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The Journal of Neuroscience, December 1, 1999, 19(23):10237-10249
Regulation of the Subcellular Distribution of m4 Muscarinic
Acetylcholine Receptors in Striatal Neurons In Vivo by the
Cholinergic Environment: Evidence for Regulation of Cell Surface
Receptors by Endogenous and Exogenous Stimulation
Véronique
Bernard1,
Allan I.
Levey2, and
Bertrand
Bloch1
1 Centre National de la Recherche Scientifique,
Unité Mixte de Recherche 5541, Laboratoire
d'Histologie-Embryologie, Université Victor
Ségalen-Bordeaux 2, 33076 Bordeaux Cedex, France, and
2 Emory University Woodruff Memorial Research
Building, Atlanta, Georgia 30322
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ABSTRACT |
Our aim was to determine how the cholinergic environment
influences, in vivo, the membrane abundance and the
intracellular trafficking of the muscarinic receptor m4 (m4R).
Immunohistochemistry at light and electron microscopic level was used
to detect the subcellular localization of m4R in several populations of
striatal cholinoceptive neurons, including cholinergic neurons and
medium spiny neurons.
(1) In control rats, in cholinergic neurons, m4R is mostly restricted
to intracytoplasmic sites involved in its synthesis, especially
endoplasmic reticulum. In contrast, m4R is preferentially located at
the plasma membrane in cell bodies and dendritic shafts and spines of
medium spiny neurons. The density of m4R was greater at the membrane of
perikarya in patches (striatal areas with low acetylcholine activity)
than in matrix (striatal areas with high acetylcholine activity). (2)
Stimulation of muscarinic receptor with oxotremorine provokes
translocation of m4R from the membrane to endosomes in perikarya and
dendrites of medium spiny neurons but has no influence on the
localization of m4R in the cytoplasm of cholinergic cell bodies.
Our results suggest that the intraneuronal trafficking and the
abundance of membrane-bound m4R in vivo is under
regulation of the cholinergic environment. The m4R subcellular
compartmentalization depends on the phenotype of the cholinoceptive
neuron and on its neurochemical environment. Such regulation, by
modulating availability of receptor for endogenous and exogenous
ligands, may play key roles in the response of target neurons.
Key words:
endocytosis; G-protein-coupled receptors; patches; matrix; basal ganglia; immunohistochemistry; multivesicular bodies
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INTRODUCTION |
Classical neurotransmitters,
including acetylcholine (ACh), dopamine, or glutamate, or neuropeptides
such as substance P or neurotensin act through G-protein-coupled
receptors (GPCRs) located at the plasma membrane of target neurons. The
neuronal response to stimulation depends in part on the abundance of
receptors at the membrane. Many in vitro experiments have
shown that the subcellular localization of GPCRs, especially their
availability at the membrane, is regulated by the neurochemical
environment (Fonseca et al., 1995 ; Roettger et al., 1995; Koenig and
Edwardson, 1996; Barnes et al., 1997; Koenig et al., 1997; Marvizon et
al., 1997). Indeed, the stimulation of such receptors induces complex
events, including internalization of the membrane-bound receptors into
endocytic vesicles and their degradation and recycling to the membrane. Modifications of the quantity of receptors available at the plasma membrane and related events have been suggested to contribute to
functional responses to stimulation, including desensitization and
resensitization (Hertel et al., 1985; Pippig et al., 1995; McDonald et
al., 1998; Mundell and Kelly, 1998). The mechanisms regulating in
vivo, the subcellular distribution of receptors in neurons, are
still poorly understood (Bloch et al., 1999). In physiological
circumstances, the abundance of receptors at the plasma membrane is the
result of complex intracellular trafficking of these receptors (Koenig
and Edwardson, 1997). The nature and the density of the afferent
innervation seems to contribute to these regulations, as demonstrated
for the somatostatin sst2A receptor (Dournaud et al., 1998). In acute
conditions, receptors for various neurotransmitters (dopamine,
substance P, or opiates) have been shown to undergo subcellular
redistribution after pharmacological stimulation, including
translocation of the receptor from the membrane to the cytoplasm,
internalization in endosomes, and recycling to the membrane (Faure et
al., 1995; Sternini et al., 1996; Marvizon et al., 1997; Bernard et
al., 1998; Dumartin et al., 1998; Bloch et al., 1999). By regulating
the quantity of the receptors available for stimulation, such phenomena
may play key roles in the neuronal response to modifications of the
neurochemical environment in physiological, experimental, or
pathological conditions.
To better understand the subcellular trafficking of classical
neurotransmitter receptors in vivo, we have investigated if the cholinergic environment may influence the compartmentalization of
ACh receptors in striatal cholinoceptive neurons in physiological and
experimental conditions. The cholinergic environment may be defined by
anatomical and neurochemical criteria, including the density of
cholinergic terminals, AChE and choline acetyltransferase (ChAT)
activity, density of cholinergic receptors, and choline uptake sites.
ACh, which is produced by striatal cholinergic neurons, regulates the
activity of various neuronal populations, including cholinergic neurons
themselves (Kemel et al., 1992; Stoof et al., 1992; Bernard et al.,
1993; Nisenbaum et al., 1994; Wang and McGinty, 1996a,b, 1997). In the
striatum, ACh action is mediated through three G-protein-coupled
muscarinic receptors expressed in several neuronal populations: in
medium spiny neurons as heteroreceptors (m1R and m4R) and also in
cholinergic neurons, as autoreceptors (m1R, m2R, and m4R) (Levey et
al., 1991; Bernard et al., 1992, 1998; Hersch et al., 1994; Ince et
al., 1997).
We have recently shown, using immunohistochemical approaches at light
and electron microscopic levels, that the muscarinic m2 receptor (m2R)
is expressed at the plasma membrane of striatal cholinergic
interneurons in basal conditions and that acute cholinergic stimulation
by agonists provokes m2R endocytosis, degradation, and/or neosynthesis
(Bernard et al., 1998). In the present study, we have first examined
and quantified the subcellular distribution of m4R in several striatal
neuronal populations of normal animals (medium spiny neurons and
cholinergic interneurons). Second, we have studied the effect of the
activation of muscarinic receptors on the distribution of m4R in the
different neuronal compartments and their association with the
different subcellular organelles.
