution of plasma
membrane-bound mGluR1a- and mGluR5-immunoreactive gold particles in the
SNr and SNc of rat and monkey. Note that the bulk of mGluR1a and mGluR5
immunoreactivity is extrasynaptic. Number of gold particles
(n) and dendrites (den) examined: mGluR1a in
monkey SNc (n = 662, den = 230); mGluR1a in
monkey SNr (n = 963, den = 178); mGluR5 in
monkey SNc (n = 834, den = 227); mGluR5 in
monkey SNr (n = 653, den = 200); mGluR1a in
rat SNr (n = 400, den = 81); mGluR5 in rat SNr
(n = 230, den = 127). Syn-Asym,
Synaptic in relation to an asymmetric synapse; Per-Asym,
perisynaptic to an asymmetric synapse; Syn-Sym, synaptic
to a symmetric synapse; Per-Sym, perisynaptic to an
asymmetric synapse; Extra, extrasynaptic.
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DISCUSSION |
The results of this study provide the first description of the
subsynaptic localization of group I mGluRs in the rat and monkey substantia nigra. Three major conclusions can be drawn from these data.
First, mGluR1a immunoreactivity is more strongly expressed in monkey
than in rat SNc neurons. Second, the relative proportion of plasma
membrane-bound mGluR1a and mGluR5 immunoreactivity is drastically
different in rat and monkey SNr. In general, most of the mGluR1a
labeling is attached to the plasma membrane, whereas the bulk of mGluR5
immunoreactivity is expressed in intracellular compartments. Finally,
plasma membrane-bound group I mGluRs are primarily extrasynaptic or
expressed in the main body of symmetric, GABAergic, striatonigral
synapses in rats and monkeys. On the other hand, labeling of asymmetric
synapses is primarily perisynaptic. These data raise questions about
the trafficking and internalization of group I mGluRs as well as the
potential functions of these receptors at GABAergic synapses.
Interspecies differences in mGluR1a expression between rats
and monkeys
The mGluR1a antiserum used in this study heavily labeled SNc
neurons in monkeys but not in rats, whereas SNr neurons displayed moderate to strong labeling in both species. The light mGluR1a labeling
in rat SNc neurons is consistent with recent in situ hybridization data showing that the mGluR1a mRNA level is relatively low in rat SNc neurons that, on the other hand, display strong mRNA
expression for the mGluR1d splice variant (Kosinski et al., 1998). It
is unlikely that the differential intensity of mGluR1a labeling between
the rat and monkey SNc is caused by technical differences in the tissue
processing because both species were perfused with the same fixative
and incubated with antibodies under similar conditions. Because of
technical limitations in quantifying immunoperoxidase deposits,
comparative in situ hybridization studies should be
performed to further analyze this interspecies difference in mGluR1a
expression between rats and primates. It is noteworthy that
interspecies differences in the expression of mGluR immunoreactivity
are not unique to mGluR1a in the SNc. Recent data showed that the
distribution of mGluR2 in various brain regions is different between
rats and primates (Phillips et al., 2000). At present, the functional
significance of such interspecies differences in the intensity of
mGluRs labeling is unknown, but it should be kept in mind while
extrapolating data from one species to the other.
Intracellular mGluR5 labeling
The relative paucity of plasma membrane-bound mGluR5
immunolabeling is surprising. A basal amount of internalized receptors trafficking from their site of synthesis in the cell body to dendrites would be understandable, but such a large proportion of intracellular receptors is rather unusual and strikingly different from mGluR1a, which is mostly expressed on the plasma membrane. It seems that this
phenomenon is not unique to group I mGluRs. For instance, the
muscarinic receptor m4 is primarily expressed intracellularly in
cholinergic interneurons and projection neurons in the matrix compartment of the rat striatum under basal conditions (Bernard et al.,
1999). Similarly, the somatostatin receptor sst2A displays a
preferential cytoplasmic localization in neurons that receive dense
somatostatin innervation (Dournaud et al., 1998). In general, G-protein-coupled receptors, including mGluR5, become internalized after agonist administration (Mantyh et al., 1995; Bernard et al.,
1998, 1999; Dournaud et al., 1998; Dumartin et al., 1998; Lissin et
al., 1999; Liu and Kirchgessner, 2000). However, in these conditions,
the receptors are initially localized on the plasma membrane in which,
presumably, they could be activated by neurotransmitters in the
extracellular space. Because most receptors would need to be exposed to
the extracellular environment to achieve contact with a signaling
molecule, a notable exception being hormone receptors, the mechanism of
activation of mGluR5 in SN neurons is rather puzzling. Recently, it has
been shown that a family of proteins called Homer participates in the
membrane expression and sequestration of Group I mGluRs (Brakeman et
al., 1997). The protein Homer 1b has been shown to impede the surface expression of Group I mGluRs, whereas Homer 1a increases the cell surface expression of these receptors (Ciruela et al., 1999; Roche et
al., 1999). Although Homer proteins can bind to both group I mGluR
subtypes, one may speculate that, in SN neurons, Homer 1b has a higher
affinity for mGluR5 and sequesters it in the cytoplasm to a higher
degree than mGluR1a. Future colocalization studies of Homer proteins
and group I mGluRs in SN neurons under basal and various experimental
conditions are essential to elucidate this issue.
