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Endocrinology Vol. 144, No. 4 1564-1573
Copyright © 2003 by The Endocrine Society

ARTICLE

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

Zsolt Csaba, Axelle Simon, Lone Helboe, Jacques Epelbaum and Pascal Dournaud

Institut National de la Santé et de la Recherche Médicale, Unité-549, IFR Broca-Sainte Anne, Centre Paul Broca (Z.C., A.S., J.E., P.D.), 75014 Paris, France; and Department of Medical Anatomy (L.H.), Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Pascal Dournaud, Ph.D., Institut National de la Santé et de la Recherche Médicale, Unité-549, IFR Broca-Sainte Anne, Centre Paul Broca, 2ter rue d’Alésia, 75014 Paris, France. E-mail: dournaud{at}broca.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Numerous physiological studies as well as in situ hybridization and PCR experiments concur in reporting a role for the sst2A receptor in transducing somatostatin (SRIF) actions in the rat hypothalamus. However, the distribution of this receptor protein is not known within this structure. Regional and cellular localization of the sst2A receptor was therefore examined in the rat hypothalamus using highly sensitive immunohistochemical techniques. In close correspondence with the distribution of SRIF-immunoreactive fibers, numerous hypothalamic areas displayed sst2A receptor immunoreactivity. Receptor labeling was, however, diffusely distributed over the tissue, and few immunopositive cells were apparent. Unraveling the distribution of receptor-expressing cells was achieved through acute in vivo agonist stimulation and subsequent receptor internalization. At the cellular level, double-immunolabeling experiments with synaptophysin and microtubule-associated protein 2 demonstrated that sst2A receptors were predominantly internalized in perikarya and dendrites. Double-labeling experiments with SRIF revealed that 93% of arcuate, but only 18% of periventricular, SRIF-positive neurons expressed internalized receptors. Taken together, these results demonstrate for the first time that the sst2A receptor protein is widely, but selectively, distributed in the hypothalamus, and that postsynaptic sst2A auto- and heteroreceptors are well poised to play an important role in the somatostatinergic regulation of hypothalamic endocrine and metabolic processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SRIF), a tetradecapeptide originally isolated from ovine hypothalamus on the basis of its ability to inhibit GH secretion from the pituitary (1), was subsequently shown to exert an important neuromodulatory role with diverse physiological effects on neuroendocrine, cognitive, and behavioral functions (for reviews, see Refs. 2 and 3). The numerous actions exerted by SRIF are mediated by G protein-coupled receptors designated sst1 through sst5, for which five different genes have been cloned. Pharmacological experiments have demonstrated that SRIF receptors bind the native peptide SRIF-14 and its N-terminal extended form SRIF-28 with comparable affinity (for review, see Ref. 4). The sst2 receptor exists in two variant forms in the rodent brain, sst2A and sst2B, generated by alternative splicing of sst2 mRNA (5, 6, 7). The two sst2 receptor variants exhibit the same binding properties, but differ in their cerebral localization, as only the mRNA encoding the sst2A, not the sst2B form, is present in several rat brain regions, including the hypothalamus (8).

After their pharmacological characterization, experiments were undertaken to elucidate the individual functions of SRIF receptors in mediating central somatostatinergic transmission (for review, see Ref. 9). Efforts were particularly concentrated on the hypothalamus, because a large body of physiological evidence demonstrated that SRIF modulates neuronal networks implicated in neuroendocrine and metabolic processes (for reviews, see Refs. 10 and 11). Accordingly, mRNA encoding SRIF receptors and high affinity SRIF-binding sites were visualized throughout the mediobasal hypothalamus by in situ hybridization and radioligand binding studies, respectively (for review, see Ref. 12). Surprisingly, although in situ hybridization, PCR, and receptor autoradiographic and physiological experiments all concurred in reporting a major role for the sst2A receptor in transducing SRIF signals in the mammalian hypothalamus (for reviews, see Refs. 11 and 12), the distribution of sst2A receptor-expressing cells as well as the cellular localization of this receptor (pre- and/or postsynaptic) are not known within this structure. This might be attributed at least in part to the diffuse distribution of sst2A receptor immunoreactivity in the central nervous system (13, 14, 15).

With the aim of elucidating the potential role of the sst2A receptor in the transduction of SRIF’s hypothalamic actions, the distribution of this subtype was examined by highly sensitive immunohistochemical techniques using a specific antibody directed toward the C-terminal tail of the sst2A receptor protein. Because we recently demonstrated that sst2A receptors internalized in vivo in response to acute agonist injection (16), we took advantage of this property to provoke the appearance of sst2A receptor-expressing cells through the concentration of cell surface receptors into intracytoplasmic compartments and establish their regional localization. By double-labeling experiments with synaptophysin, microtubule-associated protein 2 (MAP2), and SRIF-14, we further studied the cellular localization of the sst2A receptor and its relationship with the endogenous peptide SRIF-14.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats (270–300 g body weight; Charles River, Saint Aubin les Elbeuf, France) were housed at constant temperature (21 C) and humidity (60%) with a fixed 12-h light, 12-h dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with the NIH Guide for the Care and Use of Laboratory Animals according to the principles expressed in the Declaration of Helsinki.

