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Somatostatin (SRIF) is a neuropeptide that acts as an important regulator of both endocrine and exocrine secretion and modulates neurotransmission in the central nervous system (CNS). SRIF also regulates cell proliferation in normal tissues and tumors. The physiological actions of SRIF are mediated by a family of five G protein-coupled receptors, called somatostatin receptor (SST) SST 1 , SST 2 , SST 3 , SST 4 , SST 5 . These five receptors share similar molecular structure and signaling pathways but they display marked differences in their anatomical distribution, subcellular localization and intracellular trafficking. The SST subtypes are widely distributed in the CNS and peripheral nervous system, in many endocrine glands and tumors, particularly of neuroendocrine origin. In this review, we focus on the agonist-dependent internalization and recycling of the different SST subtypes in vivo in the CNS, peripheral organs and tumors. We also discuss the physiological, pathophysiological and potential therapeutic effects of the intracellular trafficking of SST subtypes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aims: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2)., Methods: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor., Results: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development., Conclusions: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions., (© 2020 British Neuropathological Society.)

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST 2 and SST 5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST 2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature., (Copyright © 2018 The Author(s).)

Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs., Significance Statement: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures., (Copyright © 2015 the authors 0270-6474/15/3511961-16$15.00/0.)

Somatostatin (SRIF), by acting mainly through sst2 and sst5 receptors, is a potent inhibitor of hormonal secretion by the human anterior pituitary gland. However, the pattern of protein expression of these SRIF receptors remains unknown during pituitary development. To get further insights into the physiological role of SRIF receptors in human development and pituitary function, the present study examined the developmental expression of the sst2 and sst5 receptors in the individual cell types of the anterior human pituitary. Thirteen fetal human pituitaries were investigated between 13 to 38 weeks of gestation (WG) by double-labeling immunofluorescence with antibodies raised against sst2 or sst5 receptors and GH, LH, FSH, TSH, or pro-opiomelanocortin proteins. SRIF immunoreactivity in the hypothalamus and median eminence was investigated at the same developmental ages. Immunoreactivity for the sst2 receptor was evident as early as 13 to 15 WG and onward mainly in TSH-, LH-, and FSH-expressing cells, whereas sst5 immunoreactivity was apparent at the late development stages (35-38 WG). GH-expressing cells mainly expressed sst5 immunoreactivity. SRIF-positive fibers and cells were detected as soon as 13 to 16 WG in the hypothalamus and median eminence and their densities increased with gestational age. The early appearance of hypothalamic SRIF cells and fibers suggests a physiological link between SRIF and its receptors during pituitary development. Whereas sst2 receptors might play a primary role in the differentiation and regulation of TSH, LH, and FSH cells, sst5 receptors appear to be mainly involved in GH regulation from birth onward.

Directionality of information flow through neuronal networks is sustained at cellular level by polarized neurons. However, specific targeting or anchoring motifs responsible for polarized distribution on the neuronal surface have only been identified for a few neuronal G-protein-coupled receptors (GPCRs). Here, through mutational and pharmacological modifications of the conformational state of two model GPCRs, the axonal CB1R cannabinoid and the somatodendritic SSTR2 somatostatin receptors, we show important conformation-dependent variations in polarized distribution. The underlying mechanisms include lower efficiency of conformation-dependent GPCR endocytosis in axons, compared with dendrites, particularly at moderate activation levels, as well as endocytosis-dependent transcytotic delivery of GPCRs from the somatodendritic domain to distal axonal portions, shown by using compartmentalized microfluidic devices. Kinetic modeling predicted that GPCR distribution polarity is highly regulated by steady-state endocytosis, which is conformation dependent and is able to regulate the relative amount of GPCRs targeted to axons and that axonally polarized distribution is an intermediary phenotype that appears at moderate basal activation levels. Indeed, we experimentally show that gradual changes in basal activation-dependent endocytosis lead to highly correlated shifts of polarized GPCR distribution on the neuronal surface, which can even result in a fully reversed polarized distribution of naturally somatodendritic or axonal GPCRs. In conclusion, polarized distribution of neuronal GPCRs may have a pharmacologically controllable component, which, in the absence of dominant targeting motifs, could even represent the principal regulator of sub-neuronal distribution. Consequently, chronic modifications of basal GPCR activation by therapeutic or abused drugs may lead to previously unanticipated changes in brain function through perturbation of polarized GPCR distribution on the neuronal surface.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

