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The Journal of Neuroscience, March 1, 1998, 18(5):1650-1661
Internalization of D1 Dopamine Receptor in Striatal Neurons
In Vivo as Evidence of Activation by Dopamine
Agonists
Brigitte
Dumartin,
Isabelle
Caillé,
Francois
Gonon, and
Bertrand
Bloch
Unité Mixte de Recherche Centre National de la Recherche
Scientifique 5541, Laboratoire d'Histologie-Embryologie, Institut
Fédératif de Recherches en Neurosciences Cliniques et
Expérimentales, Université Victor Ségalen-Bordeaux 2, Bordeaux, France
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ABSTRACT |
To investigate how dopamine influences the subcellular localization
of the dopamine receptors in the striatal dopaminoceptive neurons, we
have used immunohistochemistry to detect D1 dopamine receptors (D1R)
after modifications of the dopamine environment. In normal rats, D1R
are located mostly extrasynaptically at the plasma membrane of the cell
bodies, dendrites, and spines. The intrastriatal injection of the full
D1R agonist SKF-82958 and the intraperitoneal injection of the same
molecule or of amphetamine (which induces a massive release of dopamine
in the striatum) induce modifications of the pattern of D1R
immunoreactivity in the dorsal and ventral striatum. Whereas normal
rats display homogenous staining of the neuropile with staining of the
plasma membrane of the cell bodies, either treatment provokes the
appearance of an intense immunoreactivity in the cytoplasm and the
proximal dendrites. The labeling pattern is heterogeneous and more
intense in the striosomes than in the matrix. Analysis of semithin
sections and electron microscopy studies demonstrates a translocation
of the labeling from the plasma membrane to endocytic vesicles and endosomes bearing D1R immunoreactivity in the cytoplasm of cell bodies
and dendrites. Injection of D1R antagonist (SCH-23390) alone or
injection of D1R antagonist, together with amphetamine or SKF-82958, do
not provoke modification of the immunoreactivity, as compared with
normal rat.
Our results demonstrate that, in vivo, the acute
activation of dopamine receptors by direct agonists or endogenously
released dopamine provokes dramatic modifications of their subcellular distribution in neurons, including internalization in the endosomal compartment in the cytoplasm. This suggests that modifications of the
localization of neurotransmitter receptors, including extrasynaptic ones, may be a critical event that contributes to the postsynaptic response in vivo.
Key words:
dopamine receptors; receptor distribution; internalization; extrasynaptic receptors; immunohistochemistry; striatum; endosomes
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INTRODUCTION |
The actions of most
neurotransmitters are mediated by a large family of G-protein-coupled
receptors (Lohse, 1993 ). In most instances these receptors have a
preferential localization at the surface of the plasma membrane of the
neurons. Immunohistochemical studies have demonstrated that the
receptors either can be located within the body of the postsynaptic
specialization and/or can be located extrasynaptically at the surface
of the cell bodies and dendrites (Aoki et al., 1989; Baude et al.,
1993; Levey et al., 1993; Hersch et al., 1994, 1995; Yung et al., 1995;
Caillé et al., 1996). In vitro studies with cells
expressing naturally occurring receptors or with transfected cells have
demonstrated that the activation of these receptors by endogenous or
artificial ligands promotes multistep molecular and cellular events
contributing to the postsynaptic response and to the metabolism and
recycling of the receptors. This includes the activation or inhibition
of second messenger systems and the desensitization and recycling of
the receptors via phosphorylation-dephosphorylation processes (Benovic
et al., 1988; Raposo et al., 1989; Lefkowitz and Caron, 1993; Fonseca
et al., 1995; Roettger et al., 1995; Krueger et al., 1997). In
vitro models show that the latter events involve primarily the
internalization of the receptors and complex intracytoplasmic trafficking, which include the formation of endocytic vesicles and
endosomes (Fonseca et al., 1995; Roettger et al., 1995; Trogadis et
al., 1995; Koenig and Edwardson, 1997). Nevertheless, little is known
about the in vivo behavior and fate of neuroreceptors and
especially about the influence of the environment caused by neurotransmitters and related drugs on the addressing, the
localization, and the distribution of receptors. Recent data
demonstrate that the evoked release or the direct application of
neuropeptides (substance P or neurotensin) or the injection of an
opiate agonist promotes in vivo dramatic and profound
modifications of the localization of the corresponding receptors,
including translocation from the plasma membrane, internalization in
endosomes, and recycling at the membrane (Faure et al., 1995; Mantyh et
al., 1995a,b; Sternini et al., 1996; Lin et al., 1997).
