In Vivo Internalization of the Somatostatin sst2A Receptor in Rat Brain: Evidence for Translocation of Cell-Surface Receptors into the Endosomal Recycling Pathway

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Molecular and Cellular Neuroscience 17, 646 – 661 (2001) doi:10.1006/mcne.2000.0958, available online at http://www.idealibrary.com on

In Vivo Internalization of the Somatostatin sst2A Receptor in Rat Brain: Evidence for Translocation of Cell-Surface Receptors into the Endosomal Recycling Pathway Zsolt Csaba,* Ve´ronique Bernard, † Lone Helboe, ‡ Marie-The´re`se Bluet-Pajot,* Bertrand Bloch, † Jacques Epelbaum,* and Pascal Dournaud* *INSERM U549, IFR Broca-Sainte Anne, Centre Paul Broca, 75014 Paris, France; †CNRS UMR 5541, Laboratoire d’Histologie-Embryologie, Universite´ Victor Se´galen-Bordeaux 2, 33076 Bordeaux Cedex, France; and ‡Department of Medical Anatomy, Panum Institut, University of Copenhagen, DK-2200 Copenhagen, Denmark

To determine whether cellular compartmentalization of somatostatin receptors can be regulated in vivo, we examined the immunocytochemical distribution of the sst2A receptor (sst2AR) after stereotaxical injections of somatostatin analogs into the rat parietal cortex. Whereas CH275, a sst1R agonist, failed to induce changes in the diffuse sst2AR immunostaining pattern characteristic of control animals, somatodendritic profiles displaying intracytoplasmic immunoreactive granules became apparent short-term after injection of either somatostatin or the sst2R agonist octreotide. Confocal microscopy revealed that 90% of sst2AR-immunoreactive endosome-like organelles displayed transferrin receptor immunoreactivity. At the electron microscopic level, the percentage of sst2AR immunoparticles dramatically decreased at the plasmalemma of perikarya and dendrites after octreotide injection. Conversely, it significantly increased in endosomes-like organelles. These results demonstrate that sst2ARs undergo, in vivo, rapid and massive internalization into the endocytic recycling compartment in response to acute agonist stimulation and provide important clues toward elucidating somatostatin receptor signaling in the mammalian brain.

INTRODUCTION Somatostatin (SRIF) was originally identified as a cyclic 14-amino-acid peptide in hypothalamic extracts (Brazeau et al., 1973). A second bioactive form with an N-terminal extension of 14 amino acids, SRIF-28, was later found in the gut (Pradayrol et al., 1980). Recently,

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further complexity of the somatostatinergic system has been elucidated by the molecular cloning of a new brain-selective cDNA encoding cortistatins (CST), 29and 14-amino-acid neuropeptides, which exhibit strong structural similarity with SRIF (de Lecea et al., 1996). In the central nervous system (CNS), SRIF neuronal pathways are widespread but particularly abundant in limbic structures (Johansson et al., 1984), whereas CST expressing neurons are mostly restricted to the neocortex and the hippocampal formation (de Lecea et al., 1997). Perturbation of somatostatinergic transmission has been demonstrated in several neurological disorders (Epelbaum et al., 1988; Mouradian et al., 1991; Fox et al., 1997; Vezzani and Hoyer, 1999). Somatostatin and cortistatin peptides bind with high affinity to a family of G protein-coupled receptors (GPCR) encoded by five individual genes (sst1–sst5). Six receptor proteins have been studied so far, since the sst2 gene generates two splice variants, sst2A and sst2B (Vanetti et al., 1993). Following their pharmacological characterization, effort has been undertaken to elucidate the individual functions of somatostatin receptors in the mammalian CNS. Although the recent localization of the different proteins in the brain (reviewed in Dournaud et al., 2000; see also Handel et al., 1999; Schreff et al., 2000) has provided new insights into SRIF neuronal signaling, far less is known about the cellular regulation of individual receptors upon agonist stimulation. In vitro experiments using recombinant receptors have provided evidence that some (Hukovic et al., 1996; 1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

In Vivo Internalization of the sst2A Receptor

Nouel et al., 1997; Roosterman et al., 1997; Roth et al., 1997; Stroh et al., 2000), but not all (Roosterman et al., 1997; Roth et al., 1997; Kreienkamp et al., 1998) SRIF receptor subtypes undergo subcellular redistribution after ligand binding. In particular, the sst2A receptor (sst2AR) subtype, which is likely to account for an important facet of somatostatinergic neurotransmission (Dournaud et al., 1996, 1998), rapidly and efficiently internalizes in various cell lines (Hukovic et al., 1996; Nouel et al., 1997; Roosterman et al., 1997; Roth et al., 1997). As for other GPCRs (Koenig and Edwardson, 1997), this process has been suggested to play an important role for the recruitment of alternative signaling pathways (Sarret et al., 1999; Boudin et al., 2000) and for receptor desensitization (Hipkin et al., 1997, 2000). Taken together, such in vitro observations suggest that changes in cellular sst2A receptor compartmentalization may be of critical importance for SRIF signaling in vivo, in a physiological context. In keeping with such an hypothesis, we recently demonstrated that cerebral regions receiving a dense SRIF innervation display low proportions of plasma membrane-associated vs intracellular sst2A receptors, as compared to poorly SRIF innervated regions (Dournaud et al., 1998). We thus hypothesized that endogenous SRIF release induces cell-surface receptor internalization. To test this assumption and to further delineate intracellular sst2AR pathways in vivo, the cellular distribution of the sst2AR was studied by immunocytochemistry following agonist injection in the rat parietal cortex. This region was selected because of its dense but diffuse receptor immunostaining, which is likely to reflect predominance of plasma membrane-associated receptors (Dournaud et al., 1998).