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MATERIALS AND METHODS |
Animals and tissue preparation
Sprague Dawley male adult rats (Center d'élevage Janvier,
Le Genest St. Isle, France; 200-300 gm) were used in this study. Environmental conditions for housing of the rats and all procedures that were performed on them were in accordance with the guidelines of
the French Agriculture and Forestry Ministry (decree 87849, license
01499), with the approval of the Centre National de la Recherche
Scientifique, and in accordance with the policy on the use of animals
in Neuroscience research issued by the Society for Neuroscience.
The rats received the following treatments: (1) Several groups of rats
were treated with a single injection of oxotremorine, a muscarinic
receptor agonist (Table 1). (2) One group
of rats was pretreated with atropine, a muscarinic receptor antagonist, 15 min before oxotremorine to block the effect of the agonist. (3)
Control animals were treated with saline as a single injection or in
association with oxotremorine or atropine. All drugs were injected
subcutaneously (0.1 ml/100 gm). The animals were usually euthanized 45 min after the last injection of each drug. To examine the time course
of the effect of oxotremorine, some animals were allowed to survive
from 10 min to 24 hr (Table 1). All drugs were diluted in 0.9% NaCl.
Oxotremorine-free base and atropine sulfate salt were obtained from
Sigma (St. Louis, MO).
The rats were deeply anesthetized with sodium chloral hydrate and then
perfused transcardially with 50-100 ml of 0.9% NaCl followed by 250 ml of fixative consisting of 2% paraformaldehyde (PFA) with 0.2%
glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4, 4°C) at a rate of ~15 ml/min. The brain was quickly removed and
left overnight in 2% PFA at 4°C. Sections from neostriatum were cut
on a vibrating microtome at ~70 µm and collected in PBS (0.01 M phosphate, pH 7.4). To enhance the penetration of
the immunoreagents in the pre-embedding procedures, the sections were equilibrated in a cryoprotectant solution (PB 0.05 M, pH
7.4, containing 25% sucrose and 10% glycerol) and freeze-thawed by freezing in isopentane cooled in liquid nitrogen and thawing in PBS.
The sections were washed and stored in PBS until use.
Immunohistochemistry
The m4R was detected by immunohistochemistry using a monoclonal
antibody raised in mouse against a fusion protein derived from a
sequence of the receptor corresponding to the third intracytoplasmic loop [purchased from Chemicon (Temecula, CA) in collaboration with
A. I. Levey]. The µ-opioid receptor (µ-opiate
receptor) was detected using a rabbit polyclonal antiserum (coded
IS-7/15; kindly provided by Dr. P. Ciofi) raised against a human
serum albumin-glutaraldehyde conjugate of a synthetic peptide
corresponding to the intracytoplasmic C-t decapeptidic sequence of rat
µ-opiate receptor-1A (NLEAETAPLP) that is absent in rat µ-opiate
receptor-1B (Zimprich et al., 1995). Preabsorption of IS-7/15 with
10-6 M decapeptide totally abolished staining. The
cholinergic neurons containing m4R immunoreactivity were identified by
their expression of ChAT. ChAT was detected using a polyclonal
antibody raised in goat (Chemicon).
Immunoperoxidase detection of m4R at the light microscopic
level. Sections of striatum were treated for the detection of m4R by immunoperoxidase using the tyramide signal amplification (TSA) method (New England Nuclear, Boston, MA). After perfusion-fixation as
described above, 70-µm-thick sections were cut on a vibratome and
incubated in 4% normal goat serum (NGS) for 30 min and then in the
antibody against m4R (1:20,000), supplemented with 1% NGS for 15 hr at
room temperature (RT). The sections were then washed in PBS and
incubated in goat anti-mouse IgG coupled to biotin (Amersham; 1:200 in
PBS for 90 min). After washing in PBS, the sections were incubated in
streptavidin-horseradish peroxidase (strept-HRP) (1:100 in PBS for 30 min), then in biotinyltyramide (1:50 in amplification diluent for 7 min), and again in strept-HRP (1:100 in PBS for 30 min). After washing
[2× PBS, 1× Tris buffer (TB) 0.05 M, pH 7.6], the
immunoreactive sites were revealed by incubation in
3,3-diaminobenzidine (DAB; Sigma, 0.05% in TB) in the presence of
H2O2 (0.0048%). The
reaction was stopped by several washes in TB. The specificity of the
labeling techniques was proven by the absence of m4R labeling when the
primary or secondary antibody was omitted. The sections were then
processed for inclusion in resin and for visualization of the labeling
on semithin sections.
Double detection of m4R and neuronal markers by combination of
immunogold and immunoperoxidase methods at the ultrastructural level. m4R was analyzed specifically in cholinergic neurons by combining m4R and ChAT detection. The m4R was analyzed in medium spiny
neurons of patches and matrix compartments by combining m4R and
µ-opiate receptor detection. The µ-opiate receptor immunoreactivity was specifically restricted to patch neurons (Arvidsson et al., 1995).