Dopamine is a fast-acting neurotransmitter inducing effects via
G-protein-coupled receptors (Seeman and Van Tol, 1994; Sokoloff and
Schwartz, 1995; Jaber et al., 1996). Dopamine receptors are direct or
indirect targets for many molecules, including neuroleptics, and
indirect dopamine agonists, such as amphetamines and cocaine (Jackson
and Westlind-Danielson, 1994; Seeman and Van Tol, 1994). Among these
receptors the D1 receptor isotype (D1R) mediates dopamine actions via
the activation of adenylate cyclase (Gingrich and Caron, 1993; Sokoloff
and Schwartz, 1995). The striatum, a major site for dopamine action, is
densely innervated by dopamine fibers. Immunohistochemical analysis
demonstrates that the cell bodies, dendrites, and spines of the
postsynaptic striatal neurons display D1R primarily extrasynaptically
located at the plasma membrane (Levey et al., 1993; Hersch et al.,
1995; Caillé et al., 1996).
To understand the cellular response mediated by dopamine, we have
searched whether the dopamine environment may influence the
distribution and the localization of D1R in striatal neurons in
vivo by using immunohistochemistry. We demonstrate here that the
injection of the D1R agonist SKF-82958 and the release of dopamine
induced by amphetamine provoke dramatic and acute modification of D1R
localization, especially its internalization in the cytoplasm of cell
bodies and dendrites via endocytosis. Our results demonstrate that
modifications of the neurotransmitter environment can influence directly the intracellular trafficking of the corresponding receptor in vivo. They also bring morphological evidence that
extrasynaptic D1R can react after ligand binding and can undergo
internalization and recycling under direct stimulation.
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MATERIALS AND METHODS |
Animals and tissue preparation
Adult male Wistar rats (250-300 gm; Centre d'élevage
Janvier, Le Genest St. Isle, France) were used in this study. They were maintained under standard housing conditions, and experiments were
performed in accordance with the guidelines of the French Agriculture
and Forestry Ministry (decree 87849, license 01499) and with the
approval of Centre National de la Recherche Scientifique. They were
treated either by the D1R full agonist SKF-82958 or by amphetamine that
induces a massive release of dopamine via action on the dopamine
transporter.
Intraperitoneal injection of SKF-82958. Rats
(n = 10) received a single injection of SKF-82958 (2 mg/kg) (Research Biochemicals, Natick, MA) dissolved in 0.9 gm/l NaCl.
After an appropriate survival time (20-60 min), the rats were
anesthetized deeply with chloral hydrate and processed for
immunohistochemical detection of D1R, as described below. Control
experiments included normal rats maintained in the same housing
conditions (n = 2), rats intraperitoneally injected
with saline (n = 3; survival time, 40 min), rats having received an injection of the D1R antagonist SCH-23390 (0.5 mg/kg, Research Biochemicals, Natick, MA) dissolved in 20% acetic acid (10 mg/ml) and diluted (0.25 mg/ml) in 0.9 gm/l NaCl (n = 4; survival time, 40 min), and rats having received combined injections
of SCH-23390 and SKF-82958 (n = 5; survival time, 40 min).
Intrastriatal injection of SKF-82958. Rats
(n = 12) were anesthetized with urethane (1.15 gm/kg)
and mounted in a stereotaxic frame. Glass micropipettes (tip external
diameter, 30 µm) were filled through the tip by negative pressure and
implanted into the dorsal striatum (bregma, + 1.2 mm; lateral, 2.0 mm;
depth, 4.2 mm). SKF-82958 (1 mg/ml in saline: NaCl 9 gm/l, KCl 0.2 gm/l, and CaCl2 1.3 mM) was injected by air
pressure (500 nl in 2 min). Animals were allowed to survive from 4 min
to 5 hr after the end of the injection. Control injections of the same
volume of vehicle solution were made simultaneously in the
contralateral striatum.
Intraperitoneal injection of amphetamine. Rats
(n = 11) received a single injection of 5 mg/kg
D-amphetamine sulfate (Coopérative Pharmaceutique
Française, Melun, France) and were allowed to survive from 20 min
to 4 hr after the end of the injection. Control groups received saline
injection (n = 3), SCH-23390 injection alone
(n = 4), or a combined injection of SCH-23390 (0.5 mg/kg) and amphetamine (n = 3).
Tissue preparation. After an appropriate survival time, the
animals were processed for tissue preparation. Rats were anesthetized deeply with chloral hydrate and perfused transcardially with 50-100 ml
of 0.9% NaCl and 400 ml of fixative (2% paraformaldehyde and 0.1%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were removed, stored in 2% paraformaldehyde overnight, and cut into 50 µm frontal sections with a vibratome. The sections were collected,
cryoprotected in PBS solution (0.01 M, pH 7.4) containing 30% saccharose, and freeze-thawed in isopentane to improve the penetration of immunoreagents. Then the sections were preincubated for
1 hr in PBS with 0.2% BSAc (Aurion, the Netherlands) (PBS-BSAc); at
room temperature before treatment with the D1R antiserum.