RESULTS Regional and Cellular Distribution of the sst2AR Immunostaining after Somatostatin Agonist Injections To assess the impact of agonist activation of the sst2A receptor on its regional and cellular distribution, SRIF-14 and the selective agonist octreotide (SMS201995, Bauer et al., 1982) for the sst2AR (Hoyer et al., 1994; Patel, 1997) were stereotaxically injected in the parietal cortex (Fig. 1), an area previously shown to display dense and homogeneously distributed sst2AR immunoreactive signals (Dournaud et al., 1996, 1998; Schindler et al., 1997). Control animals were injected

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FIG. 1. Regional distribution of sst2AR immunoreactive pattern illustrated in a coronal rat brain section (left panel). Right panel depicts the anatomical landmarks at the corresponding level. Sst2AR immunolabeling predominates in the deep layers of the cortex, dorsal endopiriform nucleus (DEn), basolateral amygdaloid nucleus (BL), medial habenula (MHb), and in the CA1-2 area and dentate gyrus (DG) of the hippocampal formation. Site of injections in the parietal cortex (Par) is indicated by arrows. 1– 6, cortical layers.

with either saline or the sst1 receptor selective ligand, CH-275 (Liapakis et al., 1996). Light microscopic examination of the sst2AR immunoreactivity in nontreated animals (Fig. 1) revealed the same regional distribution and immunostaining patterns as reported previously (Dournaud et al., 1996, 1998; Schindler et al., 1997). Immunoreactivity was totally abolished when the immune serum was either preadsorbed by an excess of the antigenic fusion protein or replaced by the preimmune serum, demonstrating the specificity of the labeling (data not shown). In the parietal cortex of noninjected animals, sst2AR immunostaining was diffusely distributed in the deeper layers but enriched in the upper part of the layer V and the deeper part of the layer VI (Fig. 2A). Somatodendritic immunoreactive profiles were only occasionally apparent, scattered in the diffuse sst2AR staining. Neither saline (Fig. 2B) nor CH-275 (Fig. 2C) injections induced changes in the sst2AR immunostaining pattern. By contrast, 15 min after the start of injection of 0.5 fmol, 0.5 pmol, or 0.5 nmol of either SRIF-14 or octreotide (Fig. 2D–2F), pronounced modifications of the sst2A receptor distribution were observed. Depending upon the sst2R agonist concentration, numerous immunoreactive cells became apparent surrounding the injection point, while a decrease in the intensity of the diffuse immunostaining was observed. High magnification microscopic examination of the

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FIG. 2. Light microscopic distribution of sst2AR immunoreactivity in the parietal cortex of saline- and somatostatin agonist-injected rats. (A) In noninjected cortices (Non-inj.) sst2AR immunostaining is diffusely distributed over the neuropil. (B, C) Neither physiological saline (NaCl) (B) nor sst1R agonist CH-275 (0.5 nmol) (C) induce change in the sst2AR immunolabeling pattern. (D–F) Fifteen minutes after intracortical injection of 0.5 fmol (D), 0.5 pmol (E), or 0.5 nmol (F) sst2R agonist octreotide (Oct), numerous immunoreactive cells become apparent around the injection site. Note that the number of immunoreactive cells correlates with octreotide concentrations. Magnified frames (bottom left) show details of the sst2AR immunostaining in control and treated rats. Scale bars, 200 ␮m.

sst2AR labeling in control animals revealed a dense and homogeneous immunoreactive network distributed over the neuropil, outlining the periphery of numerous cell bodies and processes (Fig. 3A). In sst2R agonistinjected animals, this diffuse immunoreactive pattern was no longer apparent. The sst2AR immunoreactivity was predominantly located in intracytoplasmic granules in nerve cell bodies and proximal dendrites, reminiscent of endosomal organelles (Figs. 3B–3D). Although most prominent in perikarya, sst2AR labeling was evident over multiple cross-sectioned neuronal processes throughout the neuropil.

Quantitative analysis demonstrated that the number of sst2AR-labeled somatodendritic profiles increased significantly in animals injected with 0.5 fmol, 0.5 pmol, 0.5 nmol of either SRIF-14 or octreotide as compared to noninjected, saline-injected, or CH-275-injected animals (Fig. 4). Somatodendritic profiles with immunoreactive foci of intracytoplasmic granules were apparent as early as 5 min after the start of 0.5 pmol octreotide injection and still detectable after 3 h (data not shown). Six and 24 h after 0.5 pmol injection of octreotide, the sst2AR immunostaining was similar to that of control animals, i.e., diffusely distributed throughout the neuropil (data

In Vivo Internalization of the sst2A Receptor

FIG. 3. Cellular distribution of sst2AR immunoreactivity in the parietal cortex of saline and octreotide-injected rats. (A) In a physiological saline (NaCl)-injected cortex the sst2AR labeling is densely distributed over the neuropil, outlining the periphery of cell bodies (asterisks). (B–D) Fifteen minutes after injection of 0.5 pmol octreotide (Oct) intensely immunopositive neurons become evident. Note that immunoreactive signals are predominantly associated with intracytoplasmic granules in somata and dendrites (arrows). Scale bars, 20 ␮m.