The neuropeptide somatostatin (SRIF) is an important modulator of neurotransmission in the central nervous system and acts as a potent inhibitor of hormone and exocrine secretion. In addition, SRIF regulates cell proliferation in normal and tumorous tissues. The six somatostatin receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, and sst5), which belong to the G protein-coupled receptor (GPCR) family, share a common molecular topology: a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an amino-terminus outside the cell, and a carboxyl-terminus inside the cell. For most of the GPCRs, intracytosolic sequences, and more particularly the C-terminus, are believed to interact with proteins that are mandatory for either exporting neosynthesized receptor, anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Accordingly, most of the SRIF receptors can traffic not only in vitro within different cell types but also in vivo. A picture of the pathways and proteins involved in these processes is beginning to emerge.

The neuropeptide somatostatin has been suggested to play an important role during neuronal development in addition to its established modulatory impact on neuroendocrine, motor and cognitive functions in adults. Although six somatostatin G protein-coupled receptors have been discovered, little is known about their distribution and function in the developing mammalian brain. In this study, we have first characterized the developmental expression of the somatostatin receptor sst2A, the subtype found most prominently in the adult rat and human nervous system. In the rat, the sst2A receptor expression appears as early as E12 and is restricted to post-mitotic neuronal populations leaving the ventricular zone. From E12 on, migrating neuronal populations immunopositive for the receptor were observed in numerous developing regions including the cerebral cortex, hippocampus and ganglionic eminences. Intense but transient immunoreactive signals were detected in the deep part of the external granular layer of the cerebellum, the rostral migratory stream and in tyrosine hydroxylase- and serotonin- positive neurons and axons. Activation of the sst2A receptor in vitro in rat cerebellar microexplants and primary hippocampal neurons revealed stimulatory effects on neuronal migration and axonal growth, respectively. In the human cortex, receptor immunoreactivity was located in the preplate at early development stages (8 gestational weeks) and was enriched to the outer part of the germinal zone at later stages. In the cerebellum, the deep part of the external granular layer was strongly immunoreactive at 19 gestational weeks, similar to the finding in rodents. In addition, migrating granule cells in the internal granular layer were also receptor-positive. Together, theses results strongly suggest that the somatostatin sst2A receptor participates in the development and maturation of specific neuronal populations during rat and human brain ontogenesis.

Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) is of particular importance, especially when modifications of the neurochemic environment occur as in pathological or therapeutic circumstances. In the central nervous system, although some GPCRs were reported to internalize in vivo, little is known about their trafficking downstream of the endocytic event. To address this issue, distribution and expression pattern of the major somatostatin receptor subtype, the somatostatin type 2 (sst2), was monitored in the hippocampus using immunofluorescence, autoradiographic and immunogold experiments from 10 minutes to 7 days after in vivo injection of the receptor agonist octreotide. We then analyzed whether postendocytic trafficking of the receptor was dependent upon integrity of the microtubule network using colchicine-injected animals. Together, our results suggest that upon agonist stimulation, dendritic receptors are retrogradely transported through a microtubule-dependent mechanism to a trans Golgi domain enriched in the t-SNARE syntaxin 6 and trans Golgi network 38 proteins, before recycling. Because we show that the exit rate from the trans Golgi apparatus back to the plasma membrane (hours) is slower than the entry rate (minutes), the neuronal postendocytic trafficking of sst2 receptor is likely to have functional consequences in several neurological diseases in which an increase in somatostatin release occurs.

