 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 1, 1998, 18(3):1056-1071
Interrelationships between Somatostatin sst2A Receptors and
Somatostatin-Containing Axons in Rat Brain: Evidence for Regulation of
Cell Surface Receptors by Endogenous Somatostatin
Pascal
Dournaud1,
Hélène
Boudin1,
Agnes
Schonbrunn3,
Gloria
S.
Tannenbaum1, 2, and
Alain
Beaudet1
Departments of 1 Neurology and Neurosurgery and
2 Pediatrics, McGill University, Montreal, Quebec H3A 2B4,
Canada and 3 Department of Integrative Biology and
Pharmacology, University of Texas, Houston Medical School, Houston,
Texas 77225
 |
ABSTRACT |
Using an antipeptide antibody, we reported previously on the
distribution of the somatostatin sst2A receptor subtype in rat brain.
Depending on the region, immunolabeled receptors were either confined
to neuronal perikarya and dendrites or distributed diffusely in tissue.
To investigate the functional significance of these distribution
patterns, we examined the regional and cellular relationships between
somatostatin axons and sst2A receptors in the rat CNS, using
double-labeling immunocytochemistry. Light and confocal microscopy
revealed a significant correlation (p < 0.02) between the distribution of somatodendritic sst2A receptor
immunoreactivity and that of somatostatin terminal fields, both
quantitatively and qualitatively. Furthermore, in regions of
somatodendritic labeling, a subpopulation of sst2A-immunoreactive cells
was also immunopositive for somatostatin, suggesting that a subset of
sst2A receptors consists of autoreceptors. By contrast, in regions
displaying diffuse sst2A labeling only moderate to low densities of
somatostatin terminals were observed, and no significant relationship
was found between terminal density and receptor immunoreactivity. At
the electron microscopic level, areas expressing somatodendritic sst2A labeling were found by immunogold cytochemistry to display low proportions of membrane-associated, as compared with intracellular, receptors. Conversely, in regions displaying diffuse sst2A receptor labeling, receptors were predominantly associated with neuronal plasma
membranes, a finding consistent with the high density of sst2 binding
sites previously visualized in these areas by autoradiography. Double-labeling studies demonstrated that in the former but not in the
latter regions, sst2A-immunoreactive somata and dendrites were heavily
contacted by somatostatin axon terminals. Taken together, these results
suggest that the low incidence of membrane-associated receptors
observed in regions of somatodendritic sst2A labeling may be caused by
downregulation of cell surface receptors by endogenous somatostatin,
possibly through ligand-induced receptor internalization.
Key words:
somatostatin; receptors; immunohistochemistry; electron
microscopy; internalization; receptor-transmitter mismatch
| |
INTRODUCTION |
Somatostatin (SRIF) is a
tetradecapeptide originally isolated from hypothalamus (Brazeau et al.,
1973 ) and subsequently found to be distributed throughout the neuraxis
as well as in various peripheral organs (Reichlin, 1983). Within the
brain, SRIF acts as a neuromodulator with widespread physiological
effects on neuroendocrine, cognitive, and behavioral functions
(Tannenbaum, 1985; Epelbaum et al., 1994). Accordingly, deficits in
SRIF and SRIF receptors have been documented in a number of
neurological disorders, including epilepsy (Schwarzer et al., 1996) and
Alzheimer's disease (Epelbaum et al., 1994). Mammalian SRIF
biosynthesis involves the post-translational proteolytic processing of
a single pro-SRIF precursor into the bioactive peptides SRIF-14 and
SRIF-28, the latter being a 14-residue NH2 terminally
extended form of the tetradecapeptide (Pradayrol et al., 1980; Schally
et al., 1980). Immunohistochemical studies have demonstrated the
presence of multiple populations of SRIF-containing nerve cells bodies
in mammalian brain, which give rise to extensive networks of
SRIF-immunoreactive fibers and axon terminals (Johannson et al., 1984).
Except for a single SRIF-28-selective neuronal system arising from the
nucleus of the solitary tract and projecting to the magnocellular
hypothalamic nuclei (Sawchenko et al., 1988), SRIF-immunoreactive
neurons contain both SRIF-14 and SRIF-28, in a proportion of ~3 to
1.
Five types of SRIF receptors, designated sst1-sst5 (Bruno et al.,
1992; O'Carroll et al., 1992; Yamada et al., 1992; Yasuda et al.,
1992), mediate the diverse functional effects of SRIF in the CNS.
Experiments on transfected cells have shown that the proteins encoded
by the cloned SRIF receptor genes display high affinity for both
SRIF-14 and SRIF-28 (Patel et al., 1995; Reisine and Bell, 1995;
Schindler et al., 1996). They also bind with high-affinity cortistatin
(Fukusumi et al., 1997), a neuropeptide with considerable homology with
SRIF but derived from a different gene product (De Lecea et al., 1996).
All five SRIF receptors are linked to guanine nucleotide binding
proteins (G-proteins) that mediate the responses of diverse cellular
effectors (Gu et al., 1995; Hoyer et al., 1995; Gu and Schonbrunn,
1997). In human and rodent tissues, the sst2 receptor was shown to
exist in two isoforms, sst2A and sst2B, generated through alternative
splicing of the sst2 mRNA at the 3 end of the coding segment (Vanetti
et al., 1992). These two variants, which differ only in length and in
their amino acid sequence at the C terminus, exhibit indistinguishable
binding properties but vary in G-protein coupling and desensitization (Vanetti et al., 1993).
Autoradiographic receptor binding studies have shown a widespread but
selective distribution of SRIF binding sites in mammalian brain (Martin
et al., 1991; Krantic et al., 1992; Moyse et al., 1992; Schoeffter et
al., 1995; Holloway et al., 1996). Recently, the development of
antisera against selective peptide sequences of the sst2A receptor has
allowed us (Dournaud et al., 1996) and others (Schindler et al., 1997)
to document the distribution of this receptor protein in rat brain by
immunocytochemistry. This distribution conformed for the most part to
that of SRIF binding sites detected using either nonselective (for
review, see Krantic et al., 1992) or sst2-preferring (Reubi and Maurer,
1985; Krantic et al., 1989, 1990; Martin et al., 1991; Schoeffter et
al., 1995; Holloway et al., 1996) ligands, suggesting that other SRIF
receptor subtypes are expressed within the same regions (Dournaud et
al., 1996; Schindler et al., 1997). However, there were marked regional differences in the light microscopic distribution of sst2A protein: in
some areas, immunolabeled receptors were selectively associated with
neuronal perikarya and dendrites, whereas in others they appeared
diffusely distributed throughout the tissue (Dournaud et al., 1996). In
the present study, we sought to investigate the functional significance
of these differential labeling patterns by correlating the regional,
cellular, and subcellular distribution of sst2A receptors with that of
SRIF-containing axon terminals in rat brain using light, confocal, and
electron microscopy.
| |
MATERIALS AND METHODS |
Antibodies
The R-88 rabbit polyclonal antibody, generated against a unique
sequence located in the C-terminal tail of the sst2A receptor, and the
rat monoclonal antibody (mAb) 354 (Chemicon, Temecula, CA), raised
against SRIF-14, were used as primary antibodies. Biochemical and
immunohistochemical characterization of the respective antibodies have
been reported elsewhere (Dun et al., 1994; Dournaud et al., 1996; Gu
and Schonbrunn, 1997). We further characterized the mAb 354 antibody by
ELISA and found it to cross-react with both SRIF-28 and cortistatin, as
well as with SRIF-14, at the dilutions used in the present
experiments.
Light microscopy
Single-labeling experiments. Adult male Sprague
Dawley rats (150-200 gm body weight; n = 5) were
anesthetized with Somnotol (80 mg/kg, i.p.) and perfused
transaortically with 4% paraformaldehyde in 0.1 M
Tris-buffered saline (TBS). Brains were cryoprotected by overnight
immersion in a 30% sucrose solution and frozen in liquid isopentane at
 45°C. The brains were sectioned at a thickness of 30 µm on a
freezing microtome. Adjacent serial sections throughout the brain were
processed for sst2A receptor and SRIF immunohistochemistry.
Sections were preincubated for 30 min in TBS containing 3% normal goat
serum (NGS) and incubated for 16 hr at room temperature (RT) in 1:2000
rabbit anti-sst2A antibody or 1:100 rat anti-SRIF antibody dilutions
containing 0.3% Triton X-100. Sections were then rinsed in 0.1 M TBS and sequentially incubated for 45 min in biotinylated
goat anti-rabbit IgG or biotinylated goat anti-rat IgG (Jackson
ImmunoResearch, West Grove, PA) diluted 1:100 in 0.1 M TBS.