649 munolabeling, on the basis of the light microscopic experiments described above. In control animals, TfR immunoreactivity was detected in most, if not all, cortical neurons as well as in brain capillaries in agreement with previous immunocytochemical studies (Hill et al., 1985; Keith et al., 1998). Serial optical sectioning at high magnification demonstrated that the bulk of the sst2AR immunostaining remained localized at the periphery of the cell (Fig. 5A), presumably at the plasma membrane, whereas the TfR immunoreactivity was confined to small intracytoplasmic granules (Fig. 5B). Examination of overlaid images revealed little or no overlap of the two markers (Fig. 5C), indicating that sst2ARs are distributed in a cellular compartment different from the TfR one in control animals. Fifteen minutes after the octreotide injection, profound modifications of the sst2AR localization were observed (Figs. 5D, 5G, and 5J), whereas the TfR immunoreactive distribution remained unchanged (Figs. 5E, 5H, and 5K). The sst2AR immunoreactive rim present at the periphery of the cells was no longer observed and bright punctate accumulations of the antigen appeared within the perikarya, sparing the nucleus (Figs. 5D, 5G,

not shown). According to their size, shape, and laminar distribution, the bulk of sst2AR immunoreactive profiles revealed by the sst2R agonist injection is likely to correspond to pyramidal cells in the layer V and to interneurons in layer VI. Subcellular Distribution of the sst2AR Immunostaining after Treatment by Octreotide Confocal microscopy. As a first step toward the characterization of the intracytoplasmic pools expressing sst2A receptors after agonist treatment, we examined by confocal laser scanning microscopy whether these vesicular structures were also immunopositive for the transferrin receptor (TfR), a marker of early endosomes. Double-labeling experiments were conducted on cortical sections from animals injected in the parietal cortex with either saline or 0.5 pmol octreotide, and sacrified 15 min after the start of the injection. Time and agonist concentration were selected to obtain significant but short-term accumulation of intracellular sst2AR im-

FIG. 4. Quantitative analysis of sst2AR immunoreactive somatodendritic profiles in layers V–VI of the parietal cortex in saline- and somatostatin agonist-injected rats. Neurons displaying intracellular immunostaining were counted in 30-␮m coronal sections, on every other section taken within 300 ␮m of the injection point. Sections from nontreated animals were also analyzed. Results are expressed as mean ⫾ SEM. Statistical analysis (nonparametric Mann–Whitney U test) reveals that neither saline (NaCl) nor CH-275 injection induced significant changes as compared to nontreated animals (Non-inj.) (P ⬎ 0.05). The number of somatodendritic profiles significantly increased in animals injected with 0.5 fmol, 0.5 pmol, 0.5 nmol of either SRIF-14 or octreotide (Oct) as compared to noninjected animals and saline- or CH-275-injected animals. (***P ⬍ 0.0001).

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FIG. 5. Internalized sst2ARs colocalize with the transferrin receptor (TfR). Confocal microscopic sections of cells from layer VI of the parietal cortex illustrating the localization of sst2AR (green) and TfR (red) in control (A–C) and 0.5 pmol octreotide-injected (D–L) rats. (A) In control animals, the sst2AR immunolabeling is observed at the periphery of the cell. (B) TfR immunoreactivity is confined to small intracytoplasmic granules. (C) Merged image of A and B reveals little overlap of the two signals. (D, G, J) In octreotide-injected rats sst2AR immunoreactivity is confined to small spherical particles within the perikarya and proximal dendrites, sparing the nucleus. (E, H, K) No obvious change in the TfR immunoreactivity is observed in octreotide-injected cortices. (F, I, L) Merged optical sections of D, G, J and E, H, K illustrates the colocalization of sst2AR with TfR (yellow signal, arrows). Note that a subpopulation of TfR immunoreactive organelles does not display sst2AR immunolabeling. Scale bars, 5 ␮m.

and 5J). Double-labeling experiments demonstrated that intracellular sst2AR labeling was almost completely colocalized with TfR immunolabeling, as shown by the extensive yellow fluorescence staining present in overlaid images as compared to the green sst2AR one (Figs. 5F, 5I, and 5L). Yellow fluorescence signals were distributed in the cytoplasm of cell bodies and dendrites, with local accumulation in the perinuclear region. Quantitative analysis revealed that 90% of

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sst2AR-positive organelles were also immunoreactive for the TfR, indicating that large quantities of sst2ARs were internalized in TfR-positive endosomes upon octreotide stimulation. Sixty percent of intracytoplasmic TfR expressing organelles also displayed sst2A receptor immunostaining. To evaluate further the spatial distribution of sst2ARlabeled endosomes in octreotide-treated cortical neurons, we realized three-dimensional reconstruction of high resolution scans from immunoreacted sections with the sst2AR antibody. Image analysis revealed pleiomorphic endocytic organelles although high number of spherical particles were apparent throughout the cells (Fig. 6). Numerous tubular structures extending over a distance of 1 ␮m were also visualized with this technique, according to 2-D scanned images, which often display a continuum of immunofluorescence signals over several adjacent sections. Immunoreactive organelles were evenly distributed throughout the cell body in the form of a corona around the nucleus. Dense foci of sst2AR immunostaining were also apparent in the core of proximal and cross sectioned dendritic processes (Fig. 6). Electron microscopy. To assess the short-term effect of the SRIF agonist octreotide on the subcellular compartmentalization of the sst2AR, preembedding immunogold immunocytochemical experiments were performed in the parietal cortex (layer VI) of animals sacrified 15 min after the start of injection of either saline or 0.5 pmol octreotide. In both control and agonist-treated animals, gold particles visualized at the electron microscopic level were predominantly associated with neuronal cell bodies and dendritic processes. Only occasionally were particles detected in axons and axon terminals. Neither glial nor endothelial vascular cells displayed any significant immunogold staining. Within the neuronal perikarya of control animals, a majority of immunoparticles was detected at the plasma membrane, outlining the internal side (Fig. 7), in agreement with the localization of the epitope included in the fusion protein. These gold particles were usually detected at extrasynaptic sites and represented 35.6% of the total number of immunoparticles (Fig. 8A). The remaining particles were detected in the cytoplasm in association with endosome-like organelles (8.5%), the Golgi network (20.6%), the endoplasmic reticulum (5.4%), and the nuclear membrane (3.3%) (Fig. 8A). Some immunoparticles could not be associated with one of the previous subcellular compartments (26.6%) (Fig. 8A). Fifteen minutes after intracortical injection of 0.5 pmol octreotide a marked change in the distribution of