Background and Purpose: Understanding the trafficking of G-protein-coupled receptors is of particular importance. In the central nervous system, although some G-protein-coupled receptors were reported to internalize in vivo, little is known about their trafficking downstream of the endocytic event., Methods: The distribution of the major somatostatin receptor subtype, the sst2, was monitored in the hippocampus using immunofluorescence from 10 minutes to seven days after in vivo injection of the receptor agonist octreotide., Results: From 10 min to 3 h after agonist injection, intensity of receptor immunoreactivity gradually decreased in the molecular layer of dentate gyrus and in the strata oriens and radiatum of CA1. Concomitantly, in the granular and pyramidal layers, small spherical immunofluorescent particles became apparent in perikarya, shortly after agonist stimulation (i.e. 30 min, 60 min). After longer survival times (i.e. 3 h, 6 h, 24 h), immunolabeling was confined to larger, intensely-stained intracytoplasmic vesicles. From 48 h to 7 d after agonist injection, distribution and intensity of sst2 receptor immunoreactivity became similar to that of control animals. The sst2 receptor labeling extensively colocalized with TGN38 and syntaxin 6 after OCT injection. Colocalization with trans-Golgi markers was observed as soon as 1 h after OCT injection and still present 24 h after. By contrast, colocalization with the endoplasmic reticulum marker PDI and the cis-Golgi marker GM130 was never observed., Conclusions: Our results suggest that upon agonist stimulation, dendritic receptors are retrogradely transported to a trans-Golgi network domain enriched in the t-SNARE syntaxin-6 and TGN38 proteins before recycling.

Temporal lobe epilepsy (TLE) is characterized by hippocampal sclerosis together with profound losses and phenotypic changes of different classes of interneurons, including those expressing somatostatin (SRIF). To understand the functional significance of the plasticity of SRIF transmission in TLE, unraveling the status of SRIF receptors is, however, a prerequisite. To address this issue, we characterized expression and distribution of the major SRIF receptor, the sst2 subtype, in hippocampal tissue resected in patients with TLE using complementary neuroanatomic approaches. In patients with hippocampal sclerosis, the number of cells expressing sst2 receptor mRNA as well as sst2 receptor-binding sites and immunoreactivity decreased significantly in the CA1-3, reflecting neuronal loss. By contrast, in the dentate gyrus, sst2 receptor mRNA expression was strongly increased in the granule cell layer, and sst2 receptor-binding sites and immunoreactivity was preserved in the inner but decreased significantly in the outer molecular layer. In this latter region, pronounced changes in SRIF terminal fields were observed. Decreased receptor density in the distal dendrites of granule cells is likely to reflect downregulation of sst2 receptors in response to physiopathologic release of SRIF. Because sst2 receptors have anticonvulsant and antiepileptogenic properties, this phenomenon may contribute to the etiology of TLE seizures.

GABAergic septohippocampal neurons play a major role in the generation of hippocampal theta rhythm, but modulatory factors intervening in this function are poorly documented. The neuropeptide somatostatin (SST) may be one of these factors, because nearly all hippocampal GABAergic neurons projecting to the medial septum/diagonal band of Broca (MS-DB) express SST. In this study, we took advantage of the high and selective expression of the SST receptor sst2A in MS-DB to examine its possible role on theta-related activity. Immunohistochemical experiments demonstrated that sst2A receptors were selectively targeted to the somatodendritic domain of neurons expressing the GABAergic marker GAD67 but were not expressed by cholinergic neurons. In addition, a subpopulation of GABAergic septohippocampal projecting neurons expressing parvalbumin (PV) also displayed sst2A receptors. Using in vivo juxtacellular recording and labeling with neurobiotin, we showed that a number of bursting and nonbursting neurons exhibiting high discharge rates and brief spikes were immunoreactive for PV or GAD67 and expressed the sst2A receptor. Microiontophoresis applications of SST and the sst2A agonist octreotide (OCT) showed that sst2A receptor activation decreased the discharge rate of both nonbursting and bursting MS-DB neurons and lessened the rhythmic activity of the latter. Finally, intraseptal injections of OCT and SST in freely moving rats reduced the power of hippocampal EEG in the theta band. Together, these in vivo experiments suggest that SST action on MS-DB GABAergic neurons, through sst2A receptors, represents an important modulatory mechanism in the control of theta activity.

