Localization of the Somatostatin Receptor SST2Ain Rat Brain
Using a Specific Anti-Peptide Antibody
Pascal Dournaud,1Yi Z. Gu,3Agnes Schonbrunn,3Jean Mazella,1Gloria S. Tannenbaum,1,2and
Departments of1Neurology and Neurosurgery and2Pediatrics, McGill University, Montre ´al, Que ´bec, Canada H3A 2B4,
and3Department of Pharmacology, University of Texas, Houston Medical School, Houston, Texas 77225
Biological actions of somatostatin are exerted via a family of
receptors, for which five genes recently have been cloned.
However, none of these receptor proteins has been visualized
yet in the brain. In the present study, the regional and cellular
distribution of the somatostatin sst2Areceptor was investigated
via immunocytochemistry in the rat central nervous system by
using an antibody generated against a unique sequence of the
receptor protein. Specificity of the antiserum was demon-
strated by immunoblot and immunocytochemistry on rat brain
membranes and/or on cells transfected with cDNA encoding
the different sst receptor subtypes. In rat brain sections, sst2A
receptor immunoreactivity was concentrated either in perikarya
and dendrites or in axon terminals distributed throughout the
neuropil. Somatodendritic labeling was most prominent in the
olfactory tubercle, layers II–III of the cerebral cortex, nucleus
accumbens, pyramidal cells of CA1–CA2 subfields of the hip-
pocampus, central and cortical amygdaloid nuclei, and locus
coeruleus. Labeled terminals were detected mainly in the en-
dopiriform nucleus, deep layers of the cortex, claustrum, sub-
stantia innominata, subiculum, basolateral amygdala, medial
habenula, and periaqueductal gray. Electron microscopy con-
firmed the association of sst2Areceptors with perikarya and
dendrites in the former regions and with axon terminals in the
latter. These results provide the first characterization of the
cellular distribution of a somatostatin receptor in mammalian
brain. The widespread distribution of the sst2Areceptor in
cerebral cortex and limbic structures suggests that it is involved
in the transduction of both pre- and postsynaptic effects of
somatostatin on cognition, learning, and memory.
Key words: immunohistochemistry; somatostatin; receptor;
transfected cells; electron microscopy; central nervous system
Somatostatin (SRIF) is a tetradecapeptide present throughout the
neuroaxis in which it is known to play both a neuroendocrine and
a neurotransmitter role with diverse physiological effects on hor-
mone release and cognitive and behavioral functions (for review,
see Epelbaum et al., 1994). SRIF also has been shown to have
antiproliferative actions on tumoral cells (Lamberts et al., 1991)
and to be perturbed in certain neurological disorders including
epilepsy, depression, and Alzheimer’s disease (Epelbaum et al.,
1994). The numerous functional effects of SRIF are exerted via
G-protein-coupled receptors (Koch and Schonbrunn, 1984; Koch
et al., 1985) for which five different genes recently have been
cloned (Bruno et al., 1992; O’Carroll et al., 1992; Yamada et al.,
1992; Yasuda et al., 1992). These receptors, designated sst1
through sst5(Hoyer et al., 1995), bind SRIF-14 and its N-terminal
extended form SRIF-28 with comparable affinity. The sst2recep-
tor exists in two variant forms, sst2Aand sst2B, generated by
alternative splicing of the sst2mRNA (Vanetti et al., 1992, 1994).
The two sst2receptor variants exhibit indistinguishable binding
properties but may differ in G-protein coupling and desensitiza-
tion (Reisine et al., 1993; Vanetti et al., 1993). Experiments on
transfected cells suggest that sst2, sst3, and sst5receptors are
equivalent pharmacologically to the SRIF-1 class of receptors,
previously defined on the basis of their high affinity for the
synthetic SRIF agonists MK 678 and octreotide, whereas sst1and
sst4are equivalent to the SRIF-2 class, which lacks affinity for
these compounds (for review, see Hoyer et al., 1994; Reisine and
The mRNAs for all five SRIF receptors are expressed widely in
the human and rodent central nervous system (Breder et al., 1992;
Kluxen et al., 1992; Kong et al., 1994; Pe ´rez et al., 1994; Senaris et
al., 1994; Beaudet et al., 1995). Although earlier autoradiographic
receptor binding studies have examined the overall distribution of
SRIF binding sites in mammalian brain and have distinguished
between SRIF-1 and SRIF-2 pharmacological subtypes (Krantic
et al., 1992), the relative abundance and localization of the dif-
ferent SRIF receptor proteins is unknown still. Furthermore,
nothing is known of their cellular distribution, which is critical for
understanding the modes of action of SRIF in the brain. In the
present study, we have developed an antiserum against the sst2A
receptor and used it to characterize the regional and cellular
localization of the sst2Areceptor protein in the rat brain.