In Vivo Internalization of the sst2A Receptor

FIG. 6. Somatodendritic distribution of internalized sst2ARs as visualized by confocal microscopic three dimensional reconstruction. Focal images were obtained and reconstructed from two cells of rats injected with 0.5 pmol octreotide. Note that in both cells immunoreactive signal is confined to numerous pleiomorphic and spherical structures throughout the cell, which form a corona around the nucleus and extend into the core of proximal dendrites.

the immunogold labeling between the plasma membrane and the cytoplasm was observed. The localization of sst2ARs at the plasma membrane, characteristic of

651 control conditions, was no longer apparent in octreotide-injected animals (Fig. 9). By contrast, numerous immunolabeled receptors became evident in the cytoplasmic compartment, associated with tubular and vesicular organelles resembling endosomes (Fig. 9). Gold particles were mainly associated with the outer side of the membrane of the endosomes although some particles were evident inside. Immunoreactive endosomes were evenly distributed throughout the cytoplasm (Fig. 10), with local accumulation in the subplasmalemma zone and in the vicinity of Golgi cisternae and nuclear membranes. Multivesicular bodies never display significant sst2AR immunostaining. Quantitative analysis demonstrated a significant decrease of the density of immunoparticles associated with the plasmalemma (⫺75%, P ⬍ 0.0001) in cells exposed to octreotide, together with a strong and significant increase of the number of gold particles associated with endosome-like organelles (⫹295%, P ⬍ 0.0001) (Fig. 8B), thus reflecting an intense movement of receptors from the plasma membrane to intracytoplasmic compartments. The frequency of gold particles also significantly increased in Golgi-like vesicles (⫹45%, P ⬍ 0.01) (Fig. 8B). Because vesicles budding from the Golgi network are morphologically indistinguishable from endosomal structures close to it (Pagano et al., 1989), this change can either result from increasing sst2AR immunoreactivity associated with the Golgi apparatus per se or with vesicles belonging to the endosomal compartment. By contrast, there was no change in the number of immunoparticles associated with the endoplasmic reticulum, nuclear membrane, and unidentified compartment (Fig. 8B). In dendrites of control animals, 75% of the total number of particles were localized at the plasma membrane, associated with its inner side (Figs. 11 and 12A). Quantitative analysis revealed that the average number of immunogold particles per micrometer plasma membrane length was not statistically higher in dendrites than in cell bodies (2.53 ⫾ 0.20 vs 2.17 ⫾ 0.17, P ⬎ 0.05), suggesting that the density of cell-surface receptors is comparable between these two compartments. The remaining 25% was divided between endosome-like vesicles (16%) and unidentified compartments (9%) (Fig. 12A). Fifteen minutes after octreotide injection, the vast majority of immunoparticles was detected intracellularly (Fig. 13). Receptors associated with the plasma membrane were rare. Quantitative analysis demonstrated that the density of immunoparticles associated with the plasma membrane decreased by 79% (P ⬍ 0.0001) (Fig. 12B). Conversely, the number of sst2ARs associated with endosome-like vesicles massively in-

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FIG. 7. Illustration of the subcellular distribution of sst2AR immunoreactivity in neuronal cell bodies of control rats. (A–E) A large number of gold particles are localized at the plasma membrane, in association with its inner side (arrowheads). Some immunolabeled sst2A receptors are detected in association with endosome-like vesicles (double arrows). Note in C, that a dentritic spine (ds) displays several gold particles at the plasma membrane. In E, arrows and arrowheads point to immunoparticles associated with the internal surface of the plasma membrane of a dendrite and a perikaryon, respectively. er, endoplasmic reticulum; n, nucleus. Scale bars: (A–D) 0.5 ␮m; (E) 0.2 ␮m.

creased (⫹651%, P ⬍ 0.0001) (Fig. 12B). The average number of immunoparticles associated with unidentified compartments remained unchanged as compared to saline-injected animals (Fig. 12B). No statistical differences were observed between the average number of immunogold particles per cell and dendritic profile in saline-injected animals (235 ⫾ 17 and 16 ⫾ 1, respectively) and octreotide-treated animals (222 ⫾ 18 and 17 ⫾ 1, respectively; P ⬎ 0.05 as compared to their respective controls).

DISCUSSION The present study provides a comprehensive analysis of the cellular and subcellular neuronal distribution of the somatostatin sst2A receptor upon agonist stimulation in vivo. As reported previously, sst2AR immunostaining in control rats appeared diffusely distributed over the layers V and VI of the parietal cortex when observed by light microscopy (Dournaud et al., 1996,

1998; Schindler et al., 1997). At the subcellular level, electron microscopic immunogold cytochemistry revealed that the majority of gold immunoparticles were associated with the plasma membrane of cell bodies and dendrites. Because the number of particles per plasma membrane length (i.e., density) was not different between perikarya and dendrites, our results suggest that the vast majority of the sst2ARs are plasma membrane-associated in the parietal cortex under physiological conditions. A striking finding of the present study was that intracerebral injections of either the native peptide SRIF-14 or the sst2R-preferring ligand octreotide triggered profound modifications of the sst2AR localization. Activation of sst2ARs provoked a massive redistribution of cell-surface receptors into endosomes-like vesicles as assessed by electron microscopy, resulting in redistribution of the immunostaining from diffuse to somatodendritic, at the light microscopic level. Neither sst1R agonist CH-275 nor saline injections induced endocytosis of sst2ARs, which demonstrates that internal-