They were then incubated for 45 min in avidin-biotin-peroxidase solution (ABC) (Vector Laboratories, Burlingame, CA) and subsequently incubated for 10 min in a 0.01% biotinyl-tyramide solution (DuPont, Billerica, MA), activated with 0.01%
H2O2, and reincubated in the ABC
solution. Visualization of the bound peroxidase was achieved by
reaction in a solution of 0.1 M Tris buffer containing
0.05% 3,3 diaminobenzidine (DAB), 0.04% nickel chloride, and 0.01% H2O2. Sections were mounted on gelatin-coated
slides, dehydrated in graded ethanols, delipidated in xylene,
coverslipped with Permount, and examined with a Leitz Aristoplan
microscope. Control experiments were performed by either omitting the
primary antibody or by replacing it with serum preabsorbed with 10 µM antigenic peptide (for sst2A) or SRIF-14 (for SRIF).
Sst2A receptor labeling was first defined as being either
somatodendritic (Table 1) or diffuse
(Table 2). Labeling densities were then
scored for each labeled region on a scale of + to +++++ according to
both the number of labeled elements and the density of immunoreactive
signal. Densities of SRIF-immunoreactive axon terminals detected in the
same regions on adjacent sections were assessed according to the same
scale. Distributions of sst2A receptors and SRIF axons were compared statistically using Spearman's correlation coefficient. Densities of
sst2A immunolabeling were also correlated with those of SRIF binding
sites documented previously by quantitative autoradiography using
sst2-preferring ligands (Tables 1, 2).
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of somatodendritic sst2A receptor
immunoreactivity with the density of SRIF-immunoreactive fibers and
axon terminals and autoradiographic sst2 receptor
labelinga
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Comparison of diffuse sst2A receptor labeling with the
density of SRIF immunoreactive fibers and axon terminals and
autoradiographic sst2 receptor labelinga
|
|
Double-labeling experiments. Serial frozen brain sections
were prepared as above (n = 5 rats) and coincubated for
16 hr at RT in a mixture of 1:2000 rabbit anti-sst2A antibody and 1:100 rat anti-SRIF antibody containing 0.3% Triton X-100. Sections were
rinsed in 0.1 M TBS and sequentially incubated for 45 min in biotinylated goat-anti-rabbit IgG diluted 1:200 in 0.1 M TBS and in ABC solution for 45 min. They were then
incubated as above for 10 min in the biotinyl-tyramide solution and
reincubated in ABC. After several washes, sections were coincubated for
1 hr in a mixed solution of 1:2000 rhodamine (TRITC)-conjugated
streptavidin (Jackson ImmunoResearch) and 1:100 fluorescein
(FITC)-conjugated goat anti-rat IgG (Jackson ImmunoResearch). They were
then washed in TBS and mounted with Aquamount. The absence of
cross-reactivity between the secondary antibodies was verified by
omitting one of the primary antibodies during the overnight
incubation.
Double-labeled sections were analyzed by confocal microscopy
using a Leica laser scanning microscope (CLSM) equipped with a Leica
Diaplan inverted microscope, an argon/krypton ion laser (488 nm), and a
VME bus MC 68020/68881 computer system (Leica, Wetzlar, Germany). All
image-generating and -processing operations were performed with the
Leica CLSM software package. Images were acquired simultaneously for
the two fluorophores (FITC and TRITC). For each image, 10 optical
sections separated by 0.24 µm steps were acquired, averaged over 32 scans per frame, and reconstructed. Micrographs were taken from the
image monitor using a Focus ImageCorder (Focus Graphics, Foster City,
CA).
Electron microscopy
For electron microscopic studies, rats were perfused
transaortically with 30 ml of heparin (100 U/ml heparin in 0.9% NaCl), sequentially followed by a mixture of 50 ml of 3.75% acrolein and 2%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, and by 200 ml of 2% paraformaldehyde in the same buffer. The brain was
then removed and post-fixed for 30 min in the paraformaldehyde solution. Coronal sections (40-µm-thick) were cut on a vibratome and
collected in PB. Sections were then processed either for single sst2A
immunogold labeling or for sst2A/SRIF combined
immunogold/immunoperoxidase labeling, according to protocols
established by Chan et al. (1990).
Single-labeling experiments. For single-labeling experiments
(n = 4), tissue sections were incubated in a solution
of 1% sodium borohydride in PB for 30 min to neutralize free aldehyde
groups and rinsed copiously with PB. They were then cryoprotected for 30 min in a solution of 25% sucrose and 3% glycerol in 0.05 M PB, frozen rapidly in isopentane at 70°C, transferred
to liquid nitrogen, and thawed in PB at RT. Sections were incubated for 30 min in TBS containing 3% NGS followed by 16 hr at 4°C in rabbit sst2A antibody diluted 1:350 in TBS containing 0.5% NGS. They were
then rinsed in 0.01 M PBS (0.01 M PB, pH 7.4, containing 0.9% NaCl), incubated for 2 hr in a 1:50 dilution of
colloidal gold (1 nm)-conjugated goat anti-rabbit IgG (Amersham,
Arlington Heights, IL) diluted in PBS containing 0.2% gelatin and
0.8% BSA, and fixed for 10 min in 2% glutaraldehyde in PBS. After
several washes in 0.2 M citrate buffer, pH 7.4, immunogold
was silver-enhanced by incubation for 7 min in the silver solution of
IntenSE M kit (Amersham). The reaction was stopped by washes in citrate
buffer, and sections were prepared for electron microscopy as described below.
Double-labeling experiments. For double-labeling experiments
(n = 4), vibratome-cut sections were incubated for 16 hr at 4°C in a mixture of rabbit sst2A antibody (diluted 1:350) and
rat SRIF antibody (diluted 1:25) in TBS containing 0.5% NGS. SRIF immunoreactivity was first revealed using the immunoperoxidase method.
Briefly, sections were incubated sequentially for 30 min in
biotinylated goat anti-rat IgG diluted 1:100 in 0.1 M TBS
and for 30 min in ABC solution. Visualization of the bound peroxidase was achieved by reaction in a solution of 0.1 M Tris buffer
containing 0.05% DAB and 0.01% H2O2. After
several washes in PBS, sst2A immunolabeling was visualized using the
immunogold method as described above. The absence of cross-reactivity
between the secondary antibodies was verified by omitting one of the
primary antibodies during the overnight incubation.
Electron microscopy. Both single- and double-labeled
sections were post-fixed with 2% osmium tetroxide in 0.1 M
PB for 40 min, dehydrated in graded ethanols and propylene oxide, and
flat-embedded in Epon 812 between two sheets of acetate. Ultrathin
sections (80 nm) were collected from the bed nucleus of the stria
terminalis, central and basolateral amygdaloid nucleus, and claustrum.
The ultrathin sections were then counterstained with lead citrate and
uranyl acetate and examined with a JEOL 100CX electron microscope.
Quantitative analyses. The subcellular distribution of
silver-enhanced gold particles was analyzed in sections from two
different animals. For each experiment, three blocks were cut and grids containing surface tissue sections were systematically scanned with the
electron microscope. We assumed that each silver-enhanced gold particle
that was detected corresponded to a specific labeling site, because
control experiments performed by omitting the primary antibody showed
only exceedingly low background levels. Each labeled element
(classified as such if it contained one silver grain or more) was
photographed at an original magnification of 10,000-14,000× to reach
a total number of 400 grains per experiment. Gold particles were then
classified according to the type of element with which they were
associated (dendritic, somatic, or axonal) and whether they were
associated with the cytoplasm or the plasma membrane. A gold particle
was considered to be associated with the plasma membrane when it
contacted or overlaid it. Particles that did not contact the plasma
membrane, even if close, were classified as intracellular.
Cross-sectional surfaces of immunoreactive elements were also measured
using computer-assisted morphometry to determine whether the proportion
of intracellular versus membrane-associated particles was dependent on
sampling.
To assess the proportion of SRIF-immunoreactive terminals in contact
with sst2A elements, immunoperoxidase-labeled axonal profiles
encountered in double-labeled sections from the bed nucleus of the
stria terminalis (n = 168) and the central nucleus of
the amygdala (n = 69) from two animals were analyzed
according to size, presence or not of a contact with a
sst2A-immunolabeled element, and length of contact and presence of a
synaptic specialization and immunogold particles at the site of
contact. All measurements (mean terminal diameter; length of
appositions) were performed using a computer-assisted image analysis
system (Historag; Biocom, Les Ulis, France).
| |
RESULTS |
Comparative distribution of sst2A receptors and SRIF axons at the
light microscopic level
Light microscopic examination of sections processed for sst2A
receptor immunohistochemistry revealed the same regional distributional pattern of immunoreactivity as reported previously (Dournaud et al.,
1996; Schindler et al., 1997). This immunolabeling was no longer
observed when sections were incubated with immune serum preadsorbed
with an excess of antigenic peptide. The distribution of SRIF
immunoreactivity, as observed in sections adjacent to the ones
processed for the sst2A receptor, was also similar to that published
earlier (Johannson et al., 1984). Here again, the labeling was
completely abolished when the antiserum was preincubated with 10 µM SRIF-14.