The m4R and ChAT or µ-opiate receptor immunoreactivities were
detected on the same sections by combining the pre-embedding immunogold
and immunoperoxidase techniques, respectively. Sections of striatum
were incubated in 4% NGS (m4R + µ-opiate receptor) or normal donkey
serum (NDS) (m4R + ChAT) for 30 min and then in a mixture of m4R
(1:2000) and µ-opiate receptor (1:2000) or ChAT (1:400) antibodies,
supplemented with 1% NGS or NDS for 15 hr at RT. For the simultaneous
detection of m4R and µ-opiate receptor, the sections were then
incubated in a mixture of goat anti-mouse IgGs conjugated to gold
particles (1.4 nm diameter; Nanoprobes, Stony Brook, NY; 1:100 in
PBS/BSA-C) and donkey anti-rabbit coupled to biotin (1:200) for 2 hr in
PBS/BSA-C. The sections were then washed (3× PBS) and post-fixed in
1% glutaraldehyde in PBS for 10 min. After washing (2× PBS; 2×
sodium acetate buffer, 0.1 M, pH 7.0), the diameter of the
gold immunoparticles was increased using a silver enhancement kit (HQ
silver; Nanoprobes) for 5 min at RT in the dark. The sections were
finally washed in acetate buffer and in PBS and incubated in an
avidin-biotin-peroxidase complex (ABC), (1:100; Vector Laboratories,
Burlingame, CA) for 1.5 hr at RT. After washing (2× PBS, 1× TB 0.05 M, pH 7.6), the immunoreactive sites for µ-opiate
receptor were revealed using DAB as above. For the double detection of
m4R and ChAT, the sections were first incubated in rabbit anti-goat
coupled to biotin (1:200) for 90 min in PBS and then, after washing, in
goat anti-mouse IgGs conjugated to gold particles (1:100) for 2 hr in
PBS/BSA-C. After intensification of the immunogold signal for m4R, the
sections were incubated in ABC (1:100) for 1.5 hr, and the peroxidase
reaction for ChAT was revealed as described above. Some spare sections were treated for the immunogold detection of m4R alone. The sections were then stored in PB and processed for electron microscopy.
Preparation for electron microscopy
The sections were post-fixed in osmium tetroxide (1% in PB, 0.1 M, pH 7.4) for 10 min (immunogold or immunogold and
immunoperoxidase) or 25 min (immunoperoxidase alone) at RT. After
washing (3× PB), they were dehydrated in an ascending series of
dilutions of ethanol that included 1% uranyl acetate in 70% ethanol.
They were then treated with propylene oxide (two times for 10 min) and equilibrated in resin overnight (Durcupan ACM; Fluka,
Buchs, Switzerland), mounted on glass slides, and cured at 60°C for
48 hr. Areas of interest were first visualized in the light microscope
and cut out from the slide and glued to blank cylinders of resin.
Blocks were cut out in µ-opiate receptor-immunopositive (patches) and immunonegative (matrix) areas. By another way, ChAT-positive neurons were identified in the light microscope. The selection was made to have
several ChAT-immunopositive neurons on the same block (usually four or
five). The immunoreactive areas identified on thick sections were cut
in semithin sections (1-µm-thick), then in ultrathin sections on a
Reichert Ultracut S. Ultrathin sections were collected on
pioloform-coated single slot copper grids. The sections were stained
with lead citrate and examined in a Philips CM10 electron microscope.
Quantitative analysis of the distribution of m4R in striatal
neuronal compartments
The distribution of m4R in different organelles: (1) of
perikarya of striatal cholinergic neurons, (2) of perikarya of medium spiny neurons, and (3) of dendrites in patches and matrix in NaCl- and
oxotremorine-treated animals was analyzed from immunogold-treated sections at electron microscopic level. The analysis was performed on
negatives of micrographs at a final magnification of 3900× (cholinergic neurons) or 5200× (medium spiny neurons) using the Metamorph software on a personal computer (Universal Imaging, Paris,
France). After scanning of the negative (Umax; software, Magic Scan,
version 3.1), the image was converted into a positive picture and
magnified to allow the identification of the subcellular element
showing immunoparticles. The measures were performed on four
NaCl-treated and four oxotremorine-treated rats. A mean of 10 (cholinergic neurons) and 15 (medium spiny neurons) perikarya per
animal and 30 dendritic profiles was analyzed. The immunoparticles were
identified and counted in perikarya in association with seven subcellular compartments. Six compartments are the plasma membrane, endosome-like vesicles, multivesicular bodies, the Golgi apparatus, the
endoplasmic reticulum, and the outer nuclear membrane. Some immunoparticles were classified as associated with a seventh
unidentified compartment because they were associated either with no
detectable organelles or with an organelle that could not be identified
as one of the previous ones. The distribution of the immunoparticles in
dendrites was quantified in dendrites surrounding the medium spiny
neurons on the same micrographs. All positive dendrites (with more
than two immunoparticles) on the picture were taken in account for the
quantification. The immunoparticles were localized in association with
three subcellular compartments: the plasma membrane, endosomes, and
multivesicular bodies. As in perikarya, some immunoparticles could not
be classified in association with one of the previous compartments. The
results were expressed (1) as the percentage of immunoparticles
associated with the different subcellular compartments in every section
of analyzed neurons in normal animals and (2) as the number of
immunoparticles per plasma or nuclear membrane length (in
micrometers), cytoplasmic surface (in square micrometers),
mutivesicular body, or Golgi apparatus in normal and in treated rats.
We assume here that the number of immunoparticles is proportional to
the absolute number of m4R. The values from NaCl- and
oxotremorine-treated rats were compared using a two-way ANOVA test,
with treatment and striatal compartment as between-factors and
within-factors, respectively. To show possible differences in values
between patches and matrix, post hoc comparisons were
performed using a Student's t test.
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RESULTS |
Cellular and subcellular distribution of m4R immunoreactivity in
the striatum in control rats
By combining several approaches of immunodetection of m4R at the
light and electron microscopic level, we have described three types of
neurons according to the subcellular localization of m4R: (1) medium
spiny neurons of the patches (identified by the presence of µ-opiate
receptor immunoreactivity), displaying a strong m4R immunolabeling at
the membrane, (2) medium spiny neurons of the matrix (identified by the
absence of µ-opiate receptor immunoreactivity), displaying a moderate
m4R immunolabeling at the membrane, and (3) cholinergic interneurons,
identified by ChAT immunoreactivity, showing a strong and prominent m4R
intracytoplasmic immunostaining.
Light microscopic observations
The normal striatum displayed intense immunoreactivity for m4R, as
evidenced by observation of immunoperoxidase-treated sections with the
light microscope (Fig. 1).
Immunoreactivity for m4R was detected in striatal dendrites and in
numerous perikarya. These neurons were usually medium-sized (10-12
µm in diameter) with an unindented nucleus surrounded by a thin rim
of cytoplasm. These neurons had thus characteristics of medium spiny
neurons. Two types of labeling were detected. Most of these neurons
showed a weak immunolabeling close to the plasma membrane, and thus
possibly associated with it and that was often masked by the neuropil. However, other cell bodies gathered in small clusters demonstrated a
strong labeling at the membrane (Fig. 1A,A'). No
obvious difference was observed in the labeling between neostriatum and
the nucleus accumbens and along the rostrocaudal and dorsoventral axes.