In Vivo Internalization of the sst2A Receptor

FIG. 8. Quantitative analysis of the subcellular distribution of sst2ARs in perikarya of control (NaCl) and 0.5 pmol octreotideinjected (Oct) rats using preembedding immunogold method. (A) Proportion of immunoparticles associated with different subcellular neuronal compartments in saline-injected animals. For each analyzed neuron, immunoparticles were identified and counted in association with six subcellular compartments. The proportion of particles associated with each compartment was calculated in relation to the total number of particles. The largest portion of immunoparticles is associated with the plasma membrane (35.6%). In the cytoplasm, immunoparticles are detected mostly in association with Golgi-like vesicles (Golgi) (20.6%). A smaller proportion of immunoparticles is associated with endosome-like vesicles (8.5%), endoplasmic reticulum (er) (5.4%), and the outer nuclear membrane (3.3%). A portion of immunoparticles is found not to be associated with any identified compartment (unidentified compartment) (26.6%). (B) Effect of 0.5 pmol octreotide injection on the localization of the sst2AR immunoparticles in cortical neurons. For each analyzed neuron, the number of immunoparticles associated with each compartment was counted in relation to the membrane length (in micrometers) for the plasma and nuclear membrane, and to the surface of cytoplasm (in square micrometers) for endosome-like vesicles, Golgi-like vesicles, endoplasmic reticulum, and the unidentified compartment. Results are expressed in relation to an arbitrary unit (100%) of the control values. Statistical analysis (nonparametric Mann–Whitney U test) shows that the density of immunoparticles significantly decreases at the plasma membrane (***P ⬍ 0.0001) and increases in endosome-like (***P ⬍ 0.0001) and Golgi-like (**P ⬍ 0.01) vesicles. NS, nonsignificant.

ization of plasma membrane-bound receptors was strictly dependent upon agonist activation. Moreover, the number of somatodendritic profiles was positively

653 correlated with somatostatin and octreotide concentrations, which further indicated that sst2ARs internalization process is agonist dependent. Interestingly, the native peptide SRIF-14 was as potent as the long-acting analog octreotide (Bauer et al., 1982) to provoke translocation of cell-surface receptors in intracytoplasmic pools, suggesting that short-term action of SRIF-14 is not affected by endogenous enzymatic activity. Colocalization experiments with the transferrin receptor allowed us to gain insights into the nature of the endocytic pathway used by internalized sst2ARs in cortical neurons. The TfR, a recycling membrane protein, is a well characterized marker of the early endocytic compartment which is composed of sorting and recycling endosomes (Mukherjee et al., 1997). Studies in living cells have demonstrated that nearly all TfR molecules are excluded from the late endosomal compartment, which is involved in the breakdown of internalized cargo (Gruenberg and Maxfield, 1995). In agreement with Keith et al. (1998), our confocal experiments revealed that TfR immunoreactivity was confined to intracytoplasmic vesicles in cortical cells suggesting that in neurons, like in other cell types (Ghosh et al., 1994; Ghosh and Maxfield, 1995), TfRs enter the recycling compartment more rapidly than leaving it, so that at steady state the majority of the immunostaining is concentrated in endosomes. Examination of the distribution of sst2ARs, 15 min after agonist injection, revealed that virtually all the sst2AR immunolabeling colocalized with TfR expressing organelles, suggesting that endocytosed sst2ARs entered massively the recycling pathway. The presence of numerous sst2AR immunoreactive tubulovesicular structures in proximal and extended dendrites, as revealed by 3-D reconstruction of confocal series, is in agreement with previous studies that suggested the existence of an extensive early endosomal network in this neuronal compartment (Parton et al., 1992; Mundigl et al., 1993; Prekeris et al., 1999). At the electron microscopic level, intracytoplasmic immunogold particles were associated mainly with tubulovesicular structures, which have the morphologic features of early endosomes (Mukherjee et al., 1997). Together with the absence of significant amount of sst2ARs associated with multivesicular bodies, such observations are also in keeping with the localization of internalized sst2ARs within the early endosomal compartment. Moreover, quantitative analysis revealed a significant increase of the number of immunogold particles associated with Golgi-like vesicles. Given the lack of increased labeling associated with the endoplasmic reticulum, and the time required for new protein synthesis, increased Golgi network labeling is likely to reflect

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FIG. 9. Illustration of the subcellular distribution of sst2AR immunoreactivity in cell bodies of 0.5 pmol octreotide-injected rats. (A–C) Immunoparticles are mainly found intracellularly, associated with tubular and vesicular organelles (double arrows). Note that immunolabeled vesicles are often in the vicinity of the Golgi apparatus (G). Only few immunoparticles are in association with the plasma membrane (arrowheads). Note in A and C, that immunoparticles are also associated with endosome-like vesicles (asterisks) in immunoreactive dendrites (d). Scale bars, 0.5 ␮m.

targeting of sst2AR-bearing endosomes to the vicinity of the Golgi apparatus, rather than maturation of neosynthesized receptors. In support of this hypothesis, the TfR has been also shown to be localized in a so-called para-Golgi tubular network in various cell lines (Gruenberg and Maxfield, 1995). Additionally, the distribution and morphology of the recycling compartment has been described to be somewhat similar to the Golgi

apparatus, which is also largely composed of tubular elements (Pagano et al., 1989). Quantitative analysis at the electron microscopic level revealed that internalization of sst2ARs occurred rapidly since a ⬃80% decrease of cell-surface receptors was observed 15 min after acute agonist injection. Although further ultrastructural experiments are needed at various time intervals to ascertain the precise in vivo rate of the