At higher magnification of sst2A-immunolabeled sections, the
immunostaining was selectively associated with neuronal perikarya and
proximal dendrites in some regions (Table 1) but diffusely distributed
throughout tissue in others (Table 2). By contrast, in sections
immunostained for SRIF, the immunostaining was either present in
perikarya and dendrites (with a greater staining of distal dendrites
than observed with the sst2A antibody) or in small beaded fibers and
punctate axon terminals.
SRIF innervation of regions expressing somatodendritic
sst2A receptors
The highest densities of neuronal cell bodies and proximal
dendrites expressing sst2A receptors were found in the bed nucleus of
the stria terminalis, the olfactory tubercle, the central amygdaloid nucleus, the pyramidal cell layer of the CA1-CA2 fields of the hippocampus, and the nucleus accumbens (Table 1). Four of these five
regions also exhibited among the highest densities of
SRIF-immunostained terminals in the brain (Table 1). Furthermore,
within each of these regions, there was a striking overlap between the
distribution of SRIF terminal fields and that of sst2A-positive
elements. Thus, in the bed nucleus of the stria terminalis, both
SRIF-immunoreactive axon terminals and sst2A-immunoreactive neurons
were restricted to the lateral part of the nucleus (Fig.
1a,a). In the olfactory tubercle, sst2A-immunoreactive nerve cell bodies were densely distributed throughout layer II and extended into the anterior part of
the medial forebrain bundle. Similarly, SRIF-immunoreactive axon
terminals were most prominent within layer II and in the medial
forebrain bundle area (Fig. 1b,b). Furthermore, sst2A receptor-expressing neurons and SRIF-immunoreactive fibers were both
more numerous in the medial than in the lateral aspect of the tubercle.
In the central nucleus of the amygdala, high numbers of
sst2A-immunoreactive neurons were matched with high densities of SRIF
axon terminals in both lateral and medial aspects of the nucleus (Fig.
2a,b). By contrast, the
neighboring medial and cortical amygdaloid nuclei expressed only medium
densities of both sst2A-immunoreactive perikarya and SRIF fibers (Table
1). The hippocampal formation was the only structure of the limbic
system in which there was a high density of sst2A-expressing neurons in
the face of a sparse SRIF innervation. Although pyramidal cells and
their basal and apical dendrites were strongly immunoreactive for the
sst2A receptor throughout the CA1-CA2 fields, only a few
SRIF-immunoreactive fibers were visible at that level, meandering
between pyramidal cells and in the outer part of the stratum
oriens.
View larger version (142K):
[in this window]
[in a new window]
|
Figure 1.
Distribution of sst2A receptor (a,
b) and SRIF immunoreactivity (a, b) in the bed
nucleus of the stria terminalis (BST; a, a) and the
olfactory tubercle (b, b). In both regions, strong sst2A immunoreactivity is apparent in nerve cell bodies and proximal dendrites. Note the close correspondence between patterns of SRIF terminal labeling and sst2A immunoreactivity in the mediolateral subdivision of the BST (a, a) and pyramidal cell layer
(Py) of the olfactory tubercle (b, b).
ic, Internal capsule; ac, anterior commissure; Po, polymorphic cell layer;
Pl, plexiform layer. a, a, 125×
magnification; b, b, 250× magnification.
|
|
View larger version (151K):
[in this window]
[in a new window]
|
Figure 2.
Comparative distribution of SRIF immunoreactivity
(a) and sst2A immunoreactivity
(b) in the central (Cen) and
basolateral (Bla) nuclei of the amygdala. A profuse SRIF
innervation (a) as well as a high density of
sst2A-expressing neurons are evident in the Cen. By
contrast, only sparse SRIF fibers (a) and diffuse sst2A labeling (b) are visible in the
Bla. Magnification, 80×.
|
|
Medium densities of sst2A-immunoreactive neurons and dendrites
were encountered in layer II of the cerebral cortex, the medial habenula, the caudal caudoputamen, the medial and cortical amygdaloid nucleus, the locus coeruleus, the spinal trigeminal tract, and the
lateral reticular nucleus. Except for the medial habenula (Fig.
3a,b) and the locus coeruleus,
in which the SRIF innervation was very sparse, all of these regions
also showed medium densities of immunoreactive SRIF axon terminals
(Table 1). Within each individual region, there was again a close
topographic overlap between the two markers. This was best exemplified
in the parietal cortex and rostral neostriatum. In the former, both
SRIF-immunoreactive terminals and sst2A-immunoreactive nerve cell
bodies and processes were more prominent in layers I and II than in
deeper layers (Fig. 4a,a). In
the latter, SRIF-immunoreactive axons and sst2A-immunoreactive aspiny
neurons were both confined, rostrally, to the ventromedial part of the
neostriatum between the myelinated fascicles of the internal capsule
and along the edge of the globus pallidus (Fig. 4b,b). More
caudally, they were found throughout the entire nucleus, again
according to the same pattern of distribution and relative density.
View larger version (124K):
[in this window]
[in a new window]
|
Figure 3.
Comparative distributions of SRIF immunoreactivity
(a) and sst2A immunoreactivity
(b) in the habenula and paraventricular nucleus
of the thalamus. In the lateral habenula, a dense network of
SRIF-immunoreactive axons is evident in both medial
(lhm) and lateral (lhl) aspects of
the nucleus (a) in the absence of any obvious
sst2A immunoreactivity (b). By contrast, in the
medial habenula, high densities of sst2A-immunoreactive processes
(b) are detected in the face of a sparse SRIF
innervation. The paraventricular thalamic nucleus
(pv) displays low densities of either marker. Magnification, 80×.
|
|
View larger version (124K):
[in this window]
[in a new window]
|
Figure 4.
Distribution of sst2A receptor (a,
b) and SRIF immunoreactivity (a, b) in the
frontal cortex (a, a) and the neostriatum (b,
b). a, a, Dense sst2A perikaryal labeling is
evident throughout layers II-III of the frontal cortex
(a), whereas SRIF-immunoreactive positive cells
are more sparse and predominate in layers IV and V (a).
Note the overlap between SRIF-immunoreactive processes and
sst2A-immunoreactive nerve cell bodies in layers II-III. b, b, sst2A-immunoreactive neurons (b) are
detected among SRIF-immunoreactive perikarya and axons
(b) between the myelinated fascicles of the internal
capsule (IC) in the ventrolateral neostriatum. a,
a, 250× magnification; b, b, 320×
magnification.
|
|
Only low densities of sst2A-expressing neurons were detected in the
diagonal band of Broca, the hypothalamus, the superior colliculus, and
the nucleus tractus solitarius. Accordingly, only sparse
SRIF-immunoreactive fibers were detected in these regions, with the
exception of the hypothalamus, in which extensive somatostatin-fiber networks were apparent.
Overall, there was a highly significant correlation between the
distribution of somatodendritic sst2A receptors and SRIF axon terminals
(p < 0.02). By contrast, there was no
significant correlation (p < 0.3) between the
density of sst2A receptor-positive neurons/dendrites and that of SRIF
binding sites documented previously by quantitative autoradiography
using sst2-preferring ligands (Table 1).
SRIF innervation of regions displaying diffuse
sst2A immunolabeling
By contrast with the high density of SRIF innervation found in
regions expressing sst2A-immunoreactive perikarya and dendrites, only
moderate to low densities of SRIF fibers and varicosities were seen in
regions displaying diffuse sst2A labeling (Table 2). Thus, the
endopiriform nucleus, the claustrum, the stratum lacunosum moleculare
of the CA1 and CA2 fields of the hippocampus, the subiculum, the
basolateral amygdaloid nucleus (Fig. 2a,b), and layers V-VI
of cerebral cortex all displayed high densities of diffuse sst2A
labeling but only few SRIF-positive fibers. Furthermore, there was no
obvious relationship between the patterning of SRIF- and
sst2A-immunoreactive structures in any of these areas.
The SRIF innervation was equally sparse in regions exhibiting only
moderate intensity of sst2A diffuse labeling, such as the anterior
olfactory nucleus, the central gray, the lateral olfactory tract
nucleus, the dentate gyrus, the substantia innominata, and the nucleus
tractus solitarius (Table 2).
In the hypothalamus, the paraventricular thalamus, the zona incerta,
the substantia nigra pars compacta, and the hilus of the hippocampus,
sst2A labeling was very low. Except for the hypothalamus, these regions
also exhibited very low SRIF innervation, as illustrated in Figure 3
for the paraventricular nucleus of the thalamus. Overall, no
significant relationship was found between diffuse sst2A receptor labeling and the density of SRIF-immunoreactive fibers/axon terminals (p = 0.69). By contrast, there was a highly
significant correlation (p < 0.0001) between
the density of sst2A immunolabeling and that of SRIF binding sites
documented previously by quantitative autoradiography using
sst2-preferring ligands (Table 2).