Immunohistochemical detection of D1R
D1R was detected at the light and electron microscopic level by
using a polyclonal D1R antiserum produced and characterized as
previously described (Caillé et al., 1995). This antiserum was
generated against a fusion protein, including the C-terminal intracytoplasmic part of the receptor. Controls for specificity were
performed in previous studies and in the present one. They demonstrated
the specificity of the antiserum via Western blot and specific
localization of the immunoreactivity in areas and neurons known
specifically to express the D1R in rat and human, especially in good
correlation with in situ hybridization studies and binding
analysis (Caillé et al., 1995, 1996; Brana et al., 1996). The
specificity of the immunolabelings also was attested by the absence of
signal when primary antibody was omitted, when preimmune serum was
used, and when D1R antiserum was preadsorbed with the fusion protein.
Detection of D1R on vibratome sections by immunoperoxidase method
with tyramide signal amplification. Vibratome sections were incubated in D1R antiserum diluted 1:10,000-1:20,000 in PBS-BSAc for
15-48 hr at 4°C. Then the sections were washed (3× PBS) and incubated for 1 hr at room temperature in biotinylated goat anti-rabbit IgG (1:200 in PBS-BSAc, Amersham-UK, Little Chalfont, UK). After being
washed (3× PBS), nonspecific binding sites were blocked by incubation
for 30 min at room temperature in TNB (0.1 M Tris-HCl and
0.15 M NaCl) containing 0.5% DuPont blocking reagent (TSA indirect kit, DuPont NEN, Wilmington, DE) and incubated in
streptavidin-horseradish peroxidase (SA-HRP) 1:500 in TNB for 30 min
at room temperature. The sections were washed three times for 10 min
each in TNT buffer (0.1 M Tris-HCl and 0.15 M
NaCl with 0.05% Tween 20) and then incubated for 3-10 min in biotinyl
tyramide (1:50 in amplification diluent) (TSA indirect kit, DuPont
NEN). After being washed (3× TNT), the sections were incubated again
with SA-HRP (1:500 in TNB) for 30 min at room temperature and rinsed
again (3× TNT). Peroxidase activity was revealed with
3,3'-diaminobenzidine (DAB; 0.05% in Tris buffer, pH 7.6; Sigma,
Poole, UK) in the presence of hydrogen peroxide (0.01%). The reaction
was stopped by several washes in Tris buffer. The vibratome sections
were mounted on glass slides, dehydrated in graded ethanols, and
mounted in Eukitt for light microscopic observation.
Detection of D1R in semithin sections with the avidin-biotin
method. Vibratome sections were immunostained by the
avidin-biotin peroxidase method (ABC; Vectastain-Elite, Vector
Laboratories, Burlingame, CA), as previously described (Caillé et
al., 1995). The sections were incubated for 48 hr at 4°C with D1R
antiserum diluted 1:1000 in PBS-BSAc. After being washed (3× PBS),
they were incubated for 1 hr at room temperature in biotinylated goat anti-rabbit IgG (1:200 in PBS-BSAc). After three rinses in PBS, sections were incubated in the ABC complex (0.5% in PBS) for 1 hr. The
D1R immunoreactive sites were revealed by incubation in H2O2-DAB solution, as described above. After
several washes in Tris buffer, selected areas of interest (dorsal
striatum) were post-fixed in osmium tetroxide (1% in PBS) for 30 min,
washed in PBS, dehydrated in graded ethanol, and transferred to
propylene oxide. The sections were preimpregnated with a 1:1 mixture of Araldite and propylene oxide for 1 hr, impregnated with Araldite overnight, and flat-embedded in Araldite. Semithin sections (1-2 µm)
were cut with a Reichert ultracut S (Leica, Nusslock, Germany), collected on glass slides, dried, and mounted in Eukitt.
Detection of D1R by immunogold method at the ultrastructural
level. Vibratome sections were incubated in D1R antiserum, as described in the immunoperoxidase procedure. After being washed twice
in PBS-BSAc and twice in PBS-BSAc supplemented with 0.1% fish
gelatin (Aurion) (PBS-BSAc-gelatin), sections were incubated for 24 hr at room temperature in goat anti-rabbit IgG conjugated to ultrasmall
colloidal gold particles (0.8 nm; Aurion) diluted in PBS-BSAc-gelatin
(1:50). After several washes (3× PBS-BSAc-gelatin, 3× PBS, and 3×
2% sodium acetate), the immunogold signal was intensified by using a
silver enhancement kit (Aurion). The reaction was performed in the dark
for 15-30 min at room temperature and stopped by two washes in 2%
sodium acetate. Then the signal was intensified and stabilized
(Trembleau et al., 1994) by immersion of the sections in gold chloride
(0.05% in distilled water) for 10 min at 4°C and then in sodium
thiosulfate (0.3% in distilled water, two times for 10 min at 4°C).
After several washes in PBS, the sections were processed for electron
microscopy. They were post-fixed in osmium tetroxide and embedded in
Araldite, as described above. Ultrathin sections of immunogold-treated
material were cut. They were collected on copper grids, contrasted with
uranyl acetate and lead citrate, and observed with a Phillips CM 10 electron microscope (Phillips Electronic Instruments, Mahwah, NJ).