MATERIALS AND METHODS
Antibody preparation and immunoblot analysis. Polyclonal antibodies
were generated in New Zealand white rabbits against the peptide
CERSDSKQDKSRLNETTETQRT after conjugation to keyhole lim-
pet hemocyanin via the NH2-terminal cysteine using m-maleimidobenzoyl-
N-hydroxysuccinimide (Lerner et al., 1981). This sequence is located in the
C-terminal region of the rat sst2Areceptor (Kluxen et al., 1992) and is
Received Feb. 5, 1996; revised April 22, 1996; accepted April 24, 1996.
This work was supported by grants from the Fonds de la Recherche en Sante ´ du
Que ´bec and the Medical Research Council of Canada to G.S.T. and A.B., and from
National Institutes of Health to A.S. G.S.T. is the recipient of a “Chercheur de
Carrie `re” award from the Fonds de la Recherche en Sante ´ du Que ´bec. We thank F.
Jiang, A. Morin, L. Mulcahy, D. Nouel, Y. Wang, and E. Di Camillo for their
Correspondence should be addressed to Alain Beaudet, Montreal Neurological
Institute, McGill University, Montre ´al, Que ´bec, Canada H3A 2B4.
Dr. Dournaud’s present address: Institut National de la Sante ´ et de la Recherche
Me ´dicale, U-159, 2 Rue D’Alesia, 75014 Paris, France.
Copyright ? 1996 Society for Neuroscience 0270-6474/96/164468-11$05.00/0
The Journal of Neuroscience, July 15, 1996, 16(14):4468–4478
conserved in the mouse and human forms. Antibody specificity was deter-
mined by using CHO-K1 cells stably transfected with receptor subtypes sst1
and sst2A(provided by Dr. P.J.S. Stork), sst2B(provided by Dr. V. Hollt), sst3
(provided by Dr. Y. Patel), sst4(provided by Dr. M. Berelowitz), and sst5
(provided by Dr. S. Seino). Membranes from CHO cells were prepared as
described previously (Brown et al., 1990) and confirmed to bind specifically
Rat cortical and cerebellar membranes were prepared from female
Harlan Sprague–Dawley rats according to the protocol of Sakamoto et al.
(1988) with minor changes. In brief, brain tissue was suspended in 10 vol
of Tris buffer (50 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 200
?g/ml bacitracin, 0.5 ?g/ml aprotonin, 10 ?g/ml trypsin inhibitor, and 4
?g/ml PMSF) and homogenized with a polytron homogenizer at 900 rpm
for 20 strokes. The membrane pellet obtained by high-speed centrifuga-
tion was stored at ?70?C.
For immunoblot analysis, thawed membranes were pelleted in a mi-
crocentrifuge, resuspended in sample buffer (62.5 mM Tris-Cl, pH 6.8, 2%
SDS, 20% glycerol, and 50 mM dithiothreitol), and electrophoresed on
10% SDS-acrylamide gels according to the method of Laemmli (1970).
Proteins were transferred electrophoretically to PVDF membranes (0.2
?m; Bio-Rad, Mississauga, Ontario, Canada) in transfer buffer (10 mM
NaHCO3, 3 mM Na2CO3, 0.1% SDS, and 20% methanol). The mem-
branes were blocked with 5% nonfat dry milk in PBS (10 mM Na2HPO4,
pH 7.5, and 150 mM NaCl) for 2 hr at room temperature (RT) and then
incubated overnight at 4?C with the anti-peptide antiserum diluted to
between 1:10,000 and 1:20,000 in 1% nonfat dry milk containing 0.05%
NaN3. Immunoreactive bands were detected by incubating washed mem-
branes for 1 hr with goat anti-rabbit IgG conjugated with horseradish
peroxidase (1:10,000) (Bio-Rad) and developing with the Amersham
(Oakville, Ontario, Canada) ECL kit according to the manufacturer’s
Immunocytochemical characterization of the antiserum. COS-7 cells
were grown in DMEM containing glutamine supplemented with 44 mM
NaHCO3and 10% fetal calf serum in the presence of 50 mg/ml genta-
micin. Transient transfections were performed with 1 ?g of recombinant
plasmids for mouse sst1, sst2A, and sst2Breceptors (provided by Dr. T.
Reisine) by the DEAE-dextran precipitation procedure (Perlman et al.,
1992) onto semiconfluent COS-7 cells grown in 100 mm cell culture
dishes. Sixty hours after transfections, cells were treated or not for 24 hr
with 50 ng/ml pertussis toxin (Sigma, St. Louis, MO; Koch et al., 1985),
fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and
incubated for 16 hr at RT with the R2-88 antiserum diluted 1:4000 in 0.1
M TBS containing 1% normal goat serum (ngs) in the presence or absence
of 0.1% Triton X-100. After rinsing, cells were incubated for 1 hr at RT
with a 1:200 dilution of conjugated Texas Red-goat anti-rabbit IgG
(Jackson ImmunoResearch, West Grove, PA), washed several times in
0.1 M TBS, and examined by confocal microscopy using a Leica inverted
microscope equipped with a krypton laser. Fluorescence intensity mea-
surements were performed on 12 pertussis toxin-treated and untreated
cells using the Leica software package. Results are expressed as gray level
units on a 0–255 gray scale.