Another possibility is that some functions necessitate a large
intracellular pool of mGluR5 receptors. Although it might take many
hours for new receptors to be synthesized and transported from the cell
body to the plasma membrane, it would only require minutes for a
prefabricated pool of receptors to be inserted into the membrane
(Szekeres et al., 1998). Rubio and Wenthold (1999) recently showed that
there is a selective distribution of intracellular ionotropic glutamate
receptor subunits and mGluR1a with respect to their cellular location,
i.e., apical versus basilar dendritic regions in dorsal cochlear
neurons. They concluded that the distribution of intracellular
receptors was related to that of synaptic receptors and that there was
no evidence for a pool of intracellular receptors that function as a
receptor reserve near the postsynaptic densities. It is difficult to
extrapolate these data to the SNr because GABAergic and glutamatergic
synapses are homogeneously distributed along proximal and distal
dendrites of SNr neurons (Shink and Smith, 1995; Smith et al., 1998).
In any case, it is clear that a difference exists in the mGluR1a and
mGluR5 regulatory systems that makes these two receptor subtypes
differentially distributed on the plasma membrane of SN neurons. It is
noteworthy that similar findings were obtained in the monkey pallidum
(Hanson and Smith, 1999). Interestingly, the pattern of subcellular
distribution of the two group I mGluRs described in the present study
is consistent with recent in vitro electrophysiological data
showing that specific antagonists of mGluR1, but not mGluR5, abolish
slow EPSPs in rat SNr neurons (Marino et al., 1999). Additional
studies of the stimuli that are sufficient to cause mGluR5 receptors to
be inserted into the plasma membrane and a better characterization of
the differential roles played by the two group I mGluRs should help to
explain the functional importance of these anatomical observations.
Group I mGluRs at symmetric GABAergic synapses
Our data showed that most of the plasma membrane-bound immunogold
particles are either extrasynaptic or located in the main body of
putative GABAergic synapses in rat and monkey SNr neurons. Although
GABA immunostaining was not used to characterize the neurotransmitter
content of the presynaptic boutons at these synapses, their
ultrastructural features and pattern of innervation are consistent with
those of striatal terminals described in previous studies (Smith et
al., 1998). The pattern of group I mGluR distribution in the SN is
strikingly different from that of group I mGluRs in the cerebellum and
hippocampus in which both mGluR1a and mGluR5 are perisynaptic to
asymmetric synapses without any significant association with symmetric
synapses (Baude et al., 1993; Nusser et al., 1994; Lujan et al., 1996;
Hanson and Smith, 1999). However, group I mGluR labeling at symmetric
striatal synapses was also found in the monkey globus pallidus (Hanson
and Smith, 1999), which indicates that postsynaptic mGluRs are
associated with striatal synapses in the two major output structures of
the basal ganglia. A potential explanation for this labeling is that
mGluR1a and mGluR5 antibodies used in this study cross-react with
GABA-B receptors that were also found to be expressed at striatonigral
synapses in monkeys (Smith et al., 2000). However, this is
unlikely because the synthetic peptides used to generate both group I
mGluR antisera have no significant homologies with the amino acid
sequence of GABA-B receptor subtypes (Kaupmann et al., 1997, 1998;
White et al., 1998). Furthermore, both group I mGluR antisera label a
single band corresponding to the molecular weight of mGluR1a and mGluR5 in Western blots (Testa et al., 1998; Upstate Biotechnology). Together, these data indicate that there is a clear ordering of postsynaptic group I mGluRs at symmetric striatonigral synapses. It is
noteworthy that a mismatch between receptor localization and
neurotransmitter release is not unique to group I mGluRs. Similar
findings were obtained in the rat cerebellum in which GABA-A receptor
subunits coexist with AMPA receptors in glutamatergic mossy fiber
synapses (Nusser et al., 1998). Clarke and Bolam (1998) also showed
that the AMPA GluR2/3 subunits are expressed at GABAergic pallidosubthalamic synapses in rats.
One could hypothesize that Group I mGluRs are expressed at GABAergic
synapses to specifically modulate the functioning of GABA receptors.
Although such interactions have not yet been studied in detail,
modulation of IPSPs after group I mGluR activation was
demonstrated in various brain regions, including the rat substantia nigra (Glaum and Miller, 1993; Bonci et al., 1997; Morishita et al., 1998). On the basis of recent data showing that activation of the
metabotropic D5 dopamine receptors inhibits the function of GABA-A
receptors via direct protein-protein interactions (Liu et al., 2000),
it is possible that such mechanisms can also be involved in
mediating interactions between group I mGluRs and GABA-A
receptor subunits at striatal synapses.
Because a relatively small proportion of nigral afferents are
glutamatergic (Smith et al., 1998), the source of the transmitter that
activates group I mGluRs remains uncertain. As discussed in our
previous study (Hanson and Smith, 1999), three possibilities should be
considered: (1) extrasynaptic diffusion of glutamate from
subthalamonigral synapses (Asztely et al., 1997; Barbour and Hausser,
1997), (2) release of glutamate from astrocytes (Antanitus, 1998;
Araque et al., 1999), and (3) corelease of GABA and glutamate from
striatonigral terminals (Dubinsky, 1989; White et al.,
1994).
The extrasynaptic localization of G-protein-coupled receptors is a
general phenomenon in the CNS (Yung et al., 1995; Gracy et al.,
1997; Dournaud et al., 1998; Bernard et al., 1999). Although the functions and mechanisms of activation of these receptors still
remain to be established, their pattern of distribution indicates that
they may play much more subtle and complex modulatory functions
in neuronal activity than previously thought.
| |
FOOTNOTES |
Received Sept. 29, 2000; revised Dec. 8, 2000; accepted Dec. 22, 2000.
This work was supported by National Institutes of Health Grants R01
NS37423-03, P50 NS38399-01, and RR 00165, and a grant from the American
Parkinson's Disease Association. We acknowledge Jean-François
Paré for technical assistance and Frank Kiernan for photography.
Correspondence should be addressed to Yoland Smith, Yerkes Regional
Primate Research Center, Emory University, 954 Gatewood Road NE,
Atlanta, GA 30322. E-mail: yolands{at}rmy.emory.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