Antibodies
The sst2A receptor was immunolocalized using a fully characterized antiserum raised in rabbit against the C-terminal segment 330–369 of the human protein (16, 17, 18). Mouse monoclonal antibodies were used to detect MAP2 (M 4403, Sigma-Aldrich, St. Louis, MO), synaptophysin (MAB368, Chemicon, Temecula, CA), and SRIF-14 (V1169, Biomeda, Hayward, CA).

Stereotaxic injections of the sst2A agonist octreotide
Animals were deeply anesthetized with sodium pentobarbital (Sanofi Pharmaceuticals, Inc., Toulouse, France; 60 mg/kg, ip) and mounted in a stereotaxic frame. Injections were made with glass micropipettes (tip diameter, 30 µm) implanted into the lateral ventricle (coordinates: 0.8 mm posterior, 1.5 mm lateral, and 3.8 mm deep from bregma). Rats were injected with 1 µl of the sst2 receptor agonist octreotide (SMS 201-995; 1 nmol) using the procedure reported by Csaba et al. (16) (gift from Novartis Pharma AG, Basel, Switzerland; n = 12) or with the same volume of vehicle (0.9% NaCl; n = 3) at a rate of 0.16 µl/min. The micropipette was left in place for an additional 4-min period to reduce backflow. Fifteen minutes (n = 3) after vehicle injection and 10 min (n = 3), 15 min (n = 3), 60 min (n = 3), and 48 h (n = 3) after agonist injection, rats were deeply anesthetized with sodium pentobarbital and perfused through the ascending aorta with 600 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were cryoprotected in 30% sucrose in phosphate buffer (4 C, 24 h), frozen in liquid isopentane at -45 C, and sectioned at a thickness of 30 µm on a freezing microtome at the level of the hypothalamus.

Immunohistochemistry
Light microscopy.
Free-floating sections were processed for sst2A receptor and SRIF immunohistochemistry using the immunoperoxidase method with the tyramide amplification system as previously described (16). Briefly, sections were rinsed in 0.1 M Tris-buffered saline, pH 7.4 (TBS), containing 0.05% Tween 20. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 in TBS for 30 min. This was followed by a 30-min preincubation in 0.5% blocking reagent in TBS (TNB) supplied in the kit (TSA-Indirect, NEN Life Science Products, Boston, MA). Sections were then incubated overnight at room temperature in 1:4000 rabbit anti-sst2A receptor antiserum or 1:100 mouse anti-SRIF antiserum diluted in TNB. Sections were then rinsed in TBS containing 0.05% Tween 20 and sequentially incubated for 45 min in 1:300 biotinylated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) or 1:500 biotinylated donkey antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in TNB and for 45 min in avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, Inc.). Sections were subsequently incubated for 10 min in a 1:100 biotinyl tyramide solution (TSA-Indirect, NEN Life Science Products) and reincubated in avidin-biotinylated horseradish peroxidase complex for 45 min. Peroxidase activity was revealed with 0.05% 3,3'-diaminobenzidine (Sigma-Aldrich) in 0.05 M TBS, pH 7.6, in the presence of hydrogen peroxide (0.0008%). The reaction was stopped by several washes in TBS. Sections were mounted on gelatin-coated slides, dehydrated in graded ethanols, delipidated in xylene, and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA) for light microscopic observation.

Confocal microscopy.
For double-labeling experiments, serial frozen brain sections were prepared as described above. Sections were rinsed in TBS. This was followed by a 30-min preincubation in 5% normal goat serum (NGS) in TBS containing 0.3% Triton X-100. Sections were coincubated overnight at room temperature in 1:2000 rabbit anti-sst2A receptor antibody with 1:10 mouse anti-SRIF, 1:100 mouse anti-MAP2, or 1:100 mouse antisynaptophysin antibody diluted in TBS containing 0.5% NGS and 0.3% Triton X-100. Sections were then rinsed in TBS and incubated for 45 min in 1:300 biotinylated goat antirabbit IgG and 1:100 Alexa Fluor 488-conjugated goat antimouse IgG (green fluorescent signal, Molecular Probes, Inc., Eugene, OR) diluted in TBS containing 3% NGS and 0.3% Triton X-100. Finally, sections were incubated for 20 min in 1:6000 streptavidin Cy3 (red fluorescent signal, Jackson ImmunoResearch Laboratories, Inc.) diluted in TBS. Sections were then rinsed in TBS, mounted on glass slides, and coverslipped with a Vectashield solution (Vector Laboratories, Inc.). The absence of cross-reactivity between the secondary antibodies was verified by omitting one of the primary antibodies. Sections were analyzed by confocal laser scanning microscopy using a TCS SP2 confocal imaging system equipped with 488-nm argon and 543-nm helium-neon lasers (Leica Corp., Heidelberg, Germany). For each optical section, double-fluorescence images were acquired in sequential mode to avoid potential contamination by linkage specific fluorescence emission cross-talk.

