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Somatostatin is abundantly expressed in mammalian brain. The peptide binds with high affinity to six somatostatin receptors, sst1, sst2A and B, sst3 to 5, all belonging to the G-protein-coupled receptor family. Recent advances in the neuroanatomy of somatostatin neurons and cellular distribution of sst receptors shed light on their functional roles in the neuronal network. Beside their initially described neuroendocrine role, somatostatin systems subserve neuromodulatory roles in the brain, influencing motor activity, sleep, sensory processes and cognitive functions, and are altered in brain diseases like affective disorders, epilepsia and Alzheimer's disease.

Five somatostatin receptors (sst) have been cloned and mRNAs for the first four (sst1–4) are expressed in many brain regions. In the present work, we compared the distribution of the non-selective ligand [125I]-Tyr0-DTrp8-SRIF14 by autoradiography in 24 brain regions and pituitary in wild type, sst1- to sst4- or SRIF-gene invalidated (KO) mice. [125I]-Tyr0-DTrp8-SRIF14 binding was not significantly modified in sst1 KO mouse brain with the noticeable exception of the substantia nigra and only moderately decreased in pituitary. For sst2 KO mice, a general decrease (>75%) was observed in most regions, with the noticeable exception of the olfactory bulb and CA1 field of the hippocampus. SST3 KO brain displayed a decrease in binding in the external plexiform layer of the olfactory bulb only (−54%). For sst4 KO mice, [125I]-Tyr0-DTrp8-SRIF14 binding levels in the external plexiform (−35%) and glomerular (−39%) layers of the olfactory bulb as well as the hippocampus CA1 field (−68%) were significantly decreased. In SRIF KO mice, a significant increase in binding levels was observed in olfactory bulb, anterior olfactory nucleus, frontal cortex upper layers, lateral septum, CA1 field, zona incerta and lateral hypothalamus, substantia nigra, periaqueductal grey and parabrachial nucleus. Competition with selective ligands (CH275, octreotide or L-779,976, L-796,778, L-803,087, and octreotide or L-817,778, for sst1–5 receptors, respectively) was in accordance with these findings. Moreover, octreotide was still able to compete on residual [125I]-Tyr0-DTrp8-SRIF14 binding sites in sst2 KO pituitary. It is concluded that most [125I]-Tyr0-DTrp8-SRIF14 binding sites in mouse brain and pituitary belong to the sst2 subtype but for the olfactory bulb (sst3 and sst4 receptors), the CA1 of the hippocampus (sst4 receptors) and the pituitary (sst5 and sst1 receptors) in which other subtypes are also expressed. The overall increase in [125I]-Tyr0-DTrp8-SRIF14 binding in SRIF KO mice indicates that SRIF receptors, mostly from the sst2 subtype, are regulated by the endogenous ligand(s).

The expression of the five somatostatin receptor subtypes, sst 1 - 5 was compared on tissue containing glial tumours (glioblastomas or oligodendrogliomas), medulloblastomas, and on normal human cortex. By semiquantitativereverse transcription coupled to polymerase chain reaction, the receptor expression profiles were high in cortex and in tissue containing oligodendrogliomas. It was moderate in medulloblastomas. Tissue containing glioblastomas displayed lower expression of somatostatin receptor subtypes, sst 1 and sst 3 being mostly expressed. By 1 2 5 I-Tyr 0 DTrp 8 somatostatin-14 or 1 2 5 I-Leu 8 DTrp 2 2 Tyr 2 5 somatostatin-28 autoradiography combined with synaptophysin immunohistochemistry, it was possible to differentiate between isolated tumoral cell component infiltrating the cerebral parenchyma (cortex or white matter) and tumoral tissue (without residual parenchyma) in glioblastomas or oligodendrogliomas. Glial tumoral tissue per se presented few somatostatin receptors. By contrast, medulloblastoma tumoral cells exhibited numerous octreotide sensitive somatostatin receptors, sst 2 immunocytochemistry demonstrated immunostaining of neuronal cells and neuropile; sst 2 and sst 3 immunostaining was identified on glioblastoma proliferating vessels endothelial cells and on medulloblastomas tumoral cells. Faint sst 2 immunostaining among glial tumoral cells was due to microglia, while glioma cells did not significantly stain. In summary, medulloblastoma tumoral cells express sst 2 /sst 3 receptors at a high level while glioma cells do not. In gliomas, sst expression is restricted to endothelial cells on proliferating vessels (displaying both sst 2 and sst 3 receptors), including parenchyma and reactive microglia (only sst 2 ). The differential expression of sst 2 /sst 3 receptors on gliomas and medulloblastomas has implications for the therapy of these tumours.