Increasing evidence suggests that somatostatin may control neuronal excitability during epileptogenesis. In the hippocampus, sst2A receptors are likely to mediate somatostatin inhibitory actions but little is known about their status in kindled tissues. In the present study, sst2A receptor and somatostatin immunoreactivity were examined by confocal microscopy in the hippocampus during and after kindling acquisition. In control rats, somatostatin-positive axon terminals were mainly found in the stratum lacunosum moleculare of CA1 area and in the outer molecular layer of the dentate gyrus. sst2A receptor immunoreactivity was diffusely distributed in the strata radiatum and oriens of CA1 and in the stratum moleculare of the dentate gyrus. Immunogold electron microscopy revealed that sst2A receptors were predominantly localized postsynaptically, at the plasma membrane of dendritic shafts and spines of principal neurons. During kindling epileptogenesis, qualitative and semiquantitative analysis revealed a progressive decrease of sst2A immunoreactivity in the outer molecular layer, which was spatially associated with an increase in somatostatin immunoreactivity. No obvious changes in sst2A receptor immunoreactivity were observed in other hippocampal subfields. These results suggest that the decrease of sst2A receptor immunoreactivity in the outer molecular layer reflects receptor down-regulation in distal dendrites of granule cells in response to chronic somatostatin release. Because the sst2A receptor appears to mediate anticonvulsant and antiepileptogenic effects of somatostatin, this may represent a pivotal mechanism contributing to epileptogenesis.

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.

We recently reported that immature porcine Leydig cells express both somatostatin (SRIF) and SRIF receptor type-2 (sst-2) transcripts. The present study was therefore undertaken to assess whether SRIF might exert autocrine actions on these cells through sst2A receptor, one of the two sst2 isoforms known to exert important neuroendocrine and endocrine functions. Using a polyclonal antibody directed towards the C-terminal tail of the sst2A receptor subtype, receptor immunoreactivity was detected in a subpopulation of Leydig cells and spermatogonia. To address the physiological correlates of this expression we then studied the possible involvement of sst2 receptor in the regulation of testosterone secretion. Functional assays showed that the sst2 agonist octreotide inhibited both basal and hCG-stimulated testosterone secretion by testosterone pretreated Leydig cells. To assess whether sst2 receptor expression might be regulated by testosterone, we performed a semi-quantitative RT-PCR analysis of sst2 mRNA expression in Leydig cells cultured in the presence or in the absence of the androgen. A significant increase in sst2 receptor transcripts was observed in testosterone-treated cells. Taken together, these data suggest that SRIF can inhibit testosterone secretion through the sst2A receptor. The mechanism of the local inhibitory actions of SRIF is probably autocrine since immature porcine Leydig cells express SRIF itself and it might involve testosterone-induced increase of sst2 receptor expression in immature Leydig cells.

Modulation of glutamatergic transmission by neuropeptides is an essential aspect of neuronal network activity. Activation of the hypothalamic somatostatin sst2 receptor subtype by octreotide decreases AMPA glutamate responses, indicating a central link between a neurohormonal and neuromodulatory peptide and the main hypothalamic fast excitatory neurotransmitter. In mediobasal hypothalamic slices, sst2 activation inhibits the AMPA component of glutamatergic synaptic responses but is ineffective when AMPA currents are pharmacologically isolated. In mediobasal hypothalamic cultures, the decrease of AMPA currents induced by octreotide requires a concomitant activation of sst2 receptors with either NMDA and/or metabotropic glutamate receptors. This modulation depends on changes in intracellular calcium concentration induced by calcium flux through NMDA receptors or calcium release from intracellular stores following metabotropic glutamate receptor activation. These results highlight an unusual regulatory mechanism in which the simultaneous activation of at least three different types of receptor is necessary to allow somatostatin-induced modulation of fast synaptic glutamatergic transmission in the hypothalamus.