To build and label the composite illustrations, Adobe Photoshop (version 6.0, Adobe Systems, Mountain View, CA) was used.

Data analysis
Regional distribution of sst2A receptor-immunoreactive neurons was evaluated throughout the hypothalamus (bregma -0.8 to -4.16) of octreotide-injected rats. Immunoreactive cells of a representative animal were plotted at six rostrocaudal levels (bregma -0.8, -1.4, -1.8, -2.56, -3.14, and -4.16) (19) on a schematic drawing using a camera lucida attached to an Olympus Corp. microscope (New Hyde Park, NY). Final schematic drawings were built and labeled using Adobe Illustrator (version 10.0, Adobe Systems).

To evaluate whether SRIF neurons also expressed the sst2A receptor, four double-labeled sections from octreotide-injected animals (taken every 120 µm) from the periventricular nucleus and the rostral, middle, and caudal parts of the arcuate nucleus were analyzed per rat under epifluorescence illumination. SRIF neurons expressing intracytoplasmic sst2A receptor-labeled granules at the level of the cell body were considered double-labeled. A total number of 1498 and 1777 SRIF-immunoreactive cells were counted in the periventricular and arcuate nuclei, respectively. Results are expressed as the mean ± SD for three animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of SRIF and sst2A receptor immunoreactivity in the hypothalamus of control rats
As a first step toward characterization of sst2A receptor distribution in hypothalamic structures, tissue sections from noninjected and saline-injected rats were processed for immunohistochemical detection of both SRIF-14 and sst2A receptor. Light microscopic analysis of sst2A receptor labeling revealed a widespread, but selective, expression of the receptor throughout the hypothalamus in both noninjected and saline-injected rats. This immunolabeling was no longer observed when the immune serum was either preadsorbed by an excess of the antigenic fusion protein or replaced by the preimmune serum. SRIF-14 immunoreactivity, as observed in sections adjacent to that processed for the sst2A receptor, was present in most hypothalamic nuclei, as previously reported (20). The regional expression of both SRIF-14 and sst2A receptor immunoreactivity was equivalent in noninjected and saline-injected rats, suggesting that saline injection did not alter the labeling and/or distribution of either marker.

In close correspondence with SRIF-14 immunoreactivity (Figs. 1Go, a–c; 2Go, a–c; and 3Go, a and b), most hypothalamic regions in saline-injected animals also displayed sst2A receptor-immunoreactive signals (Figs. 1Go, d–f; 2Go, d–f; and 3Go, c and d). However, in contrast to the sections immunostained with SRIF-14, which clearly revealed positive cell profiles, processes, and axon-like terminals, receptor immunoreactivity was diffusely distributed throughout the tissue, without any apparent association with particular cell types or cellular compartments (Figs. 1Go, d–f; 2Go, d–f, and 3Go, c and d). The only exception was the arcuate nucleus, in which few sst2A receptor-immunoreactive cells were observed (Figs. 2fGo and 3dGo). Diffuse sst2A receptor immunoreactivity was detected in the periventricular, paraventricular, lateroanterior, dorsomedial, arcuate, perifornical, medial tuberal, and premammillary nuclei. In all of these nuclei, SRIF-positive cell bodies and/or axon-like terminals were encountered. Neither SRIF-14 nor diffuse sst2A immunoreactivity conformed to the borders of classical neuroanatomical nuclei, but could often be found in areas between the well established nuclei mentioned above, i.e. medial and lateral preoptic areas; anterior, lateral, dorsal, and posterior hypothalamic areas; and tuber cinereum. The only hypothalamic regions that displayed moderate to high densities of SRIF-immunoreactive axon-like terminals, but were virtually devoid of sst2A receptor immunoreactivity, were the suprachiasmatic nucleus, dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus (Fig. 2dGo), and median eminence (Fig. 2dGo).