Immunocytochemistry. Adult male Sprague–Dawley rats (150–200 gm
body weight) were anesthetized with Somnotol (MTC Pharmaceuticals;
80 mg/kg, i.p.) and perfused transaortically with 4% paraformaldehyde in
0.1 M TBS. Brains were post-fixed for 60 min in the same fixative,
cryoprotected, and frozen in liquid isopentane at ?45?C. Immunocyto-
chemical experiments were performed by using the avidin-biotinylated-
peroxidase complex (ABC) standard kit (Vector Laboratories, Burlin-
game, CA) and a biotinyl-tyramide amplification system (DuPont NEN,
Wilmington, DE). Briefly, 30 ?m sections were preincubated for 30 min
in TBS containing 3% ngs and incubated for 16 hr at RT in a 1:1000
dilution of the R2-88 antiserum containing 0.2% Triton X-100. Then
sections were rinsed in 0.1 M TBS and incubated sequentially for 45 min
in biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch) diluted
1:100 in 0.1 M TBS and in ABC solution. Then they were incubated for 10
min in a 0.01% biotinyl-tyramide solution (Adams, 1992), activated with
0.01% H2O2, and then reincubated in 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. Immunolabeling for electron microscopy was
performed as above, except that glutaraldehyde (0.2%) was added to the
fixative, and the tissue was sectioned on a Vibratome. Immunoreacted
sections taken at the level of the hippocampal formation, amygdaloid
complex, and medial habenula were post-fixed for 1 hr in 2% osmium
tetroxide, dehydrated in graded ethanols, embedded in Epon, sectioned
with an ultramicrotome, counterstained with lead citrate, and examined
with a Jeol 100CX electron microscope.
Controls. For controls, the R2-88 antiserum was adsorbed with an
excess of sst2Acarboxyl-terminal peptide, or the immune serum was
replaced by R2-88 preimmune serum. In addition, a method specificity
control was performed by omitting the antiserum from the immunohis-
tochemical staining protocol.
The anti-peptide antiserum R2-88 reacted strongly with a broad
band of ?85 kDa in CHO-K1 cells stably transfected with a rat
sst2A-receptor expression plasmid (Fig. 1). The immunoreactivity
was abolished completely when the staining was performed in the
presence of 1 ?M peptide antigen or with preimmune in lieu of
immune serum, demonstrating that it was produced by the anti-
peptide antibodies (Fig. 1). The antiserum did not react with the
parental cells, which do not express sst receptors, nor with sst
receptors from cells transfected with any of the other receptor
subtypes, demonstrating that it was specific for the sst2Areceptor
(Fig. 1). In separate experiments, R2-88 antiserum was found to
immunoprecipitate the sst2Areceptor with 60–80% efficiency and
to precipitate ?1% of any of the other receptor subtypes (Schon-
brunn et al., 1995).
To determine whether the antiserum specifically recognized the
sst2Areceptor protein in brain tissue, we compared the immuno-
reactivity present in membranes from rat cerebral cortex, which
contains high levels of sst2receptor mRNA, with that from rat
cerebellum, which contains no or only low levels of sst2receptor
mRNA (Breder et al., 1992; Kong et al., 1994; Perez et al., 1994;
cells. Membranes (40 ?g) from either parental CHO-K1 cells or from
CHO-K1 cells transfected with cDNA encoding individual sst receptor
subtypes were separated on SDS-PAGE. After transfer to PVDF mem-
branes, the proteins were immunoblotted with a 1:10,000 dilution of R2-88
immune serum (Immune), R2-88 preimmune serum (PreImmune), or
R2-88 immune serum in the presence of 1 ?M peptide antigen (Pread-
sorbed). Molecular size markers (in kDa) are shown on the right.
Western blot analysis of R2-88 immunoreactivity in CHO-K1
Dournaud et al. • CNS Localization of the SST2AReceptor J. Neurosci., July 15, 1996, 16(14):4468–4478 4469
Senaris et al., 1994). The antiserum reacted strongly with a broad
72 kDa band in cortical membranes (Fig. 2), consistent with the
results from affinity cross-linking of rat cerebrocortical sst recep-
tors (Sakamoto et al., 1988; Kimura, 1989). This staining was
prevented by 1 nM peptide antigen and was not detected in
cerebellar membranes (Fig. 2). Although several other bands were
stained weakly by the antiserum, the lack of competition by
peptide antigen indicated that their staining was caused by unre-
lated antibodies in the serum.