Colocalization of somatodendritic sst2A receptors and SRIF axon
terminals at the confocal microscopic level
To further investigate the relationships between SRIF axons and
sst2A-expressing elements in areas in which the two showed a high
degree of correspondence (i.e., areas of somatodendritic sst2A receptor
localization), the two antigens were immunolabeled in the same sections
and examined by confocal microscopy.
In these double-stained preparations, both TRITC-labeled sst2A receptor
and FITC-labeled SRIF immunostaining showed distributional patterns and
relative densities comparable to those found with the peroxidase
procedure in adjacent sections. When R2-88 or mAb 354 antibodies were
omitted during the double-labeling procedure, respective TRITC- or
FITC-fluorescence labeling was no longer observed in any of the
sections examined, providing evidence that no cross-species reaction of
secondary antisera had occurred.
Typically, somatodendritic sst2A labeling pervaded the cytoplasm of the
perikarya, sparing the nucleus, and was confined mainly to proximal
portions of primary dendrites (Fig.
5a,b,d,f). In some
regions, however, the labeling was also evident within extensive dendritic arbors such as in the spinal trigeminal tract and lateral reticular nucleus (Fig. 5c,e). There was no preferential
accumulation of sst2A immunoreactivity at the outskirts of the cells to
suggest a preferential association with the plasma membrane.
View larger version (109K):
[in this window]
[in a new window]
|
Figure 5.
Confocal microscopic images of double sst2A
receptor (red) and SRIF axonal
(green) immunolabeling in the olfactory tubercle (a), the central amygdaloid nucleus
(b), the spinal trigeminal tract
(c), the substriatal area
(d), the lateral reticular nucleus (e), and the ventrolateral neostriatum
(f). In a-e, note the
overlap between SRIF-immunoreactive axons and sst2A labeled perikarya and dendrites. In several instances, SRIF-immunoreactive varicosities are seen in close apposition with sst2A-stained elements (a-c, e; arrows). In the neostriatum
(f), a SRIF-immunoreactive soma is seen to
extend immunoreactive processes toward several sst2A-immunoreactive neurons. a, c-f, 600× magnification; b,
2000× magnification.
|
|
In all of these regions, immunoreactive SRIF fibers overlapped heavily
with sst2A receptor-expressing neurons (Fig. 5). Even in regions
displaying moderate densities of both sst2A receptor-expressing neurons
and SRIF innervation, extensive intermesh was evident between the two
markers (Fig. 5f). In most instances,
sst2A-immunoreactive perikarya and dendrites were embedded in a field
of SRIF-immunoreactive axons. In many cases, however, individual SRIF
varicose fibers or axon terminals were seen to appose
sst2A-immunoreactive elements (Fig. 5a,b,c,e).
In several brain areas, SRIF nerve cell bodies were also apparent in
regions expressing sst2A receptor immunoreactivity. There was no
obvious correlation between the number and distribution of SRIF- and
sst2A-immunoreactive cells. With conventional epifluorescence and
confocal microscopy, however, it was apparent that in the central
amygdaloid nucleus (Fig.
6a,a), the bed nucleus of the stria terminalis (Fig. 6b,b), the premammillary nucleus,
and the lateral reticular nucleus (Fig. 6c,c), a
subpopulation of cells immunoreactive for sst2A receptor was also
immunopositive for SRIF. In other structures, even when both sst2A- and
SRIF-expressing neurons were present, such as in cerebral cortex,
neostriatum, or hypothalamus, no cellular colocalization of the two
antigens was apparent. Because this study was conducted in
noncolchicine-treated animals to optimize visualization of SRIF
terminal fields, the number of SRIF-immunoreactive neurons, and thereby
the occurrence frequency of double-labeled cells, is likely to have
been underestimated. Thus we did not attempt to quantify neurons
expressing both SRIF and sst2A receptors in any of the above brain
regions.
View larger version (106K):
[in this window]
[in a new window]
|
Figure 6.
Confocal microscopic imaging of sst2A
(a-c) and SRIF (a-c) double-labeled
cells (arrows) in the central amygdaloid nucleus (a, a), bed nucleus of the stria terminalis (b,
b), and lateral reticular nucleus (c, c). Note
that in all of these areas, SRIF immunoreactivity is detected only in a
subpopulation of sst2A-expressing neurons. a, a, c, c,
650× magnification; b, b, 1200× magnification.
|
|
Electron microscopy
The ultrastructural distribution of sst2A receptors was examined
using preembedding immunogold cytochemistry in regions representative of those displaying prominent somatodendritic (bed nucleus of the stria
terminalis and central nucleus of the amygdala) or merely diffuse
(claustrum) labeling at the light microscopic level. In all of these
regions, sst2A immunoreactivity was found to be associated exclusively
with neurons. Neither glial nor endothelial vascular cells showed any
significant immunogold labeling. Within neurons, 65% of the total
number of gold particles was detected over dendrites. The remaining
35% was divided between soma, unmyelinated axons, and axon terminals,
with variable enrichment according to the region sampled. Thus, in the
bed nucleus of the stria terminalis and central nucleus of the
amygdala, 26% of gold particles was associated with neuronal somata
and 9% with unmyelinated axons and axon terminals. Conversely, in the
claustrum, 11% of the immunolabeling was associated with somata and
24% with axons and axon terminals. However, the main difference
between regions exhibiting somatodendritic (bed nucleus of the stria
terminalis and central nucleus of the amygdala) as compared with
diffuse (claustrum) labeling at the light microscopic level concerned
the proportion of intracellular versus plasma membrane-associated sst2A
receptors. In the former regions, only a small proportion of gold
particles (22%) was associated with the plasma membrane of nerve cell
bodies, dendrites, or axons/axon terminals, the remainder being
intracellular (Fig. 7a). By
contrast, in the claustrum, 70% of labeled sst2A receptors was
associated with the plasma membrane, and only 30% was intracellular
(Fig. 7b,c). These differences could not be attributed to
sampling, because the mean cross-sectional surface of labeled elements
was comparable for all three areas.
View larger version (171K):
[in this window]
[in a new window]
|
Figure 7.
Electron microscopic localization of sst2A
receptors using silver-enhanced immunogold in the central nucleus of
the amygdala (a) and claustrum (b,
c). a, A high proportion of gold particles is
found inside dendrites, often in association with irregularly shaped
vesicles (arrowheads). A few gold particles are also
associated with the plasma membrane (arrows). b,
c, Immunolabeled sst2A receptors are distributed mainly along
dendritic plasma membranes (arrows). a,
b, 28,000× magnification; c, 20,000×
magnification.
|
|
Dual electron microscopic localization of sst2A receptors and SRIF was
performed in two brain areas shown to exhibit significant overlap
between immunoreactive SRIF fibers and sst2A receptor-expressing neurons at the light microscopic level, namely the bed nucleus of the
stria terminalis and the central amygdaloid nucleus. In both of these
regions, the peroxidase reaction product, indicative of SRIF
immunoreactivity, was found almost exclusively in axon terminals. Only
sparse immunoreactive dendrites were observed. SRIF-immunoreactive axon
terminals measured 0.97 ± 0.06 µm in mean diameter and were
filled with numerous clear synaptic vesicles. In addition, 50% of them
also exhibited large dense core vesicles (one to three per terminal
profile). Direct appositions between SRIF-immunoreactive terminals and
sst2A-labeled somatodendritic elements were apparent on 23% (39 of
168) and 10% (8 of 69) of labeled terminal profiles sampled in the bed
nucleus of the stria terminalis and central amygdaloid nucleus,
respectively (Fig. 8). Contacted elements
were predominantly large dendrites and, less frequently, dendritic
branchlets and perikarya (Fig. 8). The proportion of
membrane-associated versus intracellular sst2A receptors within these
dendrites was similar to that observed in dendrites lacking any
apparent contact with SRIF elements. Membrane-associated sst2A
receptors expressed in dendrites apposed to SRIF-containing terminals
were distributed on the entire surface of the dendrites and not
specifically opposite the abutting SRIF terminal. Nonetheless, 29% of
appositions between the two types of immunolabeled elements showed a
gold particle at the site of apposition. Of the SRIF terminals
contacting sst2A somatodendritic elements, only 20% exhibited a
synaptic specialization in the plane of section. These synapses were
mainly symmetrical (Fig. 8c), but a few showed a high
postsynaptic differentiation typical of asymmetric synapses (Fig.
8a). Occasionally, SRIF-immunoreactive terminals apposed or
not to sst2A-positive dendrites established synaptic junctions with
nonlabeled neuronal elements.
View larger version (183K):
[in this window]
[in a new window]
|
Figure 8.
Combined visualization of sst2A receptors
and SRIF-containing axons in the central nucleus of the amygdala using
immunogold and immunoperoxidase labeling, respectively.
a, sst2A-immunoreactive dendritic trunk contacted by two
SRIF-immunoreactive terminals (T1, T2).