No glial labeling was observed in the striatum. Large-sized neurons corresponding to cholinergic neurons (see below), which could seldom be
identified at the light microscopic level, displayed a faint m4R
cytoplasmic labeling.
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Figure 1.
Cellular and subcellular distribution of m4R
immunoreactivity in striatal neurons in control (A, A'),
oxotremorine-treated (B, B') rats, and rat pretreated
with atropine 15 min before oxotremorine (C, C') using
the immunoperoxidase method on thick (70 µm)
(A-C) and semithin sections (1 µm)
(A'-C'). In control animals (A, A'), m4R
immunoreactivity is detected at the membrane of some cell bodies of
neurons often seen in clusters (asterisks).
Immunolabeling for m4R is also seen in dendrites
(arrowheads), but with reduced immunoreactivity. After
oxotremorine treatment (B, B'), a decrease in the amount
of neuropil labeling was observed, and a strong m4R immunoreactivity
was detected in the cytoplasm of numerous cell bodies
(asterisks). Spots of labeling are clearly identified in
perikarya and in dendrites [small arrows in
B (dendrites) and B'(perikarya)]. The
pretreatment with atropine, a muscarinic receptor antagonist, abolishes
the effect of oxotremorine: immunoreactivity for m4R is similar to the
labeling observed in control animals (C, C'). Scale
bars, 10 µm.
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Electron microscopic observations
The observation at electron microscopic level confirmed that m4R
was mostly detected in cell bodies and dendritic shafts and spines
(Figs. 2,
3). Part of the immunoparticles were
associated with the internal side of plasma membranes (Figs. 2, 3). In
cell bodies, dendrites, and spines, most immunoparticles were detected at extrasynaptic sites, albeit they could be localized rarely in
association with postsynaptic specializations of synapses (Figs. 2, 3).
Dendrites and spines displaying m4R immunoreactivity were postsynaptic to boutons forming usually asymmetrical synapses, and more
rarely, symmetrical synapses (Fig. 3A,C,E). Very few immunoparticles for m4R were visible in boutons.
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Figure 2.
Subcellular distribution of m4R
immunoreactivity in the striatum in the matrix and patches of control
rats using the pre-embedding immunogold method with silver
intensification. The matrix and patch areas were identified by the
absence (A) or presence
(B), respectively, of immunoreactivity for
µ-opiate receptor (MOR), detected by the
immunoperoxidase method. In B, the MOR immunoreaction
product was visible close to the membrane of the cell body and in a
dendrite (d). A, B,
Immunopositive cell body with an unindented nucleus
(n) and thin rim of cytoplasm are characteristic
of striatal medium spiny neurons. The immunoparticles are associated
mostly with the internal side of the plasma membrane
(arrowheads). The density of immunoparticles is higher
in the patches (B) than in the matrix neurons
(A). Some immunoparticles are associated with the
outer nuclear membrane (flat arrows) and the
internal side of the membrane of dendrites (d).
Scale bars, 0.5 µm. n, Nucleus.
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Figure 3.
Subcellular distribution of m4R
immunoreactivity in the striatal neuropil in the matrix
(A-C) and patches (D, E) of
control rats using the pre-embedding immunogold method with silver
intensification. The matrix and patches areas were identified by the
absence (A-C) or presence (D, E), respectively, of
immunoreactivity for MOR, detected by the immunoperoxidase method. In
patches and matrix, m4R immunoparticles were located mostly at the
internal side of the membrane of dendrites (d)
(A, B, D, E) and spines (s)
(A, C, E). Most of the immunoparticles are located at
extrasynaptic sites. Some immunoparticles are located at the edges of
postsynaptic specialization of asymmetrical axodendritic
(arrow) (A) and axospinous
synapses (C), and sometimes of putative
symmetrical synapses (B) (double
arrow). b, Bouton. Scale bars, 0.25 µm.
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Distribution of m4R immunoreactivity in perikarya. The m4R
immunogold labeling was observed and quantified specifically in three
types of cell bodies: (1) in µ-opiate receptor immunopositive cell
bodies of medium spiny neurons in patches; (2) in µ-opiate receptor
immunonegative cell bodies of medium spiny neurons, in the matrix (Fig.
2); and (3) in perikarya of cholinergic neurons (immunopositive for
ChAT) (Fig. 4). In all these perikarya,
the immunoparticles were identified and counted in association with seven subcellular compartments: plasma membrane, endosome-like vesicles, multivesicular bodies, Golgi apparatus, endoplasmic reticulum, nuclear membrane, and unidentified compartments. The endosome-like vesicles were small (100-200 nm in diameter), round, or
irregular-shaped vesicles. The multivesicular bodies were large round
vesicles (500-600 nm in diameter) containing several small round-shaped vesicles with a clear content (see Fig. 7).
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Figure 4.
Subcellular distribution of
m4R immunoreactivity in striatal cholinergic neurons of control rats
(A) or rats treated with oxotremorine
(B) using pre-embedding immunogold method with
silver intensification. The cholinergic neurons were identified using
the presence of immunoreactivity for ChAT, detected by the
immunoperoxidase method. The ChAT immunoreaction product was visible
throughout the cytoplasm of the perikarya. A, The neuron
immunopositive for ChAT and m4R has a large volume of cytoplasm, one of
the characteristic features of a striatal interneuron. Some
immunoparticles are detected in the cytoplasm at the external surface
of the endoplasmic reticulum (arrows). Immunoparticles
are also associated with the Golgi apparatus (G)
and the external membrane of the nucleus (flat
arrow). Very few immunoparticles are associated with the plasma
membrane (arrowheads). B, Detail of the
m4R immunogold labeling in a ChAT-immunopositive neuron, after
treatment with oxotremorine. Most immunoparticles are clearly
associated with endoplasmic reticulum lamina (arrows).
Scale bars, 1 µm.