Visualization of sst2Areceptor in transfected cells
Approximately 20% of COS-7 cells transiently transfected with
cDNA encoding the sst2Areceptor were immunolabeled intensely
with the R2-88 antiserum (dilution, 1:4000; Fig. 3a), congruent
with the reported transfection yield in this cell line (Pollard et al.,
1984). By contrast, no immunoreaction product was detected in
cells transfected with cDNA encoding the sst1(not shown), the
sst2Breceptor (Fig. 3d), or in nontransfected cells. Immunolabel-
ing was decreased greatly in the absence of detergent and abol-
ished when the incubation was performed with either preimmune
serum or with immune serum preadsorbed with 1 ?M sst2Aanti-
genic peptide (Fig. 3b,c). Neither the number nor the labeling
density of sst2A-immunoreactive cells was modified by pretreat-
ment of the cells with pertussis toxin (mean fluorescence intensity,
94.5 ? 5.8 vs 92 ? 7.3 gray level units for treated vs untreated
Light microscopic localization of sst2Areceptor in
Rat brain sections immunoreacted with R2-88 antiserum exhib-
ited selective patterns of immunostaining, the distribution of
which closely resembled that of SRIF binding sites previously
documented by using receptor autoradiography (Fig. 4a,b). This
staining was absent in sections incubated either with preimmune
serum, immune serum preadsorbed with sst2Aantigenic peptide
(Fig. 4c), or in the absence of primary antibody. At high magni-
fication, the immunostaining was seen to be associated either with
neuronal perikarya and dendrites or with axon terminals, depend-
ing on the region examined (Table 1; Figs. 5–7). Perikaryal label-
ing pervaded the cytoplasm of the cells, sparing the nucleus;
dendritic labeling was usually confined to primary or, more rarely,
secondary branches (Figs. 6D, 7C). Axonal labeling took the form
of a fine dusting of the neuropil (Figs. 5D, 7B), within which
individually labeled punctae could be distinguished at high mag-
nification (Fig. 6B). No sst2Areceptor immunolabeling was ob-
served over glial cells.
Rostrally, numerous strongly immunoreactive nerve cell bodies
and proximal dendrites were observed throughout the pyramidal
layer of the olfactory tubercle, extending into the polymorph layer
(Fig. 5A; Table 1). More sparse and less intensely reactive nerve
cell bodies also were visible in layer II of the piriform cortex.
Dense terminal labeling, but no labeled perikarya, was apparent
in the endopiriform and anterior olfactory nuclei as well as in the
lateral olfactory tract nucleus.
Both immunoreactive perikarya and immunoreactive terminal
fields were detected throughout the neocortex, in which they
assumed specific laminar distributions (Table 1). In the anterior
cingulate and retrosplenial cortices, sst2Aimmunoreactivity was
concentrated mainly within nerve cell bodies in layer II (Fig. 5B).
In the frontal, parietal, temporal, and occipital cortices, promi-
nent perikaryal staining was observed in layers II–III (Fig. 5C).
Most of these neurons extended labeled apical dendrites up to
layer I. A few less intensely immunoreactive perikarya also were
evident in layers V–VI (Fig. 5C). Most conspicuous in the latter
Membranes from rat cerebral cortex (50 ?g), rat cerebellum (50 ?g), or
CHO-R2A cells (25 ?g) were separated on a 10% SDS-acrylamide gel.
After transfer to PVDF membranes, the proteins were immunoblotted
with a 1:20,000 dilution of R2-88 immune serum in the absence or
presence of 1 nM peptide antigen, as indicated. Molecular size markers (in
kDa) are shown on the right.
Western blot analysis of R2-88 immunoreactivity in rat brain.
stained for the sst2Areceptor. COS-7 cells transiently transfected with
cDNA encoding the sst2Areceptor exhibit intense cytoplasmic immuno-
reactivity (a). The yield of the transfection is ?20%, which explains the
presence of nonlabeled cells in the same field. No labeled cells are
apparent in preparations incubated with preimmune serum (b) or with the
R2-88 immune serum preadsorbed with an excess of peptide antigen (c).
COS-7 cells transfected with cDNA encoding the sst2Breceptor are
immunonegative, also (d). Magnification, 300?.
Confocal imaging of COS-7 cells immunocytochemically
4470 J. Neurosci., July 15, 1996, 16(14):4468–4478Dournaud et al. • CNS Localization of the SST2AReceptor
layers, however, were immunoreactive terminal fields (Fig. 5C).
Perirhinal and enthorinal cortices exhibited numerous labeled
perikarya in layers II–III and dense terminal labeling in the outer
portion of layer V and throughout layer VI (Fig. 5D).
High densities of immunoreactive perikarya and dendrites were
detected in the lateral division of the bed nucleus of the stria
terminalis (Fig. 6A; Table 1). Smaller and more sparsely distrib-
uted cells also were evident in the intermediate and ventral
divisions of the nucleus, dorsal and ventral to the anterior com-
missure, respectively (Fig. 6A). In the medial habenular nucleus,
immunoreactive cell bodies were distributed among a dense net-
work of strongly immunoreactive processes (Fig. 6B). The lateral
part of the nucleus was devoid of immunoreactivity.