Quantitative analysis of variations of D1R immunoreactivity.
Variations in D1R immunoreactivity were measured at the
ultrastructural level by using plates of immunolabeled cell bodies and
dendritic shafts of control and SKF-82958-treated rats. Morphometric
analysis was performed by using Metamorph software (Universal Imaging, Paris, France). The measures were performed in sections from control animals (n = 3; 10 cell bodies and 26 dendrites), from
animals (n = 3) having received systemic injection of
SKF-82958 (19 cell bodies and 20 dendrites), and from animals
(n = 3) having received intrastriatal injection of
SKF-82958 (19 cell bodies and 20 dendrites). Gold immunoparticles
present at the plasma membrane were identified and counted, and the
results were expressed as the number of immunoparticles per 100 µm of
membrane in dendrites and cell bodies (length of analyzed membrane was
~1500 µm for cell bodies and 1000 µm for dendrites for each
group). Immunopositive endosomes and endocytic vesicles were identified
and counted in the cytoplasm, and the results were expressed as the
number of endosomes per 100 µm2 (surface of
analyzed cytoplasm was ~12,000 µm2 for cell
bodies and 2000 µm2 for dendrites for each group).
Details of analyses and measures are described in Figure 6.
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RESULTS |
Localization of D1R immunoreactivity in normal animals
The analysis of D1R immunoreactivity in normal animals and in
animals injected with vehicle solution demonstrated the presence of D1R
at the membrane of the cell bodies and dendrites of striatal neurons,
as previously described (Caillé et al., 1996). An overview of the
immunolabeling in 50-µm-thick vibratome sections demonstrated an
intense and homogeneous labeling of the neuropile in the striatum with
few, faintly immunoreactive cell bodies (Fig.
1A,B). The labeling
generally appeared more intense in the nucleus accumbens. The detailed
analysis of vibratome and of semithin sections obtained after
embedding in Araldite (Fig. 1B,C) demonstrated that
the large majority of immunoreactivity was located at the membrane of
the dendrites and cell bodies. Such localization was confirmed by
ultrastructural examination that showed immunoreactivity as gold
particles located at the inner side of the plasma membrane of the cell
bodies, dendrites, and spines (Fig. 1D,E). The large majority of the immunoreactivity appeared extrasynaptically located. In
spines, gold particles generally were located at the edge of asymmetrical synapses. Intracytoplasmic immunoreactivity was present in low abundance; it was restricted mostly to a few vesicles
with the morphological features of endosomes. The
endoplasmic reticulum cisternae and the Golgi apparatus contained
very little immunoreactivity.
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Figure 1.
Immunohistochemical detection of D1R in normal
rats: light and electron microscopy. A shows a general
view of the striatum (vibratome section). Immunoreactivity is
distributed homogeneously in the neuropile throughout the dorsal and
the ventral striatum. B and C show high
magnification in vibratome (B) and semithin (C) sections. The labeling is located essentially
along the membrane of the cell bodies (arrows) and in
the neuropile as a thin deposit. D, E,
Electron microscopy after the immunogold technique. D
shows part of an immunoreactive cell body. D1R immunoreactivity is
located as gold particles dispersed along the plasma membrane
(arrowheads); note the absence of intracytoplasmic
staining in this neuron. E shows a sagittal section of a
dendritic shaft with gold particles restricted to the plasma membrane
(arrowheads). CC, Corpus callosum; CP, caudate putamen nucleus; AC,
accumbens nucleus; OT, olfactory tubercle;
Go, Golgi apparatus; d, dendrite;
s, spine. Scale bars: A, 250 µm;
B, C, 20 µm; D,
E, 0.5 µm.
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Localization of D1R immunoreactivity after intraperitoneal
injection of SKF-82958
Injection of SKF-82958 provoked dramatic modifications of
the aspect and localization of D1R immunoreactivity. Vibratome sections showed heterogeneous immunolabeling, with a stronger signal in areas
that displayed the aspect and location of striosomes (Fig. 2A), such as the
pericallosal striosome. Numerous cell bodies present throughout the
dorsal and ventral striatum were intensely immunoreactive in the
cytoplasm and the proximal dendrites (Fig. 2B-E).
Higher signal in the areas that may correspond to the striosomes was
attributable to a higher density of immunoreactive fibers and cell
bodies (Fig. 2A). Cell bodies were also numerous and intensely reactive in the nucleus accumbens. The analysis of semithin sections showed that the appearance of immunoreactive cell bodies was
attributable to an accumulation of intracytoplasmic deposits with D1R
immunoreactivity, with a sharp decrease or a disappearance of the
immunolabeling located at the membrane of the neurons (Fig. 2B). Similar aspects were also visible in proximal
dendrites, as compared with normal rats (Fig. 2B).
The ultrastructural analysis confirmed this pattern and demonstrated
that the intracytoplasmic immunoreactivity appeared to be associated
mostly with the cytoplasmic side of membranes, limiting vacuoles that
displayed morphological features of the endosomal compartment (Figs.