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In Vivo Internalization of the sst2A Receptor

FIG. 10. Schematic drawing illustrating the subcellular distribution of sst2ARs in neuronal cells representative of a control (NaCl) and an octreotide-treated (Oct) rat. Cell profiles were drawn from electron micrographs using a computer image analysis system. Immunoparticles associated with the plasma membrane (white dots) and endosome-like vesicles (white circles) are illustrated. Immunolabeling associated with Golgi apparatus, endoplasmic reticulum, outer nuclear membrane, and unidentified compartment is not represented. (A) Sst2ARs are mainly associated with the plasma membrane. The insert from the original micrograph shows a portion of the plasmalemma labeled with immunoparticles. (B) Sst2ARs are mainly intracellular, associated with endosomes. Only few gold particles are localized at the plasma membrane. The insert from the original micrograph shows an endosome-like vesicle labeled with immunoparticles. n, nucleus. Scale bars, 2 ␮m.

sst2AR endocytosis, this kinetics is quite similar to the rate at which TfR is internalized (t 1/2 ⫽ 5–7 min) (van der Sluijs et al., 1992). It is also congruent with the kinetics of SRIF internalization via the sst2AR expressed ectopically in COS-7 cells (Nouel et al., 1997). Interestingly, the decrease of cell-surface receptors measured in cell bodies was similar in magnitude to the one observed in dendrites, which suggests that cellular mechanisms and kinetics underlying sst2AR internalization in these two neuronal compartments are comparable.

The overlapping pathways between internalized sst2ARs and TfRs suggest that sst2ARs receptors are recycling. Accordingly, we found the labeling back to normal ⬃6 h after the agonist injection. This long-term effect might suggest that the concentration of the agonist in the extracellular space is sufficient to induce several cycles of endocytosis. Therefore, the reversibility of the effect is likely to reflect the clearance of the agonist. We cannot exclude, however, that at longer times sst2ARs could leave early endosomes to be delivered to late endosomes and lysosomes and be degraded. In this latter case, cellsurface labeling observed 6 h after agonist activation would represent a pool of newly synthesized receptors targeted to the plasma membrane. Our findings add a new member, the somatostatin sst2A receptor, to the small group of neuropeptide receptors (neurokinin 1 and ␮-opioid), which have been shown to internalize in vivo in the nervous system (Mantyh et al., 1995a,b; Sternini et al., 1996; Abbadie et al., 1997, 1999; Allen et al., 1997; Liu et al., 1997; Keith et al., 1998). Together with recent experiments demonstrating endocytosis of classical neurotransmitter receptors in the CNS (Bernard et al., 1998, 1999; Dumartin et al., 1998), these studies suggest that GPCR internalization is a common phenomenon in the mammalian brain and may have profound implications for regulation of functional receptor availability at the plasma membrane (Koenig and Edwardson, 1997; Bloch et al., 1999). In summary, we have demonstrated that the sst2A receptors undergo rapid and massive internalization in the CNS in vivo in response to acute agonist stimulation and that internalized receptors are likely to enter the endocytic recycling compartment. These findings strongly support our earlier hypothesis that endogenously released somatostatin triggers cell-surface receptor endocytosis and provide important clues toward elucidating the cellular regulation of somatostatin receptors in the central nervous system. Whether in vivo internalization of the somatostatin sst2A receptor subserves receptor desensitization or resensitization, or activation of alternative signaling pathways as it has been recently suggested in vitro (Sarret et al., 1999; Boudin et al., 2000), will represent an intriguing issue for future studies.

EXPERIMENTAL METHODS 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

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FIG. 11. Illustration of the subcellular distribution of sst2AR immunoreactivity in dendrites of control rats. (A–E) Immunoparticles are mainly in association with the internal surface of the plasma membrane (arrowheads). Note that few immunoparticles are intracellular (arrows), associated with unidentified compartments. Scale bars: (A–D) 0.5 ␮m; (E) 0.2 ␮m.

(60%) with a fixed 12-h light/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 Somatostatin sst2A receptor antibody was raised in rabbit as described (Helboe et al., 1997), against the carboxyl-terminal segment 330 –369 of human sst2A receptor. For double-labeling experiments mouse monoclonal anti-rat transferrin receptor antibody (MRC OX26) (Serotec, Oxford, UK) was used to specifically label early endosomes (Mellman, 1996). The specificity of both antibodies has been described in detail elsewhere (Hill et al., 1985; Keith et al., 1998; Helboe et al., 1997, 1999; Helboe and Moller, 1999). Stereotaxic Injections of SRIF-14, Octreotide, CH-275, or Saline Animals were deeply anesthetized with sodium pentobarbital (Sanofi, Toulouse, France) (60 mg/kg i.p.) and mounted in a stereotaxic frame. Injections were made with glass micropipettes (tip diameter 30 ␮m) implanted into the parietal cortex (bregma, ⫺2.8 mm; lateral, 6.2 mm; depth, 5.6 mm). A volume of 0.5 ␮l was

injected for each drug and vehicle (0.9% NaCl) at a rate of 0.16 ␮l/min. The micropipette was left in place for an additional 2-min period to reduce backflow. To examine the specificity of the agonist induced internalization of the sst2AR, 30 rats were injected with either saline (n ⫽ 3) or 0.5 fmol, 0.5 pmol, and 0.5 nmol of either SRIF-14 (Peninsula Laboratories, San Carlos, CA) (n ⫽ 9, 3 rats per dose), octreotide (gift from Novartis Pharma AG, Basel, Switzerland) (n ⫽ 9, 3 rats per dose), or CH-275 (gift from C. Hoeger and J. Rivier, San Diego, CA) (n ⫽ 9, 3 rats per dose), and sacrificed 15 min after the start of the injection. To examine the time-course of agonist-induced sst2AR internalization, 14 rats were injected with 0.5 pmol octreotide and allowed to survive 5, 15, 30, 60 min and 3, 6, 24 h after the start of the injection (2 rats per survival time). To examine the subcellular distribution at the confocal and the electron microscopic level of the sst2AR after agonist stimulation, 18 rats received injections of 0.5 pmol octreotide (3 for electron microscopy and 9 for confocal microscopy) or saline (3 for electron microscopy and 3 for confocal microscopy) and were sacrificed 15 min after the start of the injection. Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital and perfused through the ascending aorta with