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  		Figure 1. Comparative distribution of SRIF (a–c) and sst2A receptor (d–i) immunoreactivity in saline-injected (a–f) and octreotide-injected (g–i) rats at the anterior level of the hypothalamus. b and c, e and f, and h and i are magnifications at the corresponding regional levels of boxed areas in a, d, and g, respectively. In saline-injected animals, the regional distribution of SRIF immunoreactivity (a) is in close correspondence with that of sst2A receptor immunoreactivity (d). At higher magnification, SRIF immunoreactivity is present in numerous nerve fibers and neuronal cells, as illustrated in the lateroanterior hypothalamic nucleus (LA; b) and the periventricular nucleus (Pe; c). By contrast, sst2A receptor immunoreactivity is homogeneously and diffusely distributed over the tissue; no immunoreactive cells or fibers are apparent (e and f). At the regional level, the distribution of sst2A receptor immunoreactivity observed in octreotide-injected animals (g) is similar to that in control animals (d). However, at the cellular level, marked changes in sst2A receptor immunoreactivity are observed (h and i). Agonist injection induces the appearance of numerous somatodendritic cell profiles, as illustrated in LA (h) and Pe (i; compare e and h, and f and i). sst2A-Cont., Saline-injected animals; sst2A-Oct., octreotide-injected animals; 3V, third ventricle; f, fornix; AHA, anterior hypothalamic area, anterior part. Scale bars: a, d, and g, 1 mm; b, e, and h, 50 µm; c, f, and i, 25 µm.

 


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  		Figure 2. Comparative distribution of SRIF (a–c) and sst2A receptor (d–i) immunoreactivity in saline-injected (a–f) and octreotide-injected (g–i) rats at the tuberal level of the hypothalamus. b and c, e and f, and h and i are magnifications at the corresponding regional levels of boxed areas in a, d, and g, respectively. In saline-injected animals, the regional distribution of SRIF immunoreactivity (a) is in close correspondence with that of sst2A receptor immunoreactivity (d). Only the ventromedial hypothalamic nucleus (VMH) and the median eminence (ME), in which SRIF immunoreactivity is present (a), are devoid of sst2A immunostaining (d). At the cellular level, SRIF immunoreactivity is confined to neuronal cells and nerve fibers, as illustrated in the dorsal hypothalamic area (DA; b) and the arcuate nucleus (Arc; c), whereas sst2A receptor immunoreactivity is diffusely distributed over the tissue in both regions (e and f). Note that in the arcuate nucleus, some sst2A-immunoreactive cells are apparent (f). At the regional level, the distribution of sst2A receptor immunoreactivity in octreotide-injected animals (g) is similar to the receptor distribution observed in saline-injected animals (d). At the cellular level, agonist injection induces the appearance of numerous somatodendritic cell profiles, as illustrated in DA (h) and Arc (i; compare e and h, and f and i). Arrows in c, f, and i point to the magnified areas shown in the corresponding panels. Stars depict immunoreactive cell bodies. sst2A-Cont., Saline-injected animals; sst2A-Oct., octreotide-injected animals; 3V, third ventricle; f, fornix. Scale bars: a, d, and g, 1 mm; b, e, and h, 100 µm; c, f, and i, 100 µm.

 


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  		Figure 3. Comparative distribution of SRIF (a–b) and sst2A receptor (c–f) immunoreactivity in saline-injected (a–d) and octreotide-injected (e and f) rats at the mammillary level of the hypothalamus. b, d, and f are magnifications of boxed areas in a, c, and e, respectively. In saline-injected animals, the regional distribution of SRIF immunoreactivity (a) is in close correspondence with that of sst2A receptor immunoreactivity (c). In the arcuate nucleus, numerous SRIF-immunoreactive neurons are apparent, embedded in an extensive network of strongly immunoreactive fibers (b). Immunoreactivity of the sst2A receptor is diffusely distributed throughout the nucleus, although some immunoreactive cell bodies are visible (d). Intracerebroventricular octreotide injection (e and f) induces the appearance of numerous sst2A-immunoreactive neurons and processes (f). Arrows in b, d, and f point to the magnified areas shown in the corresponding panels. Stars depict immunoreactive cell bodies. sst2A-Cont., Saline-injected animals; sst2A-Oct., octreotide-injected animals; 3V, third ventricle. Scale bars: a, c, and e, 1 mm; b, d, and f, 100 µm.

 
Comparison of SRIF and sst2A receptor immunoreactivity in the hypothalamus of octreotide-injected rats
Regional and cellular localization of sst2A receptor immunoreactivity after intracerebroventricular injection of octreotide.
Because the diffuse distribution of receptor immunoreactivity in control animals prevented characterization of the nature and regional localization of sst2A receptor-expressing cells in the hypothalamus, we evaluated whether acute in vivo agonist-induced receptor internalization would reveal individual cell bodies and processes. Indeed, compared with saline-injected animals (Figs. 1Go, d–f; 2Go, d–f; and 3Go, c and d), pronounced modification of the aspect of sst2A receptor immunoreactivity was observed 15 min after the administration of octreotide in the lateral ventricle (Figs. 1Go, g–i; 2Go, g–i; and 3Go, e and f). Due to intracytoplasmic accumulation of immunoreactive granules, numerous somatodendritic cell profiles displaying morphological features of neurons became apparent throughout the rostrocaudal extent of the hypothalamus, whereas the diffuse immunostaining pattern, characteristic of control animals, markedly decreased. No sst2A immunolabeling was observed over glial or endothelial cells. Immunoreactive neurons were evident as early as 10 min after octreotide injection, were strongly labeled 15 min after injection (as illustrated in Figs. 1–3GoGoGo), and were still detectable after 60 min. Forty-eight hours after agonist injection, receptor immunoreactivity was similar to that in control animals, i.e. diffusely distributed over the neuropil, indicating that the agonist effect on receptor localization was reversible.