3,
4A,B). These endosomes had homogenous and clear content, had a vesicular or tubulovesicular aspect, and were limited by an irregular membrane. They appeared to be
located frequently at the periphery of the cytoplasm at the vicinity of
the plasma membrane (Figs. 3B-D, 4). Occasionally, the
presence of endocytic vesicles associated with the plasma membrane was
detected in cell bodies and dendrites (Fig. 3C,D). Such
modifications were observed at all times in the largest part of
immunoreactive neurons, with a maximal effect at 20 and 40 min (Fig.
2C-E). Electron dense vacuoles that may correspond to lysosomes did not display immunoreactivity. Golgi apparatus and endoplasmic reticulum did not show modifications, as compared with
normal rats. The examination of dendritic spines suggested that there
was no modification of the immunolabeling for the receptors located at
the periphery of synaptic clefts. Quantification of D1R
immunoreactivity at the ultrastructural level confirmed the sharp
modifications of the receptor compartmentation in the cell bodies and
dendrites (see Fig. 6). Detailed counting showed that there was an
important decline in plasma membrane immunoreactivity (Fig.
6A; 3.3 times and 3.5 times less in cell bodies and
dendrites, respectively) and a parallel increase in the density of
endosomal structures bearing D1R immunoreactivity in the cytoplasm
(Fig. 6B; 4.9 times and 3.9 times more in cell bodies
and dendrites, respectively). Control (saline-injected) animals and
animals injected with the D1 antagonist SCH-23390 did not demonstrate
modifications, as compared with normal rats (data not shown).
Simultaneous injection of SCH-23390 with SKF-82958 sharply decreased or
abolished the modifications induced by the injection of SKF-82958 alone
(see Fig. 2F).
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Figure 2.
Immunohistochemical detection of D1R after
intraperitoneal injection of SKF-82958: light microscopy.
A and B show details of the striatum 40 min after injection. In A (vibratome section), the
neuropile labeling is heterogeneous, and numerous intensely immunoreactive cell bodies are present. Areas of high signal intensity may correspond to striosomes. In B (semithin section),
the neuronal immunoreactivity appears mostly as an accumulation of dots
located in the cytoplasm (arrows). The
arrowheads point to immunoreactivity inside a dendrite.
C-E show the striatum at 20 and 40 min and at 1 hr
after injection (vibratome sections). The immunoreactive neurons
display intense labeling in the cytoplasm and proximal dendrites in
C and D. E shows that the
signal has decreased after 1 hr. Immunoreactivity of the neuropile is
less intense in C-E than in the normal or
saline-injected rat. F (vibratome section) shows the
striatum after a combined injection of D1R antagonist SCH-23390 and
SKF-82958. The labeling appears identical to that observed in the
normal or saline-injected rat in Figure 1B
(arrows). st, Striosome;
m, matrix. Scale bars in A-F, 20 µm.
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Figure 3.
Immunohistochemical detection of D1R after
intraperitoneal injection of SKF-82958 (40 min): electron microscopy
after immunogold technique. A shows detail of a
cell body. Part of the immunoreactivity is still at the plasma membrane
(arrowheads), but there is an accumulation of gold
particles in the cytoplasm (arrows). B
and C show details of the cytoplasmic labeling; it is
restricted mostly at the periphery of vesicles that have morphological
features of endosomes inside the cytoplasm (arrows in
B) or of endocytic vesicles (arrow in
C) located at the immediate vicinity of the plasma
membrane; the arrowhead in C points to
the neck linking the plasma membrane to the endocytic vesicle.
D shows a dendrite. Part of the immunoreactive material
is located at the membrane (arrowheads), but many gold
particles are present inside the dendritic shaft (large
arrows). The thin arrows point to endocytic
vesicles containing DR1 immunoreactivity at the vicinity of the plasma membrane. d, Dendrite. Scale bars, 0.5 µm.
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Figure 4.
Immunohistochemical detection of D1R after
injection of SKF-82958: electron microscopy after immunogold technique
and details of dendrites. A shows the transversal
section of a dendrite with a dendritic spine, with a synapse
(star) located at the neck of the spine. Sections
demonstrate various aspects of the receptor at the membrane
(arrowheads) and of the internalization of the receptor
with especially early phases of the formation of the endocytic vesicles
and endosomes in B and C
(arrows). A, At 40 min after
intraperitoneal injection. B, At 40 min after
intraperitoneal injection. C, At 10 min after
intrastriatal injection. d, Dendrite; s, dendritic
spine. Scale bars, 0.5 µm.