T1 is more strongly immunoreactive than
T2 and shows an asymmetric specialization at the site of
contact (small arrow). Note that most of the gold
particles are intracellular and that many of them are associated with
vesicular elements. Also note the presence of coated "endocytic"
vesicles on the postsynaptic side, close to the sites of contact
(large arrows). b, sst2A-immunopositive cell body in contact with a SRIF-immunoreactive terminal. Here again,
most of the gold particles are intracellular. Some of them are
associated with organelles implicated in protein synthesis and
maturation such as the Golgi apparatus (G) and
endoplasmic reticulum (ER). Two of the gold particles
are seen in the vicinity of the abutting SRIF terminal.
c, An intensely labeled SRIF terminal containing several
dense core vesicles (dcv) is seen in symmetric synaptic
contact with a large sst2A-immunoreactive dendrite displaying prominent
intracellular labeling. Again, note that only a few gold particles are
associated with the plasma membrane. a, 28,000× magnification; b, c, 17,000× magnification.
|
|
| |
DISCUSSION |
The present study provides a comprehensive description of the
regional and cellular relationships between SRIF axons and SRIF receptor-expressing neurons in the CNS. Although the SRIF receptor subtype visualized here, sst2A, represents only one of the six SRIF
receptors (including the sst2B receptor variant) cloned to date (Bruno
et al., 1992; O'Carroll et al., 1992; Vanetti et al., 1992; Yamada et
al., 1992; Yasuda et al., 1992), pharmacological (Reisine and Bell,
1995), and neuroanatomical (Martin et al., 1991; Pérez et al.,
1994; Senaris et al., 1994; Beaudet et al., 1995; Schoeffter et al.,
1995; Holloway et al., 1996) data suggest that it accounts for an
important facet of somatostatinergic neurotransmission in the
brain.
The immunohistochemical methods used in the present investigation
relied on the use of well characterized SRIF and sst2A receptor antibodies (Dun et al., 1994; Dournaud et al., 1996; Gu and Schonbrunn, 1997). Further support for the specificity of SRIF and sst2A
immunolabeling patterns observed here stemmed from their similarity
with those obtained in previous studies using either the same (Dun et
al., 1994; Dournaud et al., 1996) or different (Johannson et al., 1984; Schindler et al., 1997) antibodies.
As reported previously (Dournaud et al., 1996), two types of sst2A
immunostaining patterns were observed by light microscopy in serial
sections from the rat brain. In some regions, such as the bed nucleus
of the stria terminalis, the olfactory tubercle, the central amygdaloid
nucleus, or the nucleus accumbens, the immunostaining was clearly
associated with neuronal perikarya and dendrites. In others, such as in
the endopiriform nucleus, the claustrum, the basal lateral amygdaloid
nucleus, or layers V-VI of the cerebral cortex, the immunostaining
appeared diffusely distributed in tissue. Electron microscopic
immunogold cytochemistry gave the key to these discrepant light
microscopic labeling patterns. Indeed, in regions of somatodendritic
sst2A labeling, most immunoreactive receptors were intracellular and
only a small proportion were associated with plasma membranes, whereas
in regions of diffuse sst2A labeling, the vast majority of
immunoreactive receptors was associated with neuronal plasma membranes.
A second major finding of the present study was that the density of
SRIF-immunoreactive axon terminals varied according to the above
compartmentalization such that regions in which sst2A receptors were
predominantly intracellular contained high densities of SRIF axon
terminals, whereas regions in which sst2A receptors were predominantly
membrane-bound received only a sparse SRIF innervation.
The fact that regions in which sst2A receptors were mainly
intracellular contained a dense SRIF innervation suggests that endogenously released SRIF may chronically downregulate
membrane-associated receptors. In light of recent studies in which
pharmacological stimulation of neuropeptide receptors was shown to
result in their translocation from the cell surface to the cytoplasm
(Liu et al., 1997), it is tempting to speculate that this
downregulation is a consequence of ligand-induced receptor
internalization. Such a mechanism has been described for various
neuropeptide receptors in the CNS, including opioid (Sternini et al.,
1996), substance P (Mantyh et al., 1995a,b), and neurotensin (Faure et
al., 1995) receptors. The sst2A receptor has been shown to efficiently
internalize bound ligand when expressed in COS-7 cells (Nouel et al.,
1997), human embryonic kidney (HEK) cells (Roth et al., 1997),
GH4-R2.20 pituitary tumor cells (Hipkin et al., 1997), and
to a much lesser extent, Chinese hamster ovary cells (Hukovic et al.,
1996). Furthermore, bound SRIF receptor ligands recently have been
demonstrated to be internalized in neurons in primary cultures (Stroh
et al., 1997) as well as in Neuro2A neuroblastoma cells (Koening et
al., 1997), both of which express the sst2 receptor subtype. These studies indicate that internalization of the somatostatin-sst2A receptor complex is dependent on receptor occupancy and could account
for a paucity of cell surface receptors in regions where there is
substantial endogenous SRIF release.
In turn, the fact that regions in which sst2A receptors were mainly
associated with the plasma membranes received only a sparse SRIF
innervation implies that in these regions the endogenous ligand must
diffuse over some distance in the extracellular space to reach its
receptor targets. Such a mode of action has been postulated previously
for a number of neuropeptides in the CNS (for review, see Herkenham,
1987) and was documented in vivo for substance P (Mantyh et
al., 1995a,b). The present results suggest further that within these
regions the concentration of endogenous SRIF that reaches its receptor
targets may not be sufficient to induce a cell surface receptor
downregulation as extensive as in regions of dense SRIF
innervation.
In regions of membrane-associated sst2A immunolabeling, the
distribution of sst2A-immunoreactive receptors was highly correlated with that previously reported for SRIF binding sites
autoradiographically labeled with sst2-preferring ligands. By contrast,
the two distributions did not correlate in regions of predominantly
intracellular somatodendritic labeling. Admittedly, these correlations
may be confounded somewhat by the fact that all current sst2-preferring
ligands also recognize, to some extent, sst3 and sst5 subtypes (Raynor
et al., 1993; Epelbaum et al., 1994). However, it would appear from
recent literature reports that the recombinant sst2 receptor is the one
that is preferentially labeled by autoradiography when using
sst2-preferring ligands (Schoeffter et al., 1995; Holloway et al.,
1996). The most likely interpretation for these correlative data is
that sst2-preferring ligands bind mainly to membrane-associated, as opposed to intracellular, receptors. Why intracellular sst2A receptors, in contrast to other types of neuropeptide receptors such as
neurotensin (Kessler et al., 1987, Dana et al., 1989; Szigethy et al.,
1990) or  opioid (Pasquini et al., 1992) receptors, should poorly
recognize their exogenous ligand is unclear but may be related to the
conformation of neosynthesized receptors or to chemical alteration
(e.g., phosphorylation) (Hipkin et al., 1997) of internalized ones. It
could also be caused by their inclusion in small membrane-bound
compartments (such as endosomes) where they might be unaccessible to,
or already occupied by, the ligand. In any event, the fact that in
regions of "somatodendritic" light microscopic sst2A labeling
immunoreactive receptors were found by electron microscopy to be
predominantly intracellular provides an explanation for the
"mismatch" between 125I-SRIF binding and SRIF
immunoreactivity reported previously in these areas (Herkenham,
1987).
Not all brain regions showed a correlation between densities of SRIF
terminals and somatodendritic sst2A receptor labeling. Thus, in the
hypothalamus, a dense SRIF innervation was detected in the absence of
any substantial somatodendritic or diffuse sst2A receptor
immunoreactivity. Because this region is also largely deprived of sst2
binding activity (Reubi and Maurer, 1985; Krantic et al., 1989, 1990;
Martin et al., 1991; Schoeffter et al., 1995; Holloway et al., 1996),
the most likely interpretation for the observed mismatch is the
presence of one or several additional SRIF receptor subtypes, a
proposal supported by in situ hybridization data (Breder et
al., 1992; Senaris et al., 1994; Beaudet et al., 1995; Pérez and
Hoyer, 1995). Other regions, such as the medial habenula, locus
coeruleus, and pyramidal cell layer of the CA1-CA2 hippocampal fields
showed dense sst2A somatodendritic immunoreactivity but only a sparse
SRIF innervation. Two of these areas, namely the medial habenula and
the locus coeruleus, also showed high sst2 binding activity. It remains
to be confirmed by electron microscopy whether a high incidence of
membrane-associated receptors exists in these regions in contrast to
other areas of sst2A somatodendritic labeling. These findings would
show whether factors other than ligand exposure could also be involved
in the regulation of cell surface receptor density.
Confocal microscopic examination of doubled-labeled material revealed
that SRIF and sst2A receptors were colocalized within the same neurons
in several brain regions, including the bed nucleus of the stria
terminalis, the central amygdaloid nucleus, and the premammillary
nucleus. These observations suggest that SRIF may be in a position to
regulate the activity of SRIF neurons, and presumably its own release,
in several brain structures. Because our experiments were conducted in
noncolchicine-treated rats (to optimize visualization of axon
terminals), it is likely that colocalization of sst2A receptors and
SRIF is more pervasive than our results and than earlier functional
studies (Peterfreund and Vale, 1984; Epelbaum et al., 1986; Richardson
and Twente, 1986) had led us to believe.