Although sst2A-expressing cells appeared more concentrated in some hypothalamic regions than in others (Fig. 4Go), they were detected within the same nuclei and areas in which diffuse receptor immunoreactivity was visualized (see Figs. 1–3GoGoGo). The highest concentrations of sst2A-immunoreactive cells were observed in the arcuate nucleus and medial tuberal nucleus (Fig. 4Go). In the former, moderately to strongly labeled neurons were homogeneously distributed in both the rostral (Fig. 2iGo) and caudal (Fig. 3fGo) parts. In the medial tuberal nucleus, numerous cells pervaded the entire nucleus, although the ventral segment displayed the highest concentrations of sst2A receptor-expressing neurons. High densities of immunoreactive perikarya were also detected in the lateroanterior nucleus (Fig. 1Go, g and h) and in the ventrocaudal part of the tuber cinereum. A majority of nuclei and areas displayed moderate densities of immunoreactive cells, including the periventricular, medial preoptic, paraventricular, perifornical, and ventral premammillary nuclei as well as the anterior, dorsal, and posterior hypothalamic areas. Only scattered sst2A-expressing neurons were observed in other hypothalamic regions, such as the lateral and medial preoptic areas, the striohypothalamic nucleus, the rostral part of the tuber cinereum, the lateral hypothalamic area, and the terete hypothalamic and dorsal premammillary nuclei. According to the receptor distribution observed in control animals, few cells were apparent in the dorsomedial and ventromedial hypothalamic nuclei, whereas the suprachiasmatic nucleus and median eminence were devoid of immunostaining (Fig. 4Go).



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  		Figure 4. Schematic representation of the regional distribution of sst2A receptor-immunoreactive neurons in the rat hypothalamus, as revealed by intracerebroventricular octreotide injection (bregma -0.8 to -4.16; Ref. 19 ). Each dot represents a single immunoreactive neuron. Gray and black dots represent moderately and strongly immunoreactive neurons, respectively. 3V, Third ventricle; AHA, anterior hypothalamic area, anterior part; AHC, anterior hypothalamic area, central part; Arc, arcuate hypothalamic nucleus; BST, bed nucleus of the stria terminalis; DA, dorsal hypothalamic area; DM, dorsomedial hypothalamic nucleus; f, fornix; LA, lateroanterior hypothalamic nucleus; LH, lateral hypothalamic area; LPO, lateral preoptic area; ME, median eminence; MPA, medial preoptic area; MPO, medial preoptic nucleus; mt, mammillothalamic tract; MTu, medial tuberal nucleus; Pa, paraventricular hypothalamic nucleus; PaAP, paraventricular hypothalamic nucleus, anterior parvocellular part; Pe, periventricular hypothalamic nucleus; PeF, perifornical nucleus; PH, posterior hypothalamic area; PMD, premammillary nucleus, dorsal part; PMV, premammillary nucleus, ventral part; PT, paratenial thalamic nucleus; PVA, paraventricular thalamic nucleus, anterior part; SCh, suprachiasmatic nucleus; sm, stria medullaris of the thalamus; SO, supraoptic nucleus; StHy, striohypothalamic nucleus; TC, tuber cinereum area; Te, terete hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta.

 
In addition to somatodendritic cell profiles, octreotide injection triggered the appearance of numerous immunoreactive clusters and processes, which could either correspond to axon terminals or dendrites displaying punctate receptor accumulations. We therefore examined by confocal laser scanning microscopy hypothalamic sections from octreotide-injected animals double-labeled with the sst2A receptor and with either a marker of presynaptic specializations, synaptophysin, or a marker of dendritic processes, MAP2 (Fig. 5Go). Careful examination of hypothalamic nuclei and areas displaying sst2A receptor immunoreactivity revealed that synaptophysin-immunoreactive puncta closely apposed sst2A-immunopositive perikarya and fibers, but the two markers did not colocalize (Fig. 5Go, a–f). Localization of sst2A immunoreactivity in neuronal cell bodies and in proximal and distal dendrites was further confirmed by double-labeling experiments with MAP2. The sst2A-immunoreactive fibers of small diameter, not attached to their parent soma and present in most hypothalamic regions, also displayed MAP2 immunostaining (Fig. 5Go, g–l). Although we cannot exclude that a restricted number of axon terminals scattered in the hypothalamus may display sst2A receptor immunoreactivity, these results suggest that the sst2A receptor is mainly targeted to the somatodendritic neuronal compartment in the rat hypothalamus.