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Localization of D1R immunoreactivity after intrastriatal injection
of SKF-82958
Immunohistochemical analysis performed after direct intrastriatal
injection of SKF-82958 demonstrated the same general features as those
that occurred after intraperitoneal injections (Fig. 5). Careful examination of the sections
showed that the neurons and dendrites located at the vicinity of the
site of injection appeared well preserved and showed changes in the
localization of the D1R immunoreactivity at the light and electron
microscopic level identical to those observed after intraperitoneal
injection (Figs. 4C, 5A-F). These
immunoreactive neurons were detectable as early as 4 min after
injection and were very intensely reactive 10, 30, and 60 min after
injection (Fig. 5A,B). Intracytoplasmic localization of the
receptor was still detectable after 5 hr, but with a lower intensity
(Fig. 5C). Quantification at the ultrastructural level also
confirmed modifications of D1R distribution that paralleled the ones
observed after intraperitoneal injection of SKF-82958 (3.5 times and
3.9 times less immunopositive gold particles at the plasma membrane of
the cell bodies and dendrites, respectively; 5.7 and 4.8 times more
endosomal structures bearing D1R immunoreactivity in the cell bodies
and dendrites, respectively) (Fig. 6).
The injection of the vehicle solution alone in the contralateral
striatum did not provoke any modification of D1R immunoreactivity (data not shown).
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Figure 5.
Immunohistochemical detection of D1R after
intrastriatal injection of SKF-82958. A-C (vibratome
sections) show the time course of the D1R immunoreactivity at 30 min
(A), 1 hr (B), and 5 hr (C) after SKF-82958 injection. All views have
been taken at the same distance from the injection site. The neurons
are highly labeled at short times and stay immunoreactive after 5 hr.
D shows details of neurons in semithin section with
immunoreactivity internalized in the cytoplasm (10 min after
injection). E, F, Electron microscopy after immunogold technique demonstrates the presence of immunoreactive endosomes in a cell body (E) and in a dendrite
(F, arrows) at 10 min after injection.
e, Endosome; d, dendrite. Scale bars: A-D, 20 µm; E, F, 0.5 µm.
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Figure 6.
Quantitative analysis of the variations of D1R
immunoreactivity in control and SKF-82958-treated rats. Immunoreactive
particles and endosomes were counted on micrographs after
immunodetection of D1R at the ultrastructural level in control rats, in
rats having received intraperitoneal (I.P.) injection of
SKF-82958 (40 min), and in rats having received intrastriatal
(I.S.) injection of SKF-82958 (10 min).
A, Immunoreactive particles (as visible in Figs.
1D,E, 3A) were counted.
Columns in A correspond to the number of
immunoparticles per 100 µm of plasma membrane in cell bodies and
dendrites ±SEM. The number of immunoparticles strongly decreases after
SKF-82958 injection in cell bodies and dendrites. B,
Immunoreactive endosomes and endocytic vesicles (as visible in Figs.
3B-D, 5E,F) were counted.
Columns in B correspond to their density
per 100 µm2 of cytoplasm in cell bodies and
dendrites ±SEM. The injection of SKF increases the density of
immunoreactive endosomes or endocytic vesicles in cell bodies and
dendrites (*p < 0.05; Mann-Whitney nonparametric
test).
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Localization of D1R immunoreactivity after intraperitoneal
injection of amphetamine
Intraperitoneal injection of amphetamine provoked modifications of
D1R immunoreactivity at the light and ultrastructural level that
appeared similar to those previously described after the injection of
SKF-82958 (Fig. 7). These modifications
were maximal 20 and 40 min after injection (Fig. 7C,D) and
were still detectable after 90 min with a lower intensity (Fig.
7E). Immunoreactivity after 4 hr was the same as for the
controls. Injection of SCH-23390, together with amphetamine, strongly
reduced or abolished modifications of the immunolabeling (data not
shown).
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Figure 7.
Immunohistochemical detection of D1R after
intraperitoneal injection of amphetamine. A and
B show a general view of the dorsal striatum after
injection of saline (A) or amphetamine (40 min; B). As compared with A,
B shows the appearance of heterogeneous labeling with
higher signal in areas that may correspond to the striosomes and the
presence of numerous cell bodies (vibratome sections).
C-E show aspects of the immunoreactive cell bodies at
20 min (C), 40 min (D), and
90 min (E) after injection (vibratome section).
F shows detail of immunoreactive endosomal compartment (arrow) in a cell body at the ultrastructural level
after immunogold technique. The arrowhead points to the
receptor at the membrane. st, Striosome;
m, matrix. Scale bars: A,
B, 100 µm; C-E, 20 µm; F, 0.5 µm.