Characterization of both neurochemical phenotype of G protein-coupled receptor (GPCR)-expressing cells and receptor compartmentalization is a prerequisite for the elucidation of receptor functions in the central nervous system. However, it is often prevented by the diffuse and homogeneous distribution of receptor immunoreactivity. This is particularly true for the somatostatin (SRIF) sst2A receptor, which is largely distributed in the mammalian brain. By using this receptor as a model, we investigated whether receptor internalization, a biochemical property shared by numerous GPCRs, would reveal sst2A-expressing cell populations in the rat dorsolateral septum (LSD), a region in which SRIF might play an important modulatory role. Thirty minutes to 1 hour after intracerebroventricular injection of the sst2A receptor agonist octreotide, numerous sst2A-immunoreactive neurons and processes became apparent due to intracytoplasmic accumulation of intensely stained granules. Double-immunolabeling experiments with synaptophysin and MAP2 provided evidence that internalized sst2A receptors are predominantly localized in the somatodendritic compartment. Revealing sst2A receptor-expressing cell bodies permitted to analyze their neurotransmitter content. Quantitative analysis demonstrated an extensive overlap (approximately 85%) between SRIF- and sst2A-expressing neuronal populations. Additionally, numerous SRIF-immunoreactive axon-like terminals were found in close apposition with sst2A-positive cell bodies and dendrites. Taken together, these data suggest that the sst2A receptor is predominantly expressed in LSD neurons as a postsynaptic autoreceptor, thus providing novel neuroanatomic clues to elucidate SRIF neurotransmission in this region. More generally, in vivo agonist-induced internalization appears as a rapid and powerful tool for the neurochemical characterization of GPCR-expressing cell populations in the mammalian brain., (Copyright 2002 Wiley-Liss, Inc.)

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

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 CH-275, 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., (Copyright 2001 Academic Press.)

Somatostatin, and the recently discovered neuropeptide cortistatin, exert their physiological actions via a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, sst5). Following the cloning of somatostatin receptors significant advances have been made in our understanding of their molecular, pharmacological and signaling properties although much progress remains to be done to define their physiological role in vivo. In this review, the present knowledge regarding neuroanatomical localization, signal transduction pathways, desensitization and internalization properties of somatostatin receptors is summarized. Evidence that somatostatin receptors can form homo- and heterodimers and can physically interact with members of the SSTRIP/Shank/ProSAP1/CortBP1 family is also discussed., (Copyright 2001 Harcourt Publishers Ltd.)

Five somatostatin (SRIH) receptors (sst1-5) have been cloned. Recent anatomical evidence suggests that sst1 and sst2 may be involved in the central regulation of GH secretion. Given the lack of specific receptor antagonists, we used selective antisense oligodeoxynucleotides (ODNs) to test the hypothesis that one or both of these subtypes are involved in the intrahypothalamic network regulating pulsatile GH secretion. In mouse neuronal hypothalamic cultures the proportion of GHRH neurons coexpressing sst1 or sst2 messenger RNAs (mRNAs) was identical. In contrast, sst1 mRNAs were more often present than sst2 in SRIH-expressing neurons. Firstly, sst1 antisense ODN in vitro treatment abolished sst1, but not sst2, receptor modulation of glutamate sensitivity and decreased sst1, but not sst2, mRNAs. The reverse was true after treatment with sst2 antisense. Sense ODNs did not alter the effects of SRIH agonists. In a second series of experiments, nonanaesthetized adult male rats were infused for 120 h intracerebroventricularly with ODNs. Only the sst1 antisense ODN diminished the amplitude of ultradian GH pulses without modifying their frequency. In parallel, sst1 antisense ODN strongly diminished sst1 immunoreactivity in the anterior periventricular nucleus and median eminence, as well as sstl periventricular nucleus mRNA levels. The effectiveness of the sst2 antisense ODN was attested by the inhibition of hypothalamic binding of [125I]Tyr0-D-Trp8-SRIH. Scrambled ODNs had no effect on GH secretion or on sst mRNAs or SRIH binding levels. These results favor a preferential involvement of sst1 receptors in the intrahypothalamic regulation of GH secretion by SRIH.

Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin. Even if it shares several biological properties with somatostatin, the effects of cortistatin on cortical electrical activity and sleep are opposite to those elicited by somatostatin. We recently demonstrated that somatostatin could modulate glutamate sensitivity, either positively through activation of the sstl receptor subtype, or negatively through activation of the sst2 receptor subtype in hypothalamic neurons in culture which express almost exclusively these two sst subtypes. Thus, in the present study we compared the effects of cortistatin and somatostatin in hypothalamic neurons in culture, in order to define the former peptide activity on both subtypes. We first determined that the affinities of cortistatin and somatostatin were similar on cloned rat sstl and sst2 receptor subtypes in transfected cells and hypothalamic neurons membranes. We then found that cortistatin, like somatostatin, depresses the glutamate response but, unlike somatostatin, never potentiates glutamate sensitivity in hypothalamic neurons. The observed effect of cortistatin is strongly suggestive of an activation of the somatostatin sst2 receptor subtype in hypothalamic neurons in culture.

Using an antipeptide antibody, we reported previously on the distribution of the somatostatin sst2A receptor subtype in rat brain. Depending on the region, immunolabeled receptors were either confined to neuronal perikarya and dendrites or distributed diffusely in tissue. To investigate the functional significance of these distribution patterns, we examined the regional and cellular relationships between somatostatin axons and sst2A receptors in the rat CNS, using double-labeling immunocytochemistry. Light and confocal microscopy revealed a significant correlation (p < 0.02) between the distribution of somatodendritic sst2A receptor immunoreactivity and that of somatostatin terminal fields, both quantitatively and qualitatively. Furthermore, in regions of somatodendritic labeling, a subpopulation of sst2A-immunoreactive cells was also immunopositive for somatostatin, suggesting that a subset of sst2A receptors consists of autoreceptors. By contrast, in regions displaying diffuse sst2A labeling only moderate to low densities of somatostatin terminals were observed, and no significant relationship was found between terminal density and receptor immunoreactivity. At the electron microscopic level, areas expressing somatodendritic sst2A labeling were found by immunogold cytochemistry to display low proportions of membrane-associated, as compared with intracellular, receptors. Conversely, in regions displaying diffuse sst2A receptor labeling, receptors were predominantly associated with neuronal plasma membranes, a finding consistent with the high density of sst2 binding sites previously visualized in these areas by autoradiography. Double-labeling studies demonstrated that in the former but not in the latter regions, sst2A-immunoreactive somata and dendrites were heavily contacted by somatostatin axon terminals. Taken together, these results suggest that the low incidence of membrane-associated receptors observed in regions of somatodendritic sst2A labeling may be caused by downregulation of cell surface receptors by endogenous somatostatin, possibly through ligand-induced receptor internalization.

Biological actions of somatostatin are exerted via a family of receptors, for which five genes recently have been cloned. However, none of these receptor proteins has been visualized yet in the brain. In the present-study, the regional and cellular distribution of the somatostatin sst2A receptor was investigated via immunocytochemistry in the rat central nervous system by using an antibody generated against a unique sequence of the receptor protein. Specificity of the antiserum was demonstrated by immunoblot and immunocytochemistry on rat brain membranes and/or on cells transfected with cDNA encoding the different sst receptor subtypes. In rat brain sections, sst2A receptor immunoreactivity was concentrated either in perikarya and dendrites or in axon terminals distributed throughout the neuropil. Somatodendritic labeling was most prominent in the olfactory tubercle, layers II-III of the cerebral cortex, nucleus accumbens, pyramidal cells of CA1-CA2 subfields of the hippocampus, central and cortical amygdaloid nuclei, and locus coeruleus. Labeled terminals were detected mainly in the endopiriform nucleus, deep layers of the cortex, claustrum, substantia innominata, subiculum, basolateral amygdala, medial habenula, and periaqueductal gray. Electron microscopy confirmed the association of sst2A receptors with perikarya and dendrites in the former regions and with axon terminals in the latter. These results provide the first characterization of the cellular distribution of a somatostatin receptor in mammalian brain. The widespread distribution of the sst2A receptor in cerebral cortex and limbic structures suggests that it is involved in the transduction of both pre- and postsynaptic effects of somatostatin on cognition, learning, and memory.