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  		Figure 5. The sst2A receptor immunoreactivity does not colocalize with synaptophysin (syn.) immunoreactivity (b and c, and e and f; green) but colocalizes with MAP2 immunoreactivity (h and i, and k and l; green) in the hypothalamus of octreotide-injected rats as visualized in the perifornical nucleus (a–c and g–i) and the paraventricular nucleus (d–f and j–l) by confocal microscopy. Note in a, d, g, and j that sst2A receptor immunoreactivity is confined to small particles in both hypothalamic structures. a–c and d–f, Although synaptophysin-immunoreactive puncta are found in close apposition with sst2A receptor-positive elements, the two markers are not colocalized. g–i and j–l, The sst2A receptor immunoreactivity is apparent in a subpopulation of elongated processes expressing MAP2 immunoreactivity. Scale bars, 4 µm.

 
SRIF- and sst2A receptor-expressing cells in the periventricular and arcuate nuclei.
Because the periventricular and arcuate nuclei, which displayed the highest densities of SRIF-immunoreactive cells in the hypothalamus, also displayed significant densities of sst2A receptor-expressing cells, we further investigated whether SRIF-expressing neurons may also express sst2A receptor immunoreactivity in these two regions. A similar SRIF-immunoreactive pattern was observed in noninjected, saline-injected, and octreotide-injected animals, indicating that octreotide did not induce changes in SRIF immunoreactivity. Not surprisingly, in saline-injected animals confocal microscopic analysis of double-labeled sections at the level of periventricular and arcuate nuclei did not allow us to unambiguously determine whether SRIF and sst2A receptors were localized in the same neuronal population, as SRIF-immunoreactive cells were embedded in receptor immunolabeling homogeneously distributed over the surrounding neuropil in both regions. By contrast, the appearance of somatodendritic profiles expressing sst2A receptors in response to octreotide injection allowed us to analyze whether sst2A receptor-expressing cells also displayed SRIF immunoreactivity (Figs. 6Go and 7Go). Quantitative analysis in the periventricular nucleus revealed that of 1498 SRIF-expressing neurons, 17.7 ± 2.3% were also positive for the sst2A receptor (Table 1Go). In the arcuate nucleus, of 412 SRIF-immunopositive neurons in the rostral part, 527 in the middle part, and 838 in the caudal part, 90.8 ± 1.9%, 93.5 ± 2.1%, and 95.6 ± 0.5% also expressed sst2A receptor immunostaining, respectively (Table 1Go). Taken together, these data demonstrate that the vast majority of SRIF cells in all parts of the arcuate nucleus expressed the sst2A receptor, whereas only a small proportion of periventricular SRIF neurons displayed sst2A receptor immunoreactivity. Examination of individual neurons expressing sst2A receptors under high confocal microscopic resolution revealed a close overlap between the distribution of SRIF-immunoreactive fibers and that of sst2A-positive elements in both regions. As illustrated in the periventricular nucleus, numerous SRIF-immunoreactive varicosities were seen in close apposition with sst2A-positive cell bodies and dendritic branches, expressing or not expressing SRIF (Fig. 6Go, d–i). In the arcuate nucleus, SRIF-positive neurons, which were most often immunoreactive for the sst2A receptor, were contacted by somatostatinergic axon-like terminals (Fig. 7Go, d–f). Additionally, a large number of sst2A receptor-expressing arcuate neurons that also received SRIF innervation did not display SRIF immunoreactivity.



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  		Figure 6. Comparative distribution of SRIF (a and c, d and f, and g and i; green) and sst2A receptor immunoreactivity (b and c, e and f, and h and i; red) in the periventricular nucleus of octreotide-injected rats as visualized by confocal microscopy. a–c, A subpopulation of SRIF-immunoreactive neurons also expresses sst2A receptor immunoreactivity (filled arrows in c). d–f, SRIF-immunoreactive varicosities are seen in close apposition with the soma and the dendritic process of a neuronal cell displaying both SRIF and sst2A receptor immunoreactivity (open arrows in f). g–i, The soma of a periventricular neuron displaying sst2A receptor, but not SRIF, immunoreactivity is surrounded by SRIF-immunoreactive fibers. n, Nucleus. Scale bars: a–c, 20 µm; d–f, 8 µm; g–i, 2 µm.

 


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  		Figure 7. Comparative distribution of SRIF (a and c, and d and f; green) and sst2A receptor immunoreactivity (b and c, and e and f; red) in the arcuate nucleus of octreotide-injected rats as visualized by confocal microscopy. a–c, SRIF-immunoreactive neurons are also positive for sst2A receptor immunoreactivity (filled arrows in c). d–f, SRIF-immunoreactive varicosities are seen in close apposition with the soma of an arcuate neuron displaying both SRIF and sst2A receptor immunoreactivity (open arrows in f). n, Nucleus. Scale bars: a–c, 16 µm; d–f, 2 µm.