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Medium spiny neurons. In medium spiny neurons of patches,
40% of the total number of immunoparticles were associated with the
internal side of the plasma membrane. In the matrix, only 24% of
immunoparticles were associated with the membrane (Fig. 5A). The statistical analysis
demonstrated a significantly higher number of immunoparticles for m4R
per membrane length in patches than in matrix (see Fig.
8A). Immunoparticles were also detected in the
cytoplasm in association with the endoplasmic reticulum (7% patches;
10% matrix), endosomes (9% patches; 11% matrix), Golgi apparatus
(8% patches; 10% matrix), nuclear membrane (4% patches; 4% matrix),
and multivesicular bodies (0.2% patches; 0.4% matrix) (Figs.
2A, 5A). Thirty-three percent (patches)
and 40% (matrix) of immunoparticles could not be associated with one of the previous subcellular elements (Fig. 5A). No
statistical difference was detected in the number of immunoparticles
associated with any of these subcellular compartments in patches and
matrix areas (see Fig. 8A).
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Figure 5.
Quantitative analysis of the subcellular
distribution of m4R in the striatum of control rats using pre-embedding
immunogold method with silver intensification. Proportion of
immunoparticles associated with different subcellular neuronal
compartments in perikarya of medium spiny neurons
(A) and cholinergic neurons
(B), and in dendrites (C).
For each cell body or dendrite, the number of immunoparticles
associated with each compartment was counted, and the proportion in
relation to the total number was calculated. Data are the result of
countings in four control rats (15 medium spiny neurons, 10 medium
spiny neurons, and 30 dendrites per animal). In medium spiny neurons
(A), of the immunoparticles that are associated
with an identified compartment, most of them are preferentially located
at the plasma membrane, whereas in cholinergic neurons
(B), they are mostly associated with the
endoplasmic reticulum (er). The proportion of
immunoparticles at the membrane is much higher in MOR+ areas (patches)
than in MOR (matrix) areas. In the cytoplasm, the immunoparticles are
mostly detected in association with small vesicles, the Golgi
apparatus, and the endoplasmic reticulum in medium spiny neurons and
with small vesicles and the Golgi apparatus in cholinergic neurons. In
both types of neurons, a small proportion of immunoparticles are
associated with multivesicular bodies (mvb) and the
outer nuclear membrane. Some immunoparticles are not seen in
association with any of an identified compartment. In dendrites
(C), most of immunoparticles in matrix and
patches are associated with the membrane, and only a small part are
detected in association with small vesicles and with unidentified
compartments.
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Cholinergic neurons. All ChAT-immunopositive neurons that
were analyzed at electron microscopic level displayed immunogold labeling for m4R (Fig. 4). In these neurons, 97% of immunoparticles were detected in the cytoplasm. Most of the immunoparticles that were
associated with an identified compartment were associated with the
endoplasmic reticulum (25%) (Figs. 4, 5B). Eleven, 4, 2. and 0.2% of immunoparticles were associated with Golgi apparatus, endosomes, nuclear membrane, and multivesicular bodies, respectively (Figs. 4, 5B). Fifty-six percent of immunoparticles could
not be located in association with any cytoplasmic compartment (Fig. 5B). Only 3% of the total number of immunoparticles were
associated with the plasma membrane (Figs. 4A,
5B).
Distribution of m4R immunoreactivity in dendrites. The m4R
immunogold labeling was observed and quantified in dendrites in patches
and matrix areas (Figs. 3, 5C). In view of the density of
medium spiny neurons in striatum (>90% of striatal neurons), most of
the labeled dendrites that we identified were probably dendrites of
medium spiny neurons. The immunoparticles were identified and counted
in association with four subcellular compartments: plasma membrane,
endosome-like vesicles, multivesicular bodies, and unidentified
compartments (Fig. 5C). Most of the labeling was associated
with the plasma membrane in patches (83%) and matrix (89%) neurons.
Eight percent (patches) and 5% (matrix) of immunoparticles were
detected in the cytoplasm in association with endosome-like vesicles
(Fig. 5C). Nine percent (patches) and 6% (matrix) of immunoparticles could not be detected in association with one of the
previous compartments. No significant difference was shown between
values from patches and matrix (see Fig. 9).
Control for specificity of the immunohistochemical labeling.
The specificity of the labeling techniques was proven by the following data: (1) the cellular localizations were in agreement with the results previously described by immunohistochemistry using
an antibody against the same m4R or by in situ
hybridization (Bernard et al., 1992; Hersch et al., 1994). (2) The
localization of immunoparticles for m4R on the internal side of the
plasma membrane was in agreement with the localization of the epitope included in the fusion protein (third intracytoplasmic loop). (3) There
was an absence of m4R labeling at light microscopic level when the
primary or secondary antibody was omitted. (4) We have checked in
double-labeling experiments (µ-opiate receptor + m4R) that the
immunoperoxidase procedure to detect µ-opiate receptor did not
interfere with the immunogold labeling for m4R. For that, we have
checked in one control animal that the subcellular distribution of m4R
did not differ statistically in double- and in single-labeling experiments.
Cellular and subcellular distribution of m4R immunoreactivity in
the striatum after treatment with oxotremorine
The observations of the labeling immunoperoxidase- and
immunogold-reacted sections at light microscopic level showed dramatic modifications of the distribution of m4R immunoreactivity in medium spiny neurons; a decrease in the amount of neuropil labeling was observed and an intense punctiform labeling appeared in the cytoplasm of all immunoreactive cell bodies and their dendrites (Fig.
1B,B'). An intracytoplasmic dotty labeling was
detectable as early as 20 min after injection of oxotremorine with a
faint intensity, was intense at 45 min, 1 hr 30 min, and
returned weak at 3 hr after injection. Five, 7, and 24 hr after
injection, m4R immunoreactivity was similar to the labeling observed in
control animals (Fig. 2F,G). These immunoreactive
puncta probably correspond to labeled endosomes seen in the electron
microscopic level (Figs. 6,
7). Pretreatment of rats with atropine, a
muscarinic receptor antagonist, completely abolished the effect of
oxotremorine on m4R immunoreactivity (Fig. 1C,C').
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Figure 6.