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DISCUSSION |
Analysis of the modifications of D1R distribution after
DR stimulation
The results of the present study demonstrate that D1R naturally
expressed at the surface of dopaminoceptive neurons in the CNS can
undergo regulated internalization and sequestration in the cytoplasm
after in vivo activation. Our data show that the same
effects are obtained by using a locally or intraperitoneally injected
agonist that activates only D1R or by provoking a massive release of
endogenous dopamine with amphetamine that activates all dopamine
receptor subtypes in the striatum. Light microscopy studies show that
short-term action of these molecules dramatically and massively alters
the localization and distribution of the D1R immunoreactivity, whatever
the treatment. Counting of D1R immunoreactivity shows that D1R
immunoreactivity present at the surface of plasma membrane in cell
bodies and dendrites of normal rats dramatically drops after
intraperitoneal or intrastriatal injection (see Fig. 6). Similarly, the
density of endosomes bearing D1R immunoreactivity highly increases in
each experimental situation both in cell bodies and dendrites. These
modifications of the ultrastructural aspect of the neurons demonstrate
that the decrease in plasma membrane immunoreactivity is associated
with the increase of the immunoreactive intracytoplasmic vesicular
compartment that displays all of the morphological features of the
endosomal compartment (Nixon and Cataldo, 1995; Koenig and Edwardson,
1997), especially a clear content, irregular tubulovesicular aspect,
and formation of endocytic vesicles from the plasma membrane. Such
features correlate with detailed morphological and molecular in
vitro studies, which demonstrate that stimulation of
G-protein-coupled receptors causes a dramatic reorganization of their
intracellular distribution, including endocytosis and formation of
endosomes (for review, see Koenig and Edwardson, 1997). The
translocation of the D1R from the plasma membrane to intracytoplasmic
vesicles occurs in the cell bodies and dendrites as also demonstrated
in vivo for substance P, neurotensin, or opioid receptors
(Faure et al., 1995; Mantyh et al., 1995a,b; Sternini et al., 1996). As
expected from the in vitro mechanisms of the formation of
endosomes containing G-protein-coupled receptors (Fonseca et al., 1995;
Roettger et al., 1995; Goodman et al., 1996; Krueger et al., 1997), the
D1R immunoreactivity (corresponding to the detection of an
intracytoplasmic C-terminal region of the molecule; Caillé et
al., 1995) is present at the inner side of the plasma membrane in
normal animals and is translocated at the membranes of these vesicles
in stimulated animals, appearing frequently associated with their
cytoplasmic side. Indeed, in the absence of appropriate detection of
markers for the various intracytoplasmic compartments, the detailed
route of G-protein-coupled receptors endocytosed in vivo can
be hypothesized only and must await detailed molecular analysis,
coupled with immunohistochemical receptor detection. Especially, the
present ultrastructural data did not allow us to establish whether
these vesicles were associated with clathrin. Nevertheless, identical ultrastructural features strongly suggest that, in vivo, the
receptors are internalized in vesicles and transferred in the endosomal compartment. The absence of a significant amount of D1R in other vesicular compartments, especially in lysosomes, favors the hypothesis of a recycling of D1R at the plasma membrane after internalization rather than an intracellular degradation. This is supported by the fact
that the morphological studies show that D1R immunoreactivity returns
to the membrane after stimulation by SKF-82958 or amphetamine. The
persistence of a residual intracytoplasmic immunoreactivity 5 hr after
intrastriatal injection of SKF-82958 nevertheless suggests that some of
the receptor may have a specific intracellular metabolism after
internalization. The absence of detectable variations in the aspect and
abundance of D1R in the endoplasmic reticulum and Golgi apparatus after
stimulation suggests that, under our experimental conditions, D1R
neosynthesis would not contribute significantly to modifications of D1R
compartmentation after stimulation. Nevertheless, we cannot exclude
that other modes of stimulation, other time courses, or more detailed
ultrastructural study may reveal other cytoplasmic pathways for D1R,
including a degradation in lysosomes, as suggested by several in
vitro studies (Raposo et al., 1989; Nixon and Cataldo, 1995;
Roettger et al., 1995). The in vivo formation of endosomes
triggered by the stimulation of G-protein-coupled receptors is in good
correlation with the in vitro models showing similar
features for D1R, adrenergic, or peptidergic receptors (Fonseca et
al., 1995; Ng et al., 1995; Roettger et al., 1995; Trogadis et al.,
1995) and with the recent observation of in vivo substance P
receptor internalization after agonist injection (Mantyh et al., 1995b)
or glutamate receptor stimulation (Lin et al., 1997). Nevertheless, in
contrast to the observations of Mantyh and colleagues showing the
reshaping of dendrites bearing stimulated substance P receptors, we did
not observe modifications of the aspects of the dendrites of the
dopaminoceptive neurons.
The D1R present at the surface of striatal neurons are mainly
extrasynaptic, as demonstrated in previous studies (Hersch et al.,
1995; Yung et al., 1995; Caillé et al., 1996). Our results demonstrate that nonsynaptic receptors can respond to in
vivo stimulation by internalization. It can be expected from these data that the binding of agonists on these nonsynaptic receptors also
triggers the cascade of transduction for D1R, including activation of
adenylate cyclase. Our data then reinforce the hypothesis that dopamine
may act in the striatum on nonsynaptic receptors via diffusion at a
distance from release sites (Garris et al., 1994). This also indicates
that these nonsynaptic receptors can be, in vivo, direct or
indirect targets for drugs interacting with dopamine transmission (such
as direct dopamine agonists) or for psychostimulants, such as cocaine
and amphetamine. The apparent absence of modification of the
localization of perisynaptic receptors located in dendritic spines may
reflect a distinct metabolism of these receptors or a different time
course for internalization. Depending on the availability of efficient
antisera, we may explore whether the other dopamine receptors present
in the striatum also display modifications of their localization after
stimulation.