 

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  		Table 1. Estimated percentage of SRIF-immunoreactive neurons expressing sst2A receptor immunoreactivity in the periventricular and arcuate hypothalamic nuclei

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Within the hypothalamus SRIF has long been known to influence the neuroendocrine axes regulating metabolism, reproductive functions, adaptative responses to stress, and growth (10, 11). Our further understanding of the multiple neuroendocrine actions of SRIF in this structure and its implications for potential therapeutic developments awaits mapping of SRIF receptor subtypes. Surprisingly, to date studies of receptor protein expression reported only a restricted distribution of SRIF receptors in the hypothalamus. Thus, sst1 receptor immunoreactivity appears confined to a subpopulation of SRIF neurons in the periventricular nucleus and SRIF fibers in the median eminence (21); sst3 receptor expression is predominant in the ventromedial hypothalamic nucleus in neuronal cilia (22), for which a physiological function is not well established; and the sst4 (23) and sst5 (24) subtypes do not display significant immunoreactive signals in hypothalamic regions. Based on these data and on the fact that mRNAs encoding the sst2A, but not the sst2B, subtype are broadly distributed in the rat hypothalamus (8, 25), one can hypothesize that the sst2A receptor plays a key role in transducing SRIF actions in this structure. The present study strengthens this assumption by several findings: 1) sst2A receptor protein is present in most hypothalamic nuclei and areas, and its distribution correlates well with that of SRIF-immunoreactive terminal fields; 2) the cellular distribution of the sst2A receptor is regulated by agonist stimulation in vivo; 3) the sst2A receptor is in position to regulate the activity of arcuate SRIF neurons; and 4) the distribution of sst2A-expressing cells is in keeping with studies demonstrating the potency of sst2 agonists to induce hypothalamic physiological responses (26, 27, 28, 29).

The regional distribution of sst2A receptor immunoreactivity in the hypothalamus of control animals conforms remarkably closely to that of SRIF-binding sites previously documented by autoradiography using the sst2-preferring ligand [125I]Tyr3 octreotide ([125I]SMS 204–090) and so-called universal radioactive ligands such as [125I]Tyr11-SRIF-14, [125I]Leu8-D-Trp22Tyr25-SRIF-28, and [125I]Tyr0-D-Trp8-SRIF-14, which bind to all six SRIF receptor subtypes (for reviews, see Refs. 12 and 30). Thus, diffuse sst2A-immunoreactive signals were encountered in the periventricular, paraventricular, lateroanterior, dorsomedial, arcuate, perifornical, medial tuberal, and premammillary nuclei as well as in the preoptic, anterior, lateral, dorsal, and posterior hypothalamic areas, all of which have been shown to contain SRIF-binding sites. The congruence between the present results and those reported by earlier autoradiographic studies together with the fact that the hypothalamic distribution of sst1, sst3, sst4, and sst5 receptor proteins appeared very restricted strongly suggest that a very high proportion of hypothalamic SRIF-binding sites corresponds to the sst2A receptor subtype. Because SRIF radioactive ligands preferentially bind to membrane-associated, as opposed to intracellular, sst2A receptors (14), the close correspondence between diffuse sst2A immunostaining and SRIF-binding sites in the hypothalamus suggests that sst2A receptors are mainly localized at the plasma membrane, as previously demonstrated in other brain regions such as the claustrum (14) and cerebral cortex (16).

Although the distribution of sst2A receptor immunoreactivity was diffuse throughout the hypothalamus, a significant number of somatodendritic cell profiles displaying intracellular sst2A immunolabeling were apparent in the rostral, middle, and caudal parts of the arcuate nucleus, a region that displayed high densities of SRIF axon terminals. This suggests that under physiological conditions SRIF released in the arcuate nucleus can trigger translocation of cell surface receptors into intracellular compartments, as previously proposed for other brain areas (14). Intracellular localization of sst2A receptors was only encountered in the arcuate nucleus, which might indicate that the release of SRIF is higher in this particular region than in other hypothalamic areas.

Agonist-dependent regulation of the cellular localization of the sst2A receptor in the hypothalamus in vivo was further confirmed by intracerebroventricular injection of the sst2A agonist octreotide. The appearance of somatodendritic profiles was the consequence of intracytoplasmic accumulation of sst2A-immunoreactive granules. This feature is likely to reflect activation and subsequent internalization of cell surface receptors in endosomal organelles, as previously documented in vitro in different cell lines (for review, see Ref. 9) and in vivo in the parietal cortex (16). Immunoreactive cells were clearly apparent shortly after intracerebroventricular agonist injection throughout the hypothalamus, supporting the utility of this approach to unravel the nature, distribution, and chemical identity of cell populations expressing the sst2A receptor. Because the appearance of sst2A receptor-immunoreactive neurons was evident shortly (i.e. 15 min) after agonist stimulation, a time window that appears not to be compatible with sst2A protein neosynthesis, this effect is likely to be due to redistribution of receptors into intracellular compartments. Distribution of sst2A receptor-immunoreactive cells was remarkably comparable to that of neurons previously found to express sst2 mRNA (25), suggesting that the sst2A receptor is mainly targeted to the somatodendritic compartment. The extensive colocalization of sst2A receptor with MAP2 and the lack of colocalization with synaptophysin found in the present study further suggest that the sst2A receptor is mainly in position to transduce the postsynaptic actions of SRIF in the hypothalamus. According to this observation, electrophysiological recordings of rodent hypothalamic neurons in primary cultures have demonstrated that selective activation of the sst2A receptor induced a decrease in glutamate sensitivity through postsynaptic mechanisms (31).