Several small or medium-sized immunoreactive nerve cell bod-
ies were evident throughout the ventrolateral and caudal seg-
ments of the neostriatum (Fig. 6C) as well as within the core and
shell divisions of the nucleus accumbens (Table 1). Dense termi-
nal labeling, but no immunoreactive perikarya, were visible in the
claustrum. Highly arborized, densely immunoreactive cell bodies
and dendrites were observed in the dorsolateral septum along the
upper part of the third ventricle immediately beneath the corpus
callosum (Fig. 6D).
Pyramidal cells in the CA1–CA2 fields of the hippocampus
were labeled the most intensely and conspicuously in the brain
(Fig. 7A; Table 1). Prominent labeling of their basal and apical
dendrites was also evident in strata oriens and radiatum, respec-
tively, superimposed over moderate terminal labeling (Fig. 7C). A
few labeled perikarya also were observed in the stratum radiatum.
No immunoreactive cell bodies, but diffuse neuropil labeling, was
apparent in the stratum lacunosum moleculare (Fig. 7A). In
contrast, the CA3 subfield remained consistently immunonega-
tive. Diffuse terminal labeling was apparent in the molecular layer
of the dentate gyrus as well as, albeit less prominently, at the
granule cell–hilar border. Dense terminal labeling also was evi-
dent in the subiculum.
Both perikaryal and terminal labeling were detected in the
amygdaloid complex, the former in the central and the latter in
the basolateral nucleus (Fig. 7B). The medial and cortical amyg-
daloid nuclei exhibited weak-to-intense perikaryal immunostain-
ing, whereas the lateral amygdaloid nucleus was immunonegative.
Only sparse sst2Areceptor-immunoreactive nerve cell bodies
were detected within the hypothalamus, and these usually were
stained lightly except in the tuberomammillary nucleus in
which they formed a tight cluster of moderately immunoreac-
tive cells. Diffuse terminal labeling was apparent in a few
hypothalamic nuclei, including the paraventricular and the
arcuate nuclei (Table 1).
In the midbrain, selectively labeled nerve cell bodies were
apparent in the deep layers of the superior colliculi and in the
periaqueductal gray matter (Table 1). Many of these neurons
were observed to extend long and fine processes. Additionally,
moderately dense terminal labeling was evident in the dorsal and
lateral segment of the periaqueductal gray, gray layers of the
superior colliculus, pars compacta of the substantia nigra, and
ventral tegmental area.
In the pons, intensely labeled perikarya and processes were
detected throughout the locus coeruleus (Fig. 7D). Less intensely
labeled neurons also were visible in the lateral dorsal tegmental
and parabrachial nuclei. In the medulla, immunoreactive nerve
cell bodies were most evident in the dorsal motor nucleus of the
vagus and lateral reticular nucleus, and immunoreactive axons
were observed in the nucleus tractus solitarius and medial vestib-
ular nucleus. Both the cerebellar cortex and deep cerebellar nuclei
were devoid of immunostaining (Table 1).
Electron microscopic localization of sst2Areceptor in
selected limbic structures
Electron microscopy confirmed the association of immunoreac-
tivity with neuronal perikarya and dendrites in the hippocampus,
medial habenula, and central amygdaloid nucleus (Fig. 8A,B) and
with axon terminals in the medial habenula and basolateral amyg-
dala (Fig. 8C). In perikarya and processes alike, the reaction
product pervaded the cytoplasm as opposed to being confined to
the plasma membrane. Labeled dendrites often were varicose and
with R2-88 antiserum preadsorbed with an excess of the peptide antigen. a, Autoradiogram of a 20 ?m rat midbrain section incubated with
[125I]-Tyr0-DTrp8-SRIF-14 for 45 min. Reproduced from Moyse et al. (1992), with permission. b, As for [125I]SRIF binding, sst2Areceptor immunore-
activity predominates in the deep layers of the cortex (cx), the medial habenula (mh), the hippocampal formation (hi), and the amygdaloid complex (am).
c, By comparison, the preadsorbed control is virtually devoid of immunoreactivity. Magnification, 7?.
Comparative distribution of [125I]SRIF binding sites (a) and sst2Areceptor immunoreactivity (b) in rat brain. Section in c was immunoreacted
Dournaud et al. • CNS Localization of the SST2AReceptorJ. Neurosci., July 15, 1996, 16(14):4468–4478 4471
received both symmetric and asymmetric synaptic contacts from
unlabeled axon terminals (Fig. 8B). Immunoreactive axon termi-
nals were seen in synaptic contact with unlabeled dendrites (Fig.
8C) but not with other unlabeled terminals.