Functional significance of in vivo internalization
Our data show that localization and subcellular distribution of
receptors mediating the effects of a fast-acting neurotransmitter, dopamine, can be altered acutely and dramatically in vivo by
modifications of the environment of the neuron. The functional
significance of these events can be considered on the basis of the
previous in vitro studies, which show that the
internalization of receptors in endocytic vesicles and endosomes is an
early event leading to the desensitization of the receptor via
phosphorylation and sequestration in the cytoplasm (Lefkowitz and
Caron, 1993; Krueger et al., 1997). Although endocytosis and
desensitization processes can be dissociated in vitro (Ng et
al., 1995), they appear to be associated closely in the cascade of
cellular and molecular events after in vitro agonist
stimulation of a G-protein receptor (Lefkowitz and Caron, 1993; Zhang
et al., 1998). Our results strongly suggest that modifications of the
subcellular distribution of neurotransmitter receptors also might,
in vivo, be a critical and early element of the postsynaptic
response because it is detectable as early as 4 min after agonist
injection. This endocytosis may limit or modify the access of a natural
or artificial ligand to its receptor and also may contribute to
receptor resensitization or downregulation (Zhang et al., 1998). These
in vivo results must encourage us to investigate
experimental and pathological conditions involving dopamine to
determine whether modifications of dopamine receptor distribution and
localization may contribute to the alteration of dopamine transmission
in acute, but also in chronic, situations. It is known that acute and
chronic behavioral effects of psychostimulants, such as amphetamine,
are linked closely to the early induction of the c-fos gene
and modifications of the expression of neuropeptide genes in striatal
neurons (Young et al., 1991; Cole et al., 1995; Jaber et al., 1995).
Most of these mechanisms are mediated via the activation of D1R located on the striatal dopaminoceptive neurons (Young et al., 1991; Drago et
al., 1996). These data suggest that the internalization of D1R may be
considered not only as an early evidence but also as a major actor of
the postsynaptic response. Indeed, these receptors most probably are
internalized with their natural or artificial ligands, as demonstrated
in vitro (Koenig and Edwardson, 1997), and it can be
hypothesized that internalization may contribute to regulate receptor
availability for extracellular ligands. This internalization is
distributed heterogeneously in the striatum, with higher intensity in
areas that display the localization and aspect of striosomes. Technical
limitations did not allow us to demonstrate unambiguously in this study
that heterogeneity in D1R immunoreactivity after SKF-82958 and
amphetamine stimulation overlaps with patch/matrix delineation.
Nevertheless, the general aspect of the striatum after stimulation
strongly favors this hypothesis. These data correlate with the fact
that amphetamine injection induces under the same conditions the
appearance of c-Fos immunoreactivity (Graybiel et al., 1990),
preferentially in the striosome compartment.
Conclusion
Our present data, as well as those described by others,
demonstrate acute and massive internalization of postsynaptic
neurotransmitter receptors in vivo after stimulation (Faure
et al., 1995; Mantyh et al., 1995a,b; Sternini et al., 1996; Lin et
al., 1997). The present results are the first obtained for a
fast-acting classical neurotransmitter and bring new evidence for this
concept. The results reinforce the hypothesis that acute or chronic
modification of neurotransmitter receptor subcellular distribution may
constitute a common event contributing in vivo to the
postsynaptic response in the CNS under normal, experimental, and
pathological conditions.
| |
FOOTNOTES |
Received Sept. 22, 1997; revised Dec. 1, 1997; accepted Dec. 10, 1997.
This work was supported by funds from the Conseil Régional
d'Aquitaine. We thank V. Bernard, M. Jaber, and D. Choquet for kind
and expert help during image analysis and statistical analysis. We
thank C. Vidauporte for expert photographic artwork and the Service
Commun de Microscopie Electronique de l'Université Victor Segalen. We also thank M. C. Fournier for her help in the
preparation of D1R antiserum.
Correspondence should be addressed to Dr. Brigitte Dumartin,
Unité Mixte de Recherche Centre National de la Recherche
Scientifique 5541, Laboratoire d'Histologie-Embryologie,
Université Victor Segalen, 146 Rue Léo-Saignat, 33076 Bordeaux Cedex, France.
| |
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J. Llado, J. Caldero, J. Ribera, O. Tarabal, R. W. Oppenheim, and J. E. Esquerda
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V. Bernard, A. I. Levey, and B. Bloch
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
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V. Bernard, O. Laribi, A. I. Levey, and B. Bloch
Subcellular Redistribution of m2 Muscarinic Acetylcholine Receptors in Striatal Interneurons In Vivo after Acute Cholinergic Stimulation
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Top
Abstract
Introduction