In Vivo Internalization of the sst2A Receptor

FIG. 12. Quantitative analysis of the subcellular distribution of sst2ARs in dendrites of control (NaCl) or 0.5 pmol octreotide-injected (Oct) rats using preembedding immunogold method. (A) Proportion of immunoparticles associated with different subcellular neuronal compartments in saline-injected animals. For each analyzed dendrite, immunoparticles were identified and counted in association with three subcellular compartments. The proportion of particles associated with each compartment was calculated in relation to the total number of particles. Most immunoparticles are associated with the plasma membrane (75%). Only a small portion is detected in association with endosome-like vesicles (16%) and with the unidentified compartment (9%). (B) Effect of 0.5 pmol octreotide injection on the localization of the sst2AR immunoparticles in cortical dendrites. For each analyzed dendrite, the number of immunoparticles associated with each compartment was counted in relation to the membrane length (in micrometers) for the plasma membrane and to the surface of cytoplasm (in square micrometers) for endosome-like vesicles and the unidentified compartment. Results are expressed in relation to an arbitrary unit (100%) of the control values. The statistical analysis (nonparametric Mann–Whitney U test) shows that the density of immunoparticles significantly decreases at the plasma membrane (***P ⬍ 0.0001) and increases in endosome-like vesicles (***P ⬍ 0.0001). NS, nonsignificant.

600 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Brains were cryoprotected in 30% sucrose in PB (4°C, 24 h) and frozen in liquid isopentane at ⫺45°C. The brains were sectioned at a thickness of 30 ␮m on a freezing microtome and collected in PB. Light microscopy. Free floating sections were processed for sst2AR immunohistochemistry using immu-

657 noperoxidase method with tyramide amplification system as previously described (Dournaud et al., 1996, 1998). Briefly, sections were rinsed in 0.1 M Tris buffer saline, pH 7.4 (TBS), containing 0.05% Tween 20 (TBST). Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H 2O 2 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 (TSAIndirect; NEN Life Sciences Products, Boston, MA). Sections were then incubated overnight at room temperature (RT) in 1:4000 rabbit anti-sst2AR antiserum diluted in TNB. Sections were then rinsed in TBST and sequentially incubated for 45 min in 1:300 biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted in TNB and for 45 min in avidin-biotinylated horseradish peroxidase complex (ABC; Vector Laboratories). Sections were subsequently incubated for 10 min in a 1:100 biotinyl tyramide solution (TSA-Indirect; NEN Life Sciences Products) and reincubated in ABC for 45 min. Peroxidase activity was revealed with 0.05% of 3,3⬘-diaminobenzidine (DAB; Sigma Chemical, St. Louis, MO) in 0.05 M Tris buffer saline, 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 for light microscopic observation. Immunocytochemical controls consisted of adsorption of the receptor antibodies with 50 ␮g/ml of sst2AR-GST fusion proteins overnight at 4°C, and incubation with the preimmune in place of the immune serum. To quantify the number of cortical neurons displaying intracellular sst2AR immunostaining after agonist injections, we first determined the spread of injected SRIF and octreotide. Four animals received 0.5 nmol of either SRIF-14 (n ⫽ 2) or octreotide (n ⫽ 2) and were perfused 15 min after. Brains were processed for immunocytochemistry as described above using a rat monoclonal anti-SRIF-14 antibody (MAB354) (Chemicon, Temecula, CA) or a rabbit polyclonal anti-octreotide antibody (Novartis Pharma AG, Basel, Switzerland). We determined by quantitative densitometry with an image analysis program (Biocom, Les Ullis, France) that a uniformly high immunoreactive signal of either SRIF-14 or octreotide was present 0.5 mm on either side of the injection point. Because osmolarity, injected volumes and injected time periods were the same for all the solutions, we assumed this as an estimate of the effective spread of injected drugs. Immunostained neurons were counted on every other section taken within 300 ␮m of the injection point. Six sections per animal

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FIG. 13. Illustration of the subcellular distribution of sst2AR immunoreactivity in dendrites of 0.5 pmol octreotide-injected rats. (A–D) The majority of immunoparticles is found inside dendrites, mostly in association with endosome-like vesicles (double arrows). Few immunoparticles are located at the plasma membrane (arrow-heads) and in unidentified compartments (arrows). Scale bars: (A–C) 0.5 ␮m; (D) 0.2 ␮m.