The present study provides the first description of the cellular
distribution of an SRIF receptor protein in mammalian brain. The
specificity of the antiserum was established initially by Western
blot in membranes of CHO-K1 cells transfected with the cDNA
encoding each of the different sst receptor subtypes. The anti-
serum recognized a broad protein band centered near 85 kDa in
membranes from sst2A-transfected CHO-K1 cells but did not
react with CHO membranes containing any of the other receptor
subtypes. This band had the same molecular weight and migration
pattern as the photoaffinity-labeled receptor in the CHO-R2A
cells, confirming that the detected protein represents the sst2A
receptor (Gu et al., 1995). The apparent molecular weight of this
receptor protein is markedly higher than the 41.2 kDa predicted
from the amino acid sequence (Kluxen et al., 1992), which is
consistent with the hypothesis that the sst2Areceptor undergoes
extensive post-translational modifications (Patel et al., 1994; Re-
isine and Bell, 1995). Further, the broad migration pattern is
characteristic of heavily glycosylated proteins and has been ob-
served with numerous other seven transmembrane domain
The specificity of the antibody for the sst2Areceptor in rat brain
was characterized further by immunoblot in rat brain membranes.
In agreement with previous cross-linking studies showing that
affinity-labeled rat cerebrocortical SRIF receptors migrate as a
broad band with an apparent molecular weight of ?72 kDa
(Sakamoto et al., 1988; Kimura, 1989), our antibody specifically
recognized a broad protein band centered near 72 kDa. For
reasons not entirely clear, this molecular weight is considerably
lower than that of 148 kDa previously reported for the sst2
receptor detected in rat brain using an antibody directed against
the third extracellular domain of the receptor (Theveniau et al.,
1994). Our conclusion that the 72 kDa band represents the sst2A
receptor is supported by the fact that (1) antibody binding to this
band was abolished by a concentration of peptide antigen as low
as 1 nM, and (2) this band was not visible in rat cerebellar
membranes that express low or undetectable levels of sst2A
mRNA (Kong et al., 1994; Perez et al., 1994; Senaris et al., 1994).
The slightly lower apparent molecular weight of the labeled sst2A
receptor in cortex as compared with its molecular weight in
CHO-R2A cells suggests that the sst2Areceptor is glycosylated
less heavily in neurons than in transfected CHO cells.
Experiments on transfected COS-7 cells further confirmed the
specificity of the R2-88 antiserum and indicated that it provided
for selective detection of the sst2Aantigen in paraformaldehyde-
fixed tissue. The lack of staining of sst1- and sst2B-transfected cells
indicated further that the antiserum recognized specifically the
C-terminal sequence of the sst2Areceptor, as expected from the
amino acid sequence of the immunogenic peptide. The marked
decrease in staining observed in the absence of detergent con-
firmed that the C-terminal tail is intracellular, as predicted from
structural homology of the receptor with rhodopsin (Kluxen et al.,
1992). Finally, the similarity in the pattern and intensity of immu-
nostaining between cells treated or not with pertussin toxin indi-
cated that our antibody recognized G-protein-coupled (Tomura et
al., 1994) and uncoupled forms of the receptor equally well.
The regional distribution of the sst2Areceptor immunoreactiv-
Table 1. Distribution of sst2A-like immunoreactivity in rat brain*
and dendrites Axon terminals
Anterior olfactory nucleus
Lateral olfactory tract nucleus
Bed nucleus stria terminalis
Diagonal band of Broca (vertical limb)
Stratum lacunosum moleculare
Molecular layer (ventral part)
Central amygdaloid nucleus
Basolateral amygdaloid nucleus
Medial amygdaloid nucleus
Cortical amygdaloid nucleus
Medial preoptic area
Dorsal raphe nucleus
Substantia nigra (pars compacta)
Ventral tegmental area
Raphe linearis caudalis
Midbrain reticular formation
Lateral dorsal tegmental nucleus
Nucleus tractus solitarius
Medial vestibular nucleus
Lateral reticular nucleus
Dorsal motor nucleus of the vagus
Deep cerebellar nuclei
*Relative values. Data based on light microscopic observations.
4472 J. Neurosci., July 15, 1996, 16(14):4468–4478Dournaud et al. • CNS Localization of the SST2AReceptor
ity in rat brain sections was strikingly similar to that of SRIF
binding sites previously documented by using receptor autora-
diography (Martin et al., 1991; Krantic et al., 1992; Moyse et al.,
1992) (see also Fig. 3). As expected, this distribution most closely
paralleled that of the binding of SRIF agonists SMS 201–995 and
MK 678 (Martin et al., 1991; Krantic et al., 1992), which recognize
with high affinity the sst2, sst3, and sst5receptors. More surpris-
ingly, it also correlated remarkably well with the binding patterns
prominent in cell bodies in the pyramidal (Py) and polymorph (Po) cell layers. Pl, Plexiform layer; Si, substantia innominata. Magnification, 250?. B,
Anterior cingulate cortex. Numerous sst2A-immunoreactive perikarya are apparent throughout layer II. cc, Corpus callosum. Magnification, 170?. C,
Parietal cortex. Immunoreactive nerve cell bodies are numerous in layers II–III and more sparsely distributed in layer V. Fields of labeled axon terminals
are evident in deep layers, most prominently in layer V. Magnification, 270?. D, Dense perikaryal labeling is evident throughout layers II–III. Labeled
terminal fields pervade the outer segment of layer V as well as layer VI. Magnification, 220?.