Electron microscopy demonstrated the existence of direct appositions
between SRIF-immunoreactive axons and sst2A-immunoreactive neurons in
the bed nucleus of the stria terminalis and the central nucleus of the
amygdala. Although the incidence with which SRIF terminal profiles were
seen in contact with sst2A-immunoreactive elements in single thin
sections was relatively low, a simple stereological calculation (based
on the assumption that these terminals are spherical and taking into
account their diameter and length of contact with the sst2A element)
(for mathematical formula, see Beaudet and Sotelo, 1981) reveals that
in the bed nucleus of the stria terminalis up to 37% of the
SRIF-immunoreactive terminals are actually in contact with
sst2A-immunoreactive perikarya and dendrites. Furthermore, this figure
is likely to be even higher if, as likely, all sst2A-positive dendrites
do not exhibit silver-gold particles in the plane of sections in which
they are contacted by SRIF terminals. The present data therefore
indicate that in regions of dense SRIF innervation-low cell surface
sst2A receptor expression, there is an extensive investment of sst2A
receptor-expressing elements by SRIF axon terminals. Surprisingly,
however, only 20% of these terminals showed a synaptic specialization
at the site of contact. Furthermore, labeled receptors were observed
only rarely at these sites of contact, whether or not a synaptic
specialization was present. These observations suggest that SRIF may
not be the "synaptic" transmitter released by these terminals, but
a cotransmitter acting more broadly on receptors located on
extrasynaptic portions of the membrane. Nonetheless, the present
results suggest that direct contact between SRIF-containing axons and
receptive neurons may be critical for a fast, efficient action of SRIF
on sst2A receptors. Whether SRIF released from terminals located at a
distance from sst2A-expressing elements acts on the same receptors,
through diffusion in the extracellular space, or on other types of SRIF receptors present on sst2A-immunonegative elements remains to be
determined.
In summary, the major conclusions drawn from our observations are the
following. (1) Regions in which sst2A receptors are mainly
intracellular receive a dense SRIF innervation, suggesting that
endogenously released SRIF chronically downregulates
membrane-associated receptors; (2) regions in which sst2A receptors are
predominantly membrane-associated receive only a sparse SRIF
innervation, implying that the exogenous ligand must diffuse over some
distance in the extracellular space to reach its targets; and (3)
regions in which sst2A-immunoreactive receptors are associated
predominantly with plasma membranes correspond to regions shown
previously by quantitative autoradiography to be most enriched in sst2
binding sites, implying (1) that intracellular sst2A receptors are
ill-recognized by exogenous SRIF and (2) that these regions are
preferential targets for exogenously administered SRIF or its
derivatives.
| |
FOOTNOTES |
Received Aug. 8, 1997; revised Oct. 3, 1997; accepted Nov. 14, 1997.
This work was supported by grants from the Fonds de la Recherche en
Santé du Québec and the Medical Research Council of Canada
to G.S.T. and A.B., and from the National Institutes of Health to
A.S. G.S.T. is the recipient of a "Chercheur de
carrière" award from the Fonds de la Recherche en Santé
du Québec. We thank Mariette Houle and Christian Charbonneau for
expert technical assistance. We are also grateful to Dr. V. M. Pickel for her tutoring of the immunogold ultrastructural localization
technique.
P.D. and H.B. contributed equally to this work.
Correspondence should be addressed to Dr. Alain Beaudet, Montreal
Neurological Institute, McGill University, 3801 University Street,
Montreal, Quebec H3A 2B4, Canada.
Dr. Dournaud's present address: Institut National de la Santé et
de la Recherche Médicale, U-159, Centre Paul Broca, 2 Ter rue
d'Alésia, 75014 Paris, France.
| |
REFERENCES |
-
Beaudet A,
Sotelo C
(1981)
Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum.
Brain Res
206:305-329[Web of Science][Medline].
-
Beaudet A,
Greenspun D,
Raelson J,
Tannenbaum GS
(1995)
Patterns of expression of SSTR1 and SSTR2 somatostatin receptor subtypes in the hypothalamus of the adult rat: relationship to neuroendocrine function.
Neuroscience
65:551-561[Web of Science][Medline].
-
Brazeau P,
Vale W,
Burgus R,
Ling N,
Butcher M,
Rivier J,
Guillemin R
(1973)
Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone.
Science
179:77-78[Abstract/Free Full Text].
-
Breder CD,
Yamada Y,
Yasuda K,
Seino K,
Saper CB,
Bell GI
(1992)
Differential expression of somatostatin receptor subtypes in brain.
J Neurosci
12:3920-3934[Abstract].
-
Bruno JF,
Xu Y,
Song J,
Berelowitz M
(1992)
Molecular cloning and functional expression of a brain-specific somatostatin receptor.
Proc Natl Acad Sci USA
89:11151-11155[Abstract/Free Full Text].
-
Chan J,
Aoki C,
Pickel VM
(1990)
Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding.
J Neurosci Methods
33:113-127[Web of Science][Medline].
-
Dana C,
Vial M,
Leonard K,
Beauregard A,
Kitabgi P,
Vincent JP,
Rostène W,
Beaudet A
(1989)
Electron microscopic localization of neurotensin binding sites in the midbrain tegmentum of the rat.
J Neurosci
9:2247-2257[Abstract].
-
De Lecea L,
Criado JR,
Prospero-Garcia O,
Gautvik KM,
Schweitzer P,
Danielson PE,
Dunlop CLM,
Siggins GR,
Henriksen SJ,
Sutcliffe JG
(1996)
A cortical neuropeptide with neuronal depressant and sleep-modulating properties.
Nature
381:242-245[Medline].
-
Dournaud P,
Gu YZ,
Schonbrunn A,
Mazella J,
Tannenbaum GS,
Beaudet A
(1996)
Localization of the somatostatin receptor sst2A in rat brain using a specific anti-peptide antibody.
J Neurosci
16:4468-4478[Abstract/Free Full Text].
-
Dun NJ,
Dun SL,
Wong RKS,
Förstremann U
(1994)
Colocalization of nitric oxide synthase and somatostatin immunoreactivity in rat dentate hilar neurons.
Proc Natl Acad Sci USA
91:2955-2959[Abstract/Free Full Text].
-
Epelbaum J,
Tapia-Arancibia L,
Alonso G,
Astier H,
Kordon C
(1986)
The anterior periventricular hypothalamus is the site of somatostatin inhibition on its own release: an in vitro and immunocytochemical study.
Neuroendocrinology
44:255-259[Web of Science][Medline].
-
Epelbaum J,
Dournaud P,
Fodor F,
Viollet C
(1994)
The neurobiology of somatostatin.
Crit Rev Neurobiol
8:25-44[Web of Science][Medline].
-
Faure MP,
Alonso A,
Nouel D,
Gaudriault G,
Dennis M,
Vincent JP,
Beaudet A
(1995)
Somatodendritic internalization and perinuclear targeting of neurotensin in mammalian brain.
J Neurosci
15:4140-4147[Abstract].
-
Fukusumi S,
Kitada C,
Takekawa S,
Kizawa H,
Sakamoto J,
Miyamoto M,
Hinuma S,
Kitano K,
Fujino M
(1997)
Identification and characterization of a novel human cortistatin-like peptide.
Biochem Biophys Res Commun
232:157-163[Web of Science][Medline].
-
Gu WZ,
Schonbrunn A
(1997)
Coupling specificity between somatostatin receptor sst2A and G proteins: isolation of the receptorG protein complex with a receptor antibody.
Mol Endocrinol
11:527-537[Abstract/Free Full Text].
-
Gu WZ,
Brown PJ,
Loose-Mitchell DS,
Stork PJS,
Schonbrunn A
(1995)
Development and use of a receptor antibody to characterize the interaction between somatostatin subtype 1 and G proteins.
Mol Pharmacol
48:1004-1014[Abstract].
-
Herkenham M
(1987)
Mismatches between neurotransmitter and receptor localizations in brain: observations and implications.
Neuroscience
23:1-38[Web of Science][Medline].
-
Hipkin RW,
Friedman J,
Clark RB,
Eppler CM,
Schonbrunn A
(1997)
Agonist-induced desensitization, internalization, and phosphorylation of the sst2A somatostatin receptor.
J Biol Chem
272:13869-13876[Abstract/Free Full Text].
-
Holloway S,
Feniuk W,
Kidd EJ,
Humphrey PPA
(1996)
A quantitative autoradiographical study on the distribution of somatostatin sst2 receptors in the rat central nervous system using 125I-BIM-23027.
Neuropharmacology
35:1109-1120[Web of Science][Medline].