Subcellular distribution of m4R
immunoreactivity in the striatum in the matrix and patches of
oxotremorine-treated rats using the pre-embedding immunogold method
with silver intensification. The matrix and patches areas were
identified using the absence (B) or presence
(C) of µ-opiate receptor immunoreactivity,
respectively, detected by the immunoperoxidase method. In
C, the µ-opiate receptor immunoreaction product was
visible in a dendrite (d). B,
C, Immunopositive cell body with an unindented nucleus
(n) and a thin rim of cytoplasm are
characteristic of striatal medium spiny neurons. Numerous
immunoparticles are detected in the cytoplasm in association with small
vesicles (arrows). A, D,
Details of the frame in B and C,
respectively, showing endosome-like vesicles having immunoparticles
associated with them. Some immunoparticles are associated with the
plasma membrane (arrowheads) and with endoplasmic
reticulum (flat arrow). Scale bars:
A, D, 0.2 µm; B,
C, 0.5 µm.
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Figure 7.
Subcellular distribution of m4R
immunoreactivity in the striatal neuropil in the matrix and patches of
oxotremorine-rats using the pre-embedding immunogold method with silver
intensification. The matrix and patch areas were identified using the
absence (A, B) or presence (C) of
immunoreactivity for µ-opiate receptor, respectively, detected by the
immunoperoxidase method (C, d). Most of m4R
immunoparticles were detected in the cytoplasm of the dendrites in
association with endosome-like vesicles (A, C, arrows).
Immunoparticles for m4R were also detected in association with a
multivesicular body in a dendrite (A, star). Some
immunoparticles were located at the membrane of dendrites (A,
C) and a spine (B)
(arrowheads in dendrites). Scale bars, 0.25 µm.
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Perikarya of medium spiny neurons
The analysis at electron microscopic level confirmed dramatic
changes in the subcellular localization of m4R. It demonstrated an
important decrease of the density of immunoparticles located at the
plasma membrane of medium spiny neurons and modifications of the
distribution of immunoreactivity in the cytoplasm in
oxotremorine-treated rats compared to control animals, especially the
appearance of numerous labeled endosome-like vesicles (Fig. 6). The
quantitative analysis demonstrated indeed a significant decrease of the
number of immunoparticles per plasma membrane length in patches
(55%) (Fig. 8A).
There was also a decrease of the density of m4R immunoparticles at the
membrane of cell bodies of matrix (34%), but this was not
statistically significant. The total number of immunoparticles per
surface of cytoplasm significantly increased (p < 0.01) after treatment (+37% patches; +39% matrix). A strong and
significant increase was detected for the number of particles
associated with the endosome-like vesicles (+335% patches; +327%
matrix). There was also increased labeling associated with the Golgi
apparatus (+40% patches; +41% matrix). The proportion of
immunopositive multivesicular bodies significantly increased (+60%
patches; +154% matrix). There were no significant differences in the
number of immunoparticles per positive multivesicular body (+29%
patches; +36% matrix). No significant difference was shown, after
treatment, in the percentage of immunoparticles associated with the
endoplasmic reticulum, with the nuclear membrane, or with unidentified
organelles (Fig. 8A). These results should be read
keeping in mind that the baseline of immunoreactivity varies in control
animals from one subcellular compartment to another, as seen in Figure
5.
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Figure 8.
Quantitative analysis of the
subcellular distribution of m4R in the striatum of control rats and
rats treated with oxotremorine using pre-embedding immunogold method
with silver intensification. Effect of the treatment with oxotremorine
on the localization of m4R immunoparticles in cell bodies of striatal
medium spiny (A) and cholinergic
(B) neurons. For each neuron, the number of
immunoparticles associated with each compartment was counted in
relation to the membrane length (in micrometers) for the plasma and
nuclear membrane, to the surface of cytoplasm (in square micrometers)
for small vesicles, the endoplasmic reticulum (er), and
the unidentified compartment (unident.). For Golgi
apparatus, the values are expressed as the number of immunoparticles
per total number Golgi apparatus. For the multivesicular bodies
(mvb), the values are expressed as the number of
immunopositive multivesicular bodies per total number of multivesicular
bodies. Data are the result of countings in four control rats and four
treated rats in ~15 medium spiny and 10 cholinergic neurons per
animal. To be able to compare the oxotremorine effect on the quantity
of m4R in different subcellular compartments, the results are expressed
in relation to an arbitrary unit 100 of the control values in matrix.
In medium spiny neurons (A), the statistical
analysis (two-way ANOVA test, followed by Student's t
test) shows a significant difference in the labeling at the plasma
membrane between control and treated rats (p < 0.01) and an interaction between treatment and striatal compartment
(p < 0.01). The post hoc
comparisons demonstrate a higher density of immunoparticles at the
membrane in control patches than in control matrix
(p < 0.05) and in control patches than in
treated patches (p < 0.01). The analysis
demonstrates an strong increase in the labeling in small vesicles
(p < 0.0001) and a moderate increase in
multivesicular bodies and Golgi apparatus (p < 0.05), without interaction between treatment and striatal
compartment. No modification of the labeling was detected in the other
neuronal compartments. In cholinergic neurons
(B), no modification of the labeling was detected
in any of the neuronal compartment.
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Perikarya of ChAT-immunopositive neurons
The statistical analysis did not demonstrate any modification of
the subcellular distribution of m4R immunoreactivity in cholinergic neurons (Fig. 8B).
Dendrites
The observation at electron microscopic level
demonstrated a decrease of the density of immunoparticles located at
the plasma membrane of dendritic shafts and a modification of the
distribution of immunoreactivity in the cytoplasm of the dendrites in
oxotremorine-treated rats as compared to control animals (Fig. 7). The
quantitative analysis demonstrated indeed a decrease of the relative
abundance of immunoparticles at the plasma membrane in patches (54%)
and matrix (54%) (Fig. 9). Moreover,
the total number of particles significantly increased in the cytoplasm
(p < 0.001) (+329% patches; +296% matrix). A
very strong increase was detected for the frequency of particles
associated with the endosome-like vesicles (+914% patches; +359%
matrix). In dendrites, very few multivesicular bodies were seen. The
m4R immunoparticles associated with unidentified compartments also
significantly increased in patches (+22%) and matrix (+77%)
areas.