Light microscopic distribution of sst2Areceptor immunoreactivity in cerebral cortex. A, Olfactory tubercle. Perikaryal immunostaining is
Dournaud et al. • CNS Localization of the SST2AReceptor J. Neurosci., July 15, 1996, 16(14):4468–4478 4473
of less selective ligands, such as iodinated SRIF-14, which have
been shown to bind all sst subtypes (reviewed in Hoyer et al.,
1994; Reisine and Bell, 1995). This correspondence suggests that
SRIF receptors other than sst2Aare expressed either by subpopu-
lations of the same cells or by subsets of neurons within the same
At cellular and subcellular levels, sst2Areceptor immunoreac-
tivity was concentrated in perikarya and dendrites as well as in
throughout the lateral, intermediate, and ventral divisions of the bed nucleus of the stria terminalis. ic, Internal capsule; ac, anterior commissure; 3V, third
ventricle. Magnification, 220?. B, Intense immunolabeling of nerve cell bodies and intervening neuropil pervades the medial habenular nucleus (Mh).
Note the absence of labeling in the lateral division of the nucleus and the presence of immunoreactive axon terminals in the paraventricular nucleus of
the thalamus (PV). Magnification, 270?. C, Small, intensely immunoreactive spiny type I neurons are evident in between the myelinated fascicles of the
internal capsule in the ventrolateral neostriatum. ec, External capsule. Magnification, 420?. D, Dorsolateral septum. The labeling clearly is seen to
pervade the cytoplasm of neuronal perikarya and their proximal dendrites (arrows). 3V, Third ventricle. Magnification, 850?.
Distribution of sst2Areceptor immunoreactivity in the limbic system and neostriatum. A, Prominent perikaryal immunolabeling is detected
4474 J. Neurosci., July 15, 1996, 16(14):4468–4478 Dournaud et al. • CNS Localization of the SST2AReceptor
and proximal dendrites in subfields CA1–CA2 are superimposed over neuropil staining, stopping abruptly at CA3. or, Stratum oriens; py, stratum
pyramidale; ra, stratum radiatum; lm, stratum lacunosum moleculare; Hil, hilus. Magnification, 140?. B, Temporal lobe. Intense immunoreactivity is
evident in the central (Ce) and basolateral (Bla) amygdaloid nuclei. However, in the former, it is confined to nerve cell bodies and, in the latter, to axon
terminals. Note the dense terminal labeling in the dorsal endopiriform nucleus (Den) and in the deep layers of the perirhinal cortex (CX). Cp, Caudate
putamen; Pir, piriform cortex. Magnification, 100?. E, Labeled pyramidal cells in CA1. Apical dendrites are seen extending into the stratum radiatum
(arrows). Note the sparing of the nucleus. Magnification, 1300?. D, Locus coeruleus (LC). Nerve cell bodies and surrounding neuropil are equally, densely
immunoreactive. V4, 4th ventricle. Magnification, 100?.
Distribution of sst2Areceptor immunoreactivity in the hippocampus, amygdala, and pons. A, Hippocampus. Intensely labeled pyramidal cells
Dournaud et al. • CNS Localization of the SST2AReceptorJ. Neurosci., July 15, 1996, 16(14):4468–4478 4475
axon terminals profusely distributed in the neuropil. These find-
ings demonstrate that the sst2Areceptor subtype is in a position to
transduce both post- and presynaptic effects of SRIF in mamma-
lian brain. The association of sst2Areceptor immunoreactivity
with the somatodendritic arbor of neurons in the locus coeruleus,
dorsolateral septum, CA1 subfield of the hippocampus, and the
nucleus of the solitary tract observed in the present study is
consistent with the postsynaptic electrophysiological effects of
locally administrated SRIF documented within these areas (Olpe
et al., 1987; Jacquin et al., 1988; Schweitzer et al., 1990, 1993;
Twery et al., 1991; Xie and Sastry, 1992). By contrast, the exten-
sive labeling of axonal fields suggests that presynaptic effects of
SRIF in the rat brain may be more pervasive than earlier func-
tional studies had led us to believe (Go ¨thert, 1980; Tanaka and
Tsujimoto, 1981). Interestingly, labeled terminals usually were
distributed in nuclei distinct from those containing immunoreac-
tive perikarya, suggesting that neurons harboring sst2Areceptors
are mainly projection neurons.