-
Hoyer D,
Bell GI,
Berelowitz M,
Epelbaum J,
Feniuk W,
Humphrey PP,
O'Caroll AM,
Patel YC,
Schonbrunn A,
Taylor JE,
Reisine T
(1995)
Classification and nomenclature of somatostatin receptors.
Trends Pharmacol Sci
16:86-88[Medline].
-
Hukovic N,
Panetta R,
Kumar U,
Patel YC
(1996)
Agonist-dependent regulation of cloned human somatostatin receptor types 1-5 (hSSTR1-5): subtype selective internalization or upregulation.
Endocrinology
137:4046-4049[Abstract].
-
Johannson O,
Hökfelt T,
Elde PR
(1984)
Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat.
Neuroscience
13:265-339[Web of Science][Medline].
-
Kessler JP,
Moyse E,
Kitabgi P,
Vincent JP,
Beaudet A
(1987)
Distribution of neurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study.
Neuroscience
23:189-198[Web of Science][Medline].
-
Koening JA,
Edwardson JM,
Humphrey PPA
(1997)
Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization.
Br J Pharmacol
120:52-59[Web of Science][Medline].
-
Krantic S,
Martel JC,
Weissmann D,
Quirion R
(1989)
Radioautographic analysis of somatostatin receptor sub-type in rat hypothalamus.
Brain Res
498:267-278[Web of Science][Medline].
-
Krantic S,
Martel JC,
Weissmann D,
Pujol JF,
Quirion R
(1990)
Quantitative radioautographic study of somatostatin receptors heterogeneity in the rat extrahypothalamic brain.
Neuroscience
39:127-137[Web of Science][Medline].
-
Krantic S,
Quirion R,
Uhl G
(1992)
Somatostatin receptors.
In: Handbook of chemical neuroanatomy, Vol II, Neuropeptide receptors in the CNS (Björklund A,
Hökfelt T,
Kuhar MJ,
eds), pp 321-346. Amsterdam: Elsevier Science.
-
Liu H,
Mantyh PW,
Basbaum AI
(1997)
NMDA-receptor regulation of substance P release from primary afferent nociceptors.
Nature
368:721-724.
-
Mantyh PW,
Allen CJ,
Ghilardi JR,
Rogers SD,
Mantyh CR,
Liu H,
Basbaum AI,
Vigna SR,
Maggio JE
(1995a)
Rapid endocytosis of a G protein-coupled receptor: substance-P evoked internalization of its receptor in the rat striatum in vivo.
Proc Natl Acad Sci USA
92:2622-2626[Abstract/Free Full Text].
-
Mantyh PW,
De Master E,
Malhorta A,
Ghilardi JR,
Rogers SD,
Mantyh CR,
Liu H,
Basbaum AI,
Vigna SR,
Maggio JE,
Simone DA
(1995b)
Receptor endocytosis and dendrite reshaping in pineal neurons after somatosensory stimulation.
Science
268:1629-1632[Abstract/Free Full Text].
-
Martin JL,
Chesselet MF,
Raynor K,
Gonzales C,
Reisine T
(1991)
Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs.
Neuroscience
41:581-593[Web of Science][Medline].
-
Moyse E,
Beaudet A,
Bertherat J,
Epelbaum J
(1992)
Light microscopic radioautographic localization of somatostatin binding sites in the brainstem of the rat.
J Chem Neuroanat
5:75-84[Web of Science][Medline].
-
Nouel D,
Gaudriault G,
Houle M,
Reisine T,
Vincent JP,
Mazella J,
Beaudet A
(1997)
Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivates.
Endocrinology
138:296-306[Abstract/Free Full Text].
-
O'Carroll AM,
Lolait SJ,
Konig M,
Mahan LC
(1992)
Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28.
Mol Pharmacol
42:939-946[Abstract].
-
Pasquini F,
Bochet P,
Garbay-Jaureguiberry C,
Roques BP,
Rossier J,
Beaudet A
(1992)
Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat.
J Comp Neurol
326:229-244[Web of Science][Medline].
-
Patel YC,
Greenwood MT,
Panetta R,
Demchyshyn L,
Niznik H,
Srikant CB
(1995)
The somatostatin receptor family.
Life Sci
57:1249-1265[Web of Science][Medline].
-
Pérez J,
Hoyer D
(1995)
Co-expression of somatostatin sstr-3 and sstr-4 receptor messenger RNAs in the rat brain.
Neuroscience
64:241-253[Web of Science][Medline].
-
Pérez J,
Rigo M,
Kaupmann K,
Bruns C,
Yasuda K,
Bell GI,
Lubbert H,
Hoyer D
(1994)
Localization of somatostatin (SRIF) SSTR-1, SSTR-2 and SSTR-3 receptor mRNA in rat brain by in situ hybridisation.
Naunyn Schmiedebergs Arch Pharmacol
349:145-160[Web of Science][Medline].
-
Peterfreund RA,
Vale WW
(1984)
Somatostatin analogs inhibit somatostatin secretion from cultured hypothalamus cells.
Neuroendocrinology
39:397-402[Web of Science][Medline].
-
Pradayrol L,
Jornvall H,
Mutt V,
Ribet A
(1980)
N-terminally extended somatostatin 28.
FEBS Lett
109:55-58[Web of Science][Medline].
-
Raynor K,
Murphy WA,
Coy DH,
Taylor JE,
Moreau J-P,
Yasuda K,
Bell GI,
Reisine T
(1993)
Cloned somatostatin receptors: identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides.
Mol Pharmacol
43:838-844[Abstract].
-
Reichlin S
(1983)
Somatostatin.
N Engl J Med
309:1556-1563[Web of Science][Medline].
-
Reisine T,
Bell GI
(1995)
Molecular properties of somatostatin receptors.
Neuroscience
67:777-790[Web of Science][Medline].
-
Reubi JC,
Maurer R
(1985)
Autoradiographic mapping of somatostatin receptors in the rat central nervous system and pituitary.
Neuroscience
15:1183-1193[Web of Science][Medline].
-
Richardson SB,
Twente S
(1986)
Inhibition of rat hypothalamus somatostatin release by somatostatin: evidence for ultrashort loop feedback.
Endocrinology
118:2076-2082[Abstract/Free Full Text].
-
Roth A,
Kreienkamp H-J,
Nehring RB,
Roosterman D,
Meyerhof W,
Richter D
(1997)
Endocytosis of the rat somatostatin receptors: subtype discrimination, ligand specificity, and delineation of carboxy-terminal positive and negative sequence motifs.
DNA Cell Biol
16:111-119[Web of Science][Medline].
-
Sawchenko PE,
Benoit R,
Brown MR
(1988)
Somatostatin 28-immunoreactive neurons inputs to the paraventricular and supraoptic nuclei: principal origin from nonaminergic neurons in the nucleus of the solitary tract.
J Chem Neuroanat
1:81-94[Medline].
-
Schally AV,
Huang WY,
Chang RLL,
Arimura A,
Redding TW,
Millar RP,
Hunkapillar MW,
Hood LE
(1980)
Isolation and structure of pro-somatostatin precursor from pig hypothalamus.
Proc Natl Acad Sci USA
77:4489-4493[Abstract/Free Full Text].
-
Schindler M,
Humphrey PP,
Emson PC
(1996)
Somatostatin receptors in the central nervous system.
Prog Neurobiol
50:9-47[Web of Science][Medline].
-
Schindler M,
Sellers LA,
Humphrey PPA,
Emson PC
(1997)
Immunohistochemical localization of the somatostatin SST2A receptor in rat brain and spinal cord.
Neuroscience
76:225-240[Web of Science][Medline].
-
Schoeffter P,
Pérez J,
Langenegger D,
Schupbach E,
Bobirnac I,
Lubbert H,
Bruns C,
Hoyer D
(1995)
Characterization and distribution of somatostatin SS-1 and SRIF-1 binding sites in rat brain: identity with SSTR-2 receptors.
Eur J Pharmacol
289:163-173[Web of Science][Medline].
-
Schwarzer C,
Sperk G,
Samanin R,
Rizzi M,
Gariboldi M,
Vezzani A
(1996)
Neuropeptides-immunoreactivity and their mRNA expression in kindling: functional implications for limbic epileptogenesis.
Brain Res Brain Res Rev
22:27-50[Medline].
-
Senaris RM,
Humphrey PPA,
Emson PC
(1994)
Distribution of somatostatin receptors 1, 2 and 3 mRNA in rat brain and pituitary.
Eur J Neurosci
6:1883-1896[Web of Science][Medline].
-
Sternini C,
Spann M,
Anton B,
Keith Jr DE,
Bunnett NW,
Von Zastrow M,
Evans C,
Breacha NC
(1996)
Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.
Proc Natl Acad Sci USA
93:9241-9246[Abstract/Free Full Text].
-
Stroh T,
Schonbrunn A,
Vincent JP,
Beaudet A
(1997)
The somatostatin receptor sst2A in primary cortical cultures: expression and internalization.