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Figure 9.
Quantitative analysis of the subcellular
distribution of m4R in the striatal dendrites of control rats and rats
treated with oxotremorine, in MOR+ areas (patches) and in MOR
(matrix) areas, using pre-embedding immunogold method with silver
intensification. To be able to compare the oxotremorine effect on the
quantity of m4R in different subcellular compartments, the results are
expressed in relation to an arbitrary unit 100 of the control values in
matrix. For each dendrite, the number of immunoparticles associated
with each compartment was counted in relation to the plasma membrane
length (in micrometers), to the surface of cytoplasm (in square
micrometers) for small vesicles and the unidentified compartment. The
statistical analysis (two-way ANOVA test, followed by Student's
t test) demonstrates a significant decrease in the
labeling associated with the plasma membrane
(p < 0.05) and a very strong increase in
the labeling associated with small vesicles
(p < 0.01). A moderate increase was
detected for the m4R immunolabeling associated with unidentified
compartments (p < 0.05). No interaction
between treatment and striatal compartment was shown.
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DISCUSSION |
We report here that the cholinergic environment influences the
subcellular compartmentalization of m4R in striatal neurons in
physiological and experimental circumstances. In control animals, m4R
is mainly located as expected at the plasma membrane of perikarya and
dendrites of medium spiny neurons. In contrast, in cholinergic neurons,
m4R is detected mostly in the cytoplasm at its sites of synthesis. The
quantitative analysis at the electron microscopic level revealed a
greater density of m4R at the membrane of medium spiny neurons in
patches than in matrix, areas known to display low and high cholinergic
activity, respectively. Oxotremorine induces internalization of m4R in
medium spiny neurons but has no effect on the subcellular distribution
of m4R in cholinergic neurons. The quantification demonstrated a
decrease of the receptor at the plasma membrane of medium spiny neurons
in oxotremorine-treated rats. Concurrently, the m4R immunolabeling
increased in the cytoplasm, strongly in endosome-like vesicles, and
more weakly in multivesicular bodies and Golgi apparatus.
Availability of m4R at the plasma membrane of striatal neurons in
basal conditions
Our results confirm and expand previous data demonstrating m4R
expression in different classes of striatal neurons (Levey et al.,
1991; Bernard et al., 1992; Hersch et al., 1994; Ince et al., 1997).
Light and electron microscopic observations revealed that several types
of neurons displayed m4R immunoreactivity: (1) medium-sized spiny
neurons, previously identified as neurons producing substance P or
enkephalins (Bernard et al., 1992); and (2) large-sized neurons,
identified as cholinergic aspiny interneurons.
We demonstrate here that in basal conditions, a same GPCR may display
distinct subcellular localization according to the type of neuron that
expresses it (Fig. 10). We have
identified three conditions with respect to the subcellular
localization of m4R: (1) medium spiny neurons in patches with a high
density of m4R at the membrane, (2) medium spiny neurons in matrix with
a moderate density of m4R at the membrane, and (3) cholinergic neurons
with almost no m4R associated with the membrane of cell bodies, but with a high m4R density in the cytoplasm. This is of particular interest if we assume that GPCRs are expected to be located at the
plasma membrane to mediate neurotransmission and that the efficiency of
the neuronal response to stimulation depends on the abundance of
receptors at the plasma membrane.
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Figure 10.
Schematic representation of the subcellular
distribution of m4R in perikarya of striatal neurons. Three conditions
with respect to the subcellular localization of m4R have been
described: (1) cholinergic neurons with almost no m4R associated with
the plasma membrane, but with a high m4R density in the cytoplasm in
association with endoplasmic reticulum and Golgi apparatus, (2) medium
spiny neurons with a high density of m4R at the membrane in patches
(displaying low cholinergic activity), and (3) medium spiny neurons
with a moderate density of m4R at the membrane in matrix (displaying
high cholinergic activity). After treatment with oxotremorine, the
abundance of m4R at the membrane of medium spiny neurons decreases in
patches but not in matrix. In parallel, numerous m4Rs are detected in
association with endosomes in the cytoplasm of medium spiny neurons in
both compartments. The m4R distribution in cholinergic neurons is not
modified by the treatment. n, Nucleus.
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Localization of m4R at the membrane of medium spiny neurons
In our model, the difference of abundance of m4R at the membrane
of cell bodies depending on the neuronal type may be related to
differences in the cholinergic microenvironment of each neuron. Indeed,
immunohistochemical studies demonstrated that the density of
cholinergic axons and terminals was greater in striosomes than in
matrix (Graybiel et al., 1986; Hirsch et al., 1989). Moreover, the
matrix displays high acetylcholinesterase activity, in contrast with
patches (Graybiel and Ragsdale, 1978). The matrix was also shown to
display more affinity choline uptake sites than patches (Lowenstein et
al., 1989). Taken together, these data suggest that the cholinergic
activity is quantitatively more intense in the matrix than in the
patches of normal animals. This suggests that there may be a
correlation between the density of m4R at the plasma membrane of
cholinoceptive medium spiny neurons and the cholinergic tone. The more
the cholinergic activity is high, the less ACh receptors are exposed at
the membrane, and vice versa. This suggests also that in physiological
circumstances, high levels of ACh in matrix may chronically
downregulate membrane-associated receptors. These phenomena of
downregulation in matrix may result of internalization of m4R induced
by their overstimulation by ACh. This is supported by our data
demonstrating that the stimulation of muscarinic receptors induces
internalization of m4R in endosomes.
Localization of m4R in the cytoplasm of cholinergic neurons
In contrast with medium spiny neurons, m4Rs are rarely detected at
putative active sites (plasma membrane), but are stored mainly in basal
conditions in the cytoplasm of cholinergic neurons, especially at the
sites of synthesis. These m4Rs associated with the endoplasmic
reticulum and the Golgi apparatus are probably receptors in the process
of synthesis before being targeted to the membrane and are thus
unlikely functional. This suggests that the cholinergic environment
tonically downregulates m4R in these neurons. Our results are in
agreement with data showing that in regions with a dense
somatostatinergic innervation, the somatostatin receptor sst2A displays
a preferential cytoplasmic localization (Dournaud et al., 1998). In
another way, in mice displaying dopaminergic overactivity caused by the
knock-out of the dopamine transporter gene (Giros et al., 1996), the
dopamine D1 receptor, which is located at the membrane of
dopaminoceptive neurons in normal conditions, accumulates in the
cytoplasm at its sites of synthesis (.