Of interest, we found that more than 90% of SRIF neurons in the arcuate nucleus also displayed sst2A receptor immunoreactivity. SRIF-containing neurons present in this nucleus are not neurosecretory neurons and are considered the main source for the dense networks of SRIF fibers innervating this nucleus. Accordingly, double-labeled neurons were highly innervated by SRIF fibers, indicating that the sst2A receptor is in a position to serve as a postsynaptic autoreceptor. A large number of sst2A-positive, but SRIF-negative, neurons were also encountered throughout the rostrocaudal extent of the nucleus, implying that besides its autoreceptor function, the sst2A receptor can also modulate nonsomatostatinergic neurons. Through the mediation of local somatostatinergic neurotransmission, sst2A auto- and heteroreceptors are therefore likely to play an important modulatory role in arcuate neuronal populations expressing or coexpressing neuropeptides involved in the hypothalamic regulation of GH secretion and/or food intake, including SRIF itself, GHRH, galanin, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, neuropeptide Y, and agouti-related protein (for reviews, see Refs. 10 , 32 , and 33). In contrast to the arcuate nucleus, only a small proportion of SRIF-expressing neurons in the periventricular nucleus were also found to express the sst2A receptor (18%). The vast majority (78%) of SRIF neurons in this nucleus project directly to the median eminence (34). Although it remains to be established whether SRIF neurons bearing the sst2A receptor are hypophysiotropic, our results together with the study by Helboe et al. (21) suggest that the sst1, rather than the sst2A, receptor is implicated in the regulation of SRIF secretion into hypophysial portal blood.

The present findings further provide a neuroanatomical substrate to previous physiological studies demonstrating that both acute and chronic in vivo treatments with the sst2A agonist octreotide modulate cell signaling in the arcuate nucleus. Thus, acute systemic administration of GH secretagogues, a class of synthetic compounds exerting potent GH-releasing activity in multiple species, including humans, has been shown to activate a subpopulation of arcuate neurons and induce c-fos expression (35, 36, 37). This latter effect was counteracted by acute intracerebroventricular and ip injection of octreotide in rats (26) and mice (29), respectively. Interestingly, the suppression of GH secretagogue-induced c-fos activation did not occur after octreotide injection in mice lacking the sst2 receptor subtype (29). In keeping with the high density of sst2A-expressing cells in the arcuate nucleus, chronic neonatal octreotide treatment was shown to provoke a permanent decrease in both GHRH and SRIF mRNA levels in the arcuate nucleus of adult rats (27). Chronic treatment with octreotide, injected through intracardiac cannula in adult rats, was also recently demonstrated to increase the expression of sst2 mRNA in neurons of the arcuate nucleus, reflecting homologous regulatory mechanisms (28). Together with the present demonstration of high expression of the sst2A receptor protein in the arcuate nucleus, these studies argue for an important role for this receptor type in mediating the effects of SRIF on arcuate neuronal activity and gene expression.

In summary, we have demonstrated that the sst2A receptor protein is diffusely distributed in most hypothalamic structures, in close correspondence with the extensive network of SRIF terminals and fibers. Through in vivo receptor activation we have provided further evidence that sst2A receptor-immunoreactive neurons are widely, but selectively, distributed in the hypothalamus, and that this particular receptor subtype might play an important role in the somatostatinergic regulation of endocrine and metabolic processes. Defining neuronal systems that bear the sst2A receptor will represent an intriguing issue for future studies. Together with the results of the present study this will provide novel clues to unravel physiological SRIF functions in the mammalian hypothalamus.


   Acknowledgments
 
We thank Dr. D. Hoyer (Novartis Pharma AG, Basel, Switzerland) for providing the sst2 receptor agonist octreotide (SMS 201-995). We are indebted to B. Lelouvier for his help with cell mapping.


   Footnotes
 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, European Community Contract QLG-1999-0098, and a French postdoctoral fellowship from the Ministère de la Recherche (to Z.C.).

Abbreviations: MAP2, Microtubule-associated protein 2; NGS, normal goat serum; SRIF, somatostatin; TBS, Tris-buffered saline; TNB, blocking reagent in Tris-buffered saline.

Received October 21, 2002.

Accepted for publication December 30, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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