In: Göttingen neurobiology report, Proceedings of the 25th Göttingen neurobiology conference, Vol II (Elsner N,
Wässle H,
eds), p 791. Stuttgart: Georg Thieme Verlag.
-
Szigethy E,
Leonard K,
Beaudet A
(1990)
Ultrastructural localization of [125I]-neurotensin binding sites to cholinergic neurons of the rat nucleus basalis magnocellularis.
Neuroscience
36:377-391[Web of Science][Medline].
-
Tannenbaum GS
(1985)
Physiological role of somatostatin in regulation of pulsatile growth hormone secretion.
In: Somatostatin (Patel YC,
Tannenbaum GS,
eds), pp 229-259. New York: Plenum.
-
Vanetti M,
Kouba M,
Wang X,
Vogt G,
Hollt V
(1992)
Cloning and expression of a novel mouse somatostatin receptor (SSTR2B).
FEBS Lett
311:290-294[Web of Science][Medline].
-
Vanetti M,
Vogt G,
Hollt V
(1993)
The two isoforms of the mouse somatostatin receptor (mSSTR2A and mSSTR2B) differ in coupling efficiency to adenylate cyclase and in agonist-induced receptor desensitization.
FEBS Lett
331:260-266[Web of Science][Medline].
-
Yamada Y,
Post SR,
Wang K,
Tager HS,
Bell GI,
Seino S
(1992)
Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.
Proc Natl Acad Sci USA
89:251-255[Abstract/Free Full Text].
-
Yasuda K,
Rens-Domiano S,
Breder CD,
Law SF,
Saper CB,
Reisine T,
Bell GI
(1992)
Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylylcyclase.
J Biol Chem
267:20422-20428[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1831056-16$05.00/0
This article has been cited by other articles:
|
|
|
|
 
T. Stroh, M. R. van Schouwenburg, A. Beaudet, and G. S. Tannenbaum
Subcellular Dynamics of Somatostatin Receptor Subtype 1 in the Rat Arcuate Nucleus: Receptor Localization and Synaptic Connectivity Vary in Parallel with the Ultradian Rhythm of Growth Hormone Secretion
J. Neurosci.,
June 24, 2009;
29(25):
8198 - 8205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Lelouvier, G. Tamagno, A. M. Kaindl, A. Roland, V. Lelievre, V. Le Verche, C. Loudes, P. Gressens, A. Faivre-Baumann, Z. Lenkei, et al.
Dynamics of Somatostatin Type 2A Receptor Cargoes in Living Hippocampal Neurons
J. Neurosci.,
April 23, 2008;
28(17):
4336 - 4349.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-H. Bassant, A. Simon, F. Poindessous-Jazat, Z. Csaba, J. Epelbaum, and P. Dournaud
Medial Septal GABAergic Neurons Express the Somatostatin sst2A Receptor: Functional Consequences on Unit Firing and Hippocampal Theta
J. Neurosci.,
February 23, 2005;
25(8):
2032 - 2041.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Vasilaki, T. Papadaki, G. Notas, G. Kolios, N. Mastrodimou, D. Hoyer, M. Tsilimbaris, E. Kouroumalis, I. Pallikaris, and K. Thermos
Effect of Somatostatin on Nitric Oxide Production in Human Retinal Pigment Epithelium Cell Cultures
Invest. Ophthalmol. Vis. Sci.,
May 1, 2004;
45(5):
1499 - 1506.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. H. Mills, R. K. Sohn, and P. E Micevych
Estrogen-Induced {micro}-Opioid Receptor Internalization in the Medial Preoptic Nucleus Is Mediated via Neuropeptide Y-Y1 Receptor Activation in the Arcuate Nucleus of Female Rats
J. Neurosci.,
January 28, 2004;
24(4):
947 - 955.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Reubi
Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy
Endocr. Rev.,
August 1, 2003;
24(4):
389 - 427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Csaba, A. Simon, L. Helboe, J. Epelbaum, and P. Dournaud
Targeting sst2A Receptor-Expressing Cells in the Rat Hypothalamus through in Vivo Agonist Stimulation: Neuroanatomical Evidence for a Major Role of this Subtype in Mediating Somatostatin Functions
Endocrinology,
April 1, 2003;
144(4):
1564 - 1573.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. J. Hofland and S. W. J. Lamberts
The Pathophysiological Consequences of Somatostatin Receptor Internalization and Resistance
Endocr. Rev.,
February 1, 2003;
24(1):
28 - 47.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. B. Binder, B. Kinkead, M. J. Owens, and C. B. Nemeroff
Neurotensin and Dopamine Interactions
Pharmacol. Rev.,
December 1, 2001;
53(4):
453 - 486.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Klisovic, M. S. O'Dorisio, S. E. Katz, J. W. Sall, D. Balster, T. M. O'Dorisio, E. Craig, and M. Lubow
Somatostatin Receptor Gene Expression in Human Ocular Tissues: RT-PCR and Immunohistochemical Study
Invest. Ophthalmol. Vis. Sci.,
September 1, 2001;
42(10):
2193 - 2201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Sinchak and P. E Micevych
Progesterone Blockade of Estrogen Activation of {micro}-Opioid Receptors Regulates Reproductive Behavior
J. Neurosci.,
August 1, 2001;
21(15):
5723 - 5729.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. W. Hubert, M. Paquet, and Y. Smith
Differential Subcellular Localization of mGluR1a and mGluR5 in the Rat and Monkey Substantia Nigra
J. Neurosci.,
March 15, 2001;
21(6):
1838 - 1847.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Reubi, B. Waser, Q. Liu, J. A. Laissue, and A. Schonbrunn
Subcellular Distribution of Somatostatin sst2A Receptors in Human Tumors of the Nervous and Neuroendocrine Systems: Membranous Versus Intracellular Location
J. Clin. Endocrinol. Metab.,
October 1, 2000;
85(10):
3882 - 3891.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. Ferone, R. Pivonello, P. M. Van Hagen, M. Waaijers, J. Zuijderwijk, A. Colao, G. Lombardi, A. J. J. C. Bogers, S. W. J. Lamberts, and L. J. Hofland
Age-related decrease of somatostatin receptor number in the normal human thymus
Am J Physiol Endocrinol Metab,
October 1, 2000;
279(4):
E791 - E798.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Boudin, P. Sarret, J. Mazella, A. Schonbrunn, and A. Beaudet
Somatostatin-Induced Regulation of SST2A Receptor Expression and Cell Surface Availability in Central Neurons: Role of Receptor Internalization
J. Neurosci.,
August 15, 2000;
20(16):
5932 - 5939.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Schreff, S. Schulz, M. Handel, G. Keilhoff, H. Braun, G. Pereira, M. Klutzny, H. Schmidt, G. Wolf, and V. Hollt
Distribution, Targeting, and Internalization of the sst4 Somatostatin Receptor in Rat Brain
J. Neurosci.,
May 15, 2000;
20(10):
3785 - 3797.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Dumartin, M. Jaber, F. Gonon, M. G. Caron, B. Giros, and B. Bloch
Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice
PNAS,
February 15, 2000;
97(4):
1879 - 1884.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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
J. Neurosci.,
December 1, 1999;
19(23):
10237 - 10249.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zitzer, H.-H. Honck, D. Bachner, D. Richter, and H.-J. Kreienkamp
Somatostatin Receptor Interacting Protein Defines a Novel Family of Multidomain Proteins Present in Human and Rodent Brain
J. Biol. Chem.,
November 12, 1999;
274(46):
32997 - 33001.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Sarret, D. Nouel, C. Dal Farra, J.-P. Vincent, A. Beaudet, and J. Mazella
Receptor-mediated Internalization Is Critical for the Inhibition of the Expression of Growth Hormone by Somatostatin in the Pituitary Cell Line AtT-20
J. Biol. Chem.,
July 2, 1999;
274(27):
19294 - 19300.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zitzer, D. Richter, and H.-J. Kreienkamp
Agonist-dependent Interaction of the Rat Somatostatin Receptor Subtype 2 with Cortactin-binding Protein 1
J. Biol. Chem.,
June 25, 1999;
274(26):
18153 - 18156.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.J. Hofland, Q. Liu, P.M. van Koetsveld, J. Zuijderwijk, F. van der Ham, R.R. de Krijger, A. Schonbrunn, and S.W.J. Lamberts
Immunohistochemical Detection of Somatostatin Receptor Subtypes sst1 and sst2A in Human Somatostatin Receptor Positive Tumors
J. Clin. Endocrinol. Metab.,
February 1, 1999;
84(2):
775 - 780.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
H. Boudin, D. Pelaprat, W. Rostene, V. M. Pickel, and A. Beaudet
Correlative Ultrastructural Distribution of Neurotensin Receptor Proteins and Binding Sites in the Rat Substantia Nigra
J. Neurosci.,
October 15, 1998;
18(20):
8473 - 8484.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
Gene