were analyzed in each group. Values were compared using the nonparametric Mann–Whitney U test. P ⬍ 0.05 was considered statistically significant. Confocal microscopy. For double-labeling experiments, serial frozen brain sections were prepared as above. Sections were rinsed in TBS. This was followed by a 30-min preincubation in 5% normal donkey serum (NDS) in TBS containing 0.3% Triton X-100. Sections were coincubated for 72 h at 4°C in 1:400 anti-sst2AR antibody with 1:50 anti-TfR antibody diluted in TBS containing 0.5% NDS and 0.3% Triton X-100. Sections were then rinsed in TBS and incubated for 45 min in 1:100 FITC-conjugated anti-rabbit IgG and 1:100 Cy3conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted in TBS containing 5% NDS and 0.3% Triton X-100. Sections were then rinsed in TBS, mounted on glass slides and coverslipped with a Vectashield solution (Vector Laboratories). 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-4D confocal imaging system equipped with an Argon-Krypton ion laser (488 and 568 nm) (Leica Instrument, 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.” The signal was treated with line averaging to integrate the signal collected over eight frames to reduce noise. A focal series was collected for each specimen. Images illustrating colocalization between sst2AR

and TfR correspond to the reconstruction of two adjacent optical sections separated by a 0.7-␮m step. To assess the proportion of double-labeled intracellular organelles, focal series of 12 sections (step 0.7 ␮m) were collected from six cells immunoreacted with the TfR and sst2AR antibodies. Merged optical sections for both signals were analyzed from the top surface to the bottom of the cell. Intracellular organelles (n ⫽ 223) displaying yellow signals were considered as doublelabeled. For three-dimensional (3-D) reconstruction experiments, single-labeled sections for the sst2AR were used. The confocal pinhole was closed to a minimum to yield the thinnest possible optical section. Focal series of up to 70 sections apart were collected for each specimen and a step of 300 nm between consecutive optical sections was chosen. 3-D reconstruction was achieved with AMIRA 2.1.1 software (TGS Inc., France) using an isosurface algorithm. Electron microscopy. Immunocytochemical procedures for the detection of the sst2AR at the ultrastructural level were performed according to Bernard et al. (1998, 1999). Rats were transaortically perfused with 50 –100 ml of 0.9% NaCl, followed by 400 ml of 4% paraformaldehyde with 0.05% glutaraldehyde in PB at 4°C at a rate of ⬃20 ml/min. Brains were postfixed overnight in 4% paraformaldehyde at 4°C. Coronal sections were cut on a vibratome at 50 ␮m and collected in 0.01 M phosphate buffer saline, pH 7.4 (PBS). Sections were equilibrated in 25% sucrose and 10% glycerol in 0.05 M PB, frozen rapidly in isopentane, cooled in liquid

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nitrogen, and thawed in PBS at RT. Sections were then incubated in 4% normal goat serum (NGS) in PBS for 30 min and incubated for 16 h at RT in anti-sst2AR antibody diluted 1:400 in PBS containing 1% NGS. After washing in PBS they were incubated for 2 h in a 1:100 dilution of colloidal gold (0.8 nm in diameter) conjugated goat anti-rabbit IgG (Aurion, Wageningen, The Netherlands) in PBS containing 2% of bovine serum albumin-c and 0.2% of cold water fish gelatin. Sections were then washed in PBS and postfixed in 0.1% glutaraldehyde in PBS for 10 min. After repeated washing in PBS and 0.1 M sodium acetate buffer, pH 7.0, gold labeling was intensified using a silver enhancement kit (HQ Silver; Nanoprobes, Stony Brook, NY) for 5–10 min in the dark at RT. Sections were finally washed in acetate buffer and then in PB. Immunogold-treated sections were post-fixed in 1% osmium tetroxide in PB for 10 min at RT. After washing three times in PB, they were dehydrated in an ascending series of ethanol, which included 1% uranyl acetate in 70% ethanol. They were then treated with propylene oxide twice for 10 min, equilibrated overnight in Durcupan ACM (Fluka, Buchs, Switzerland), mounted on glass slides, and cured at 60°C for 48 h. Areas of interest were cut out from the slide and glued to blank cylinders of resin. The immunoreactive neurons identified on the thick sections were cut in semithin sections (1 ␮m) and then in ultrathin sections on a Reichert Ultracut S microtome. Ultrathin sections were collected on pioloform-coated single-slot grids. Sections were stained with lead citrate and examined with a Philips CM10 electron microscope. The subcellular distribution of sst2A receptor in cortical layer VI was analyzed according to Bernard et al. (1998, 1999), in 3 NaCl- and 3 octreotide-injected rats. A mean of 10 neurons and 20 dendrites per animal was analyzed. In perikarya, immunoparticles were identified and counted in association with six subcellular compartments. Five compartments are the plasma membrane, endosome-like vesicles, the Golgi network, the endoplasmic reticulum and the outer nuclear membrane. Some immunoparticles were classified as a sixth unidentified compartment, because they were associated with either no detectable organelles or an organelle that could not be unambiguously identified. In dendrites surrounding immunopositive neurons, immunoparticles were identified and counted in association with the plasma membrane, endosome-like vesicles, and unidentified compartment. The results are expressed as (1) the percentage of immunoreactive particles associated with the different subcellular compartments in perikarya and dendrites in control animals

and (2) the number of immunoparticles per membrane length (micrometers) or cytoplasmic surface (square micrometers) in control and treated rats. Values from NaCl- and octreotide-treated rats were compared using the nonparametric Mann–Whitney U test. P ⬍ 0.05 was considered statistically significant.

ACKNOWLEDGMENTS This study was supported by the “Institut National de la Sante´ et de la Recherche Me´dicale” and CEE contract QLG-1999-0098. Z. Csaba was funded by a “Poste vert” postdoctoral fellowship from Inserm. We are grateful to G. Ge´raud (Institut Jacques Monod, Service Imagerie, Universite´ Paris VII, France) for confocal sampling and imaging and to the Electron Microscopy Center of the University Victor Se´galen-Bordeaux 2. We also thank Dr. D. Hoyer (Novartis Pharma AG, Basel, Switzerland) for providing the anti-octreotide antibody and E. Doudnikoff for her expert technical work.

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