In both COS-7 cells and rat brain sections, sst2Areceptor immu-
noreactivity was found by high resolution microscopy (confocal for
the former and electron for the latter) to be associated not only with
the inner plasma membrane but also with the cytoplasm of labeled
cells. Similar intracytoplasmic localizations have been reported for
other types of G-protein-coupled receptors (Levey et al., 1991, 1993;
Sesack et al., 1994; Arvidsson et al., 1995) and may be attributed, in
part, to artifactitious diffusion of the peroxidase complex from the
membrane (Novikoff et al., 1972). This factor alone, however, would
not explain the cytoplasmic labeling of COS-7 cells that were stained
by immunofluorescence. It is therefore likely that part of the immu-
nolabeling observed in the cytoplasm of both neurons and COS-7
cells represents neosynthesized, transported, and/or recycled recep-
tors. These intracellular sites are likely to be in a different functional
ligand-binding state than membrane-associated ones, because re-
gions shown here to contain sst2A receptor-immunoreactive
contain only low densities of SRIF binding sites (e.g., pyramidal cell
layer of the hippocampus) (Martin et al., 1991; Krantic et al., 1992;
Moyse et al., 1992). By contrast, regions containing immunoreactive
axon terminals, in which sst2Areceptors are likely to be mainly in
membrane-associated form, were found to be intensely labeled by
receptor autoradiography (Martin et al., 1991; Krantic et al., 1992;
Moyse et al., 1992).
As a whole, the distribution of sst2Areceptor-immunoreactive
perikarya correlated well with that of neurons previously found to
pervades the entire cytoplasm but spares the nucleus. Magnification, 8000?. B, Large varicose dendrite in the medial habenula. Both the upper and the
lower dilatations are in synaptic contact (double arrows) with unlabeled terminals. Note the four neighboring cross-sectioned immunoreactive dendrites
(d). One of these dendrites (*) is labeled less intensely than the others and is apposed directly to the large varicose one. Magnification, 11,500?. C,
Labeled axon terminal (Lt) detected among unlabeled ones (Ut) in the basolateral amygdala. The labeled terminal forms asymmetric synaptic contacts
(double arrows) with two immunonegative dendrites. Magnification, 32,000?.
Electron microscopic detection of sst2Areceptor protein in rat brain. A, Neuronal perikaryon in the medial habenula. The reaction product
4476 J. Neurosci., July 15, 1996, 16(14):4468–4478Dournaud et al. • CNS Localization of the SST2AReceptor
express sst2receptor mRNA by in situ hybridization in either
mouse (Breder et al., 1992) or rat (Pe ´rez et al., 1994; Senaris et al.,
1994; Beaudet et al., 1995) brain. In fact, all areas found to
contain sst2Areceptor-immunoreactive nerve cell bodies previ-
ously had been shown to express high levels of sst2receptor
mRNA. However, a number of areas had been reported to express
high levels of sst2receptor mRNA and showed either no or only
low numbers of immunoreactive cells in the present study, such as
layers IV and VI of the cerebral cortex, the basolateral amygdal-
oid nucleus, the claustrum, the endopiriform nucleus, and the
hypothalamus. Interestingly, all of these areas exhibited moderate
to high concentrations of sst2Areceptor-immunoreactive termi-
nals, suggesting that the receptor protein might have been ad-
dressed specifically to axons of sst2Areceptor-expressing cells that
were arborizing locally. It is also possible that regions expressing
high levels of sst2mRNA but lacking perikaryal sst2Areceptor
immunoreactivity selectively expressed the sst2Bsplice variant,
which does not contain the immunogenic peptide sequence. In-
deed, both sst2A-and sst2B-expressing cells would have been rec-
ognized by the probes used in published in situ hybridization
studies. This latter interpretation seems particularly likely in the
case of the hypothalamus, in which the sst2Bsplice variant has
been suggested by Northern blotting to be expressed predomi-
nantly (Patel et al., 1993; Kong et al., 1994) and in which SRIF
binding sites have been localized to nerve cell bodies by autora-
diography (Epelbaum et al., 1989; McCarthy et al., 1992). This
interpretation could imply that different splice variants of the sst2
receptor are involved in the transduction of the neural and neu-
roendocrine functions of SRIF.
In summary, the present results demonstrate that the sst2A
receptor is associated with both somatodendritic and axonic ele-
ments, suggesting that it is involved in the transduction of both
pre- and postsynaptic effects of SRIF in the mammalian brain.
The widespread distribution of the sst2Areceptor in cerebral
cortex and limbic structures suggests that this receptor plays a
critical role in mediating SRIF effects on cognition, expression of
emotional behavior, learning, and memory. These findings, to-
gether with the development of more subtype-specific SRIF ana-
logs, should provide pharmacological strategies for the treatment
of neurological and psychiatric disorders involving alterations in
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