The m4Rs synthesized in cholinergic neurons may also be addressed to
sites of the membrane that, for technical limitations, we cannot
identify in this study, e.g., at the membrane of dendrites or of the
postsynaptic specialization as suggested for subunits of the channel
AMPA and NMDA receptors (Dumartin et al., Bernard et al., 1997; Bernard and Bolam,
1998).
Effect of the stimulation of muscarinic receptors on the
subcellular distribution of m4R in striatal neurons
We report here that the stimulation of muscarinic receptors
induced dramatic modifications of the subcellular distribution of m4R
in medium spiny neurons. The quantitative analysis at subcellular level
demonstrates a decrease of the abundance of m4R at the membrane of
perikarya and dendrites of medium spiny neurons in patches and matrix,
although the decrease was not significant in cell bodies in matrix. In
parallel, we have shown a strong increase of the m4R immunolabeling in
association with endosomes both in perikarya and dendrites. This
suggests that the stimulation of muscarinic receptors provokes
internalization of membrane-bound m4R through endocytosis. Endocytotic
mechanisms of neurotransmitter receptors after activation have been
widely described in vitro (Koenig and Edwardson, 1997),
including for muscarinic receptors (Koenig and Edwardson, 1996; Barnes
et al., 1997; Vogler et al., 1999). We have recently demonstrated that
m2Rs also undergo endocytosis in striatal cholinergic neurons after
stimulation by oxotremorine in vivo (Bernard et al., 1998).
We report here that the same stimulation induces endocytosis of another
muscarinic receptor, m4R, in another type of neuron, i.e., medium spiny
neurons. However, in contrast with m2R, the stimulation does not modify
the subcellular compartmentalization of m4R in cholinergic neurons.
This was expected because this receptor is not exposed, in contrast
with m2R, to the membrane in basal conditions and is therefore probably
not functional. Nevertheless, we cannot exclude that endocytosis occurs
specifically in dendrites of cholinergic neurons that we could not
identify here.
In dendrites of patches and matrix, we have also shown a decrease of
m4R at the membrane and a high increase of the quantity of receptors
associated with endosomes; suggesting that endocytosis occurs all along
the somatodendritic field. In perikarya of the matrix area, the
decrease of the abundance of m4R at the membrane after stimulation is
not statistically significant, whereas we show a strong increase of the
quantity of m4R associated with endosomes. This may be explained by the
fact that part of endosomes in perikarya originate in dendrites and are
transported to cell bodies. This is supported by data showing that
early endosomes are predominantly located in dendrites, in contrast
with late endosomes that are present in cell bodies in hippocampal
cultured neurons, in basal conditions (Parton et al., 1992). This is
also in agreement with the internalization of the neurotensin receptor and its ligand in endosomes that are mobilized from dendrites and
accumulate in perikarya (Faure et al., 1995). These results suggest
complex relationships between the different neuronal compartments that
contribute to regulate overall neuronal quantity of membrane-bound receptors (Bloch et al., 1999).
Our data also demonstrate that endocytosis is associated with other
intracellular events induced by the stimulation of m4R. Indeed, we have
also demonstrated an increase of m4R associated with multivesicular
bodies and the Golgi apparatus in cell bodies. Because multivesicular
bodies are thought to have function of lysosomes, part of internalized
m4R may thus undergo a process of degradation (van Deurs et al., 1993).
Moreover, maturation of m4R in Golgi apparatus may be a quick event
consecutive to stimulation to compensate the loss of m4R at the
membrane by maturation of new receptors. These two phenomena seem to be
a classical reaction, at least in this type of stimulation, because
they were observed also for m2R (Bernard et al., 1998).
Functional implications
The present results demonstrates that in vivo, in
physiological and experimental conditions, the neurochemical
environment regulates the subcellular localization of neurotransmitter
receptors, especially the abundance of these receptors at the plasma
membrane. The regulation of the distribution of neurotransmitter
receptors may be a means to adapt the response of the postsynaptic
neuron to variations of the neurochemical environment, including
modifications of the levels of the endogenous ligand in basal
conditions. In humans, such a regulation may occur in pathology. The
motor disorders observed in Parkinson's disease are thought to be
caused, at least in part, by cholinergic overactivity (Calne, 1993).
The modification of the availability of muscarinic receptors that may
result of the overstimulation of muscarinic receptors on efferent
neurons could modify the function of these neurons, including
excitability, neurotransmitter synthesis or release, or gene
expression. Availability of receptors and thus, the transmission of the
neuronal message, may also be modified by molecules interacting with
these receptors, as suggested by the fact that
L-dopa provokes internalization of D1R in
Parkinson's disease patients (Muriel et al., 1999). The same
methodology could be expanded to other brain areas, which could help to
better understand the physiological relevance of regulations of the
subcellular localization of receptors and the role of perturbations of
the neuronal activity caused by cholinergic dysfunctions, especially in
the cortex in Alzheimer's disease.
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FOOTNOTES |
Received July 6, 1999; revised Sept. 8, 1999; accepted Sept. 15, 1999.
We thank Dr. P. Ciofi (U.378 Institut National de la Santé, et de
la Recherche Médicale-Institut François Magendie, 1, rue
Camille Saint-Saëns, 33077 Bordeaux, France) for providing the
anti-µ opiate receptor-1A. We also thank Evelyne Doudnikoff for her
expert technical work, Claude Vidauporte for the photographic artwork,
and the Electron Microscopy Centre of the University Victor
Ségalen-Bordeaux 2.
Correspondence should be addressed to Dr. Véronique Bernard,
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5541, Laboratoire d'Histologie-Embryologie, Université Victor Ségalen-Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux Cedex, France. E-mail:
Veronique.Bernard{at}umr5541.u-bordeaux2.fr.
| |
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