In vitro studies using various cell systems have provided conflicting results regarding homologous regulation of somatostatin (SRIH) receptors, and information on whether SRIH regulates the expression of its own receptors in vivo is lacking. In the present study we examined, by in situ hybridization, the effects of pretreatment with the sst2-preferring SRIH analog, octreotide, in vivo, on mRNA levels of two SRIH receptor subtypes, sst1 and sst2, in rat brain and pituitary. 125I-[DTrp8]-SRIH binding was also measured in these regions. Three hours after the iv injection of 50 µg octreotide to conscious adult male rats, there was a 46% increase (p < 0.01) in the labeling density of sst2 mRNA-expressing cells in the hypothalamic arcuate nucleus compared to normal saline-pretreated controls, but not in any of the other brain regions examined. Computer-assisted image analysis revealed that 3 h exposure to octreotide significantly (p < 0.01) augmented both the number and labeling density of sst2 mRNA-expressing cells in the arcuate nucleus, compared to those in saline-treated controls. By contrast, within the anterior pituitary gland, in vivo exposure to octreotide did not affect the expression of sst2 mRNA. No changes in sst1 mRNA-expressing cells were observed after octreotide treatment in any of the regions measured, indicating that the observed effects were homologous, i.e. specific of the receptor subtype stimulated. Octreotide pretreatment was also without effect on the density of 125I-[DTrp8]-SRIH binding in either the arcuate nucleus or pituitary. These results demonstrate, for the first time, that SRIH preexposure in vivo upregulates the expression of a subtype of its own receptors, sst2, within the central nervous system. They further suggest that pretreatment with SRIH in vivo does not cause sst2 receptor desensitization in arcuate nucleus and pituitary. Such homologous regulatory mechanisms may play an important role in the neuroendocrine control of growth hormone (GH) secretion by the arcuate nucleus.

Somatostatin was originally characterised as a hypothalamic neurohormone responsible for the inhibition of pituitary Growth Hormone secretion. In mammals two genes encode for somatostatin-related peptides, somatostatin 14 and 28, and cortistatins, respectively. All peptides bind with similar affinities to the five cloned somatostatin receptors (sst), which belong to the GPCR family. Despite numerous studies, no clear behavioural function has yet been attributed to somatostatin-related peptides. This is due to the lack of good pharmacological tools (selective antagonists) and animal models. This review will focus on the recent development of such tools.

Somatostatin (SRIF) controls many physiological and pathological processes in the central nervous system but the respective roles of the five receptor isotypes (sst1-5) that mediate its effects are yet to be defined. In the present study, we attempted to identify functions of the sst2 receptor using mice with no functional copy of this gene (sst2 KO mice). In contrast with control 129Sv/C57Bl6 mice, sst2 mRNA was no longer detectable in the brain of sst2 KO mice; 125I-labeled Tyr0DTrp8-SRIF14 binding was also greatly reduced in almost all brain structures except for the hippocampal CA1 area, demonstrating that sst2 accounts for most SRIF binding in mouse brain. Invalidation of this subtype generated an increased anxiety-related behaviour in a number of behavioural paradigms, while locomotor and exploratory activity was decreased in stress-inducing situations. No major motor defects could be detected. sst2 KO mice also displayed increased release of pituitary ACTH, a main regulator of the stress response. Thus, somatostatin, via sst2 receptor isotype pathways, appears involved in the modulation of locomotor, exploratory and emotional reactivity in mice.

Somatostatin binding sites have been localized and quantified in the sheep brain using 125I-Tyr0-DTrp8-somatostatin, by quantitative high resolution light microscopic autoradiography. Sections were analyzed by densitometry on radioautographic film, and subsequently on slides coated with photoemulsion. Specific somatostatin binding sites were concentrated in the medial habenula, superior colliculus, dorsal motor nucleus of the vagus nerve, inferior olive, spinal trigeminal nucleus, and cerebellum. In competition experiments, octreotide, a sst2/sst3/sst5 selective agonist only partially displaced 125I-Tyr0-DTrp8-somatostatin in the three cerebellar layers while it was fully active as compared to somatostatin 14 and 28 in the deeper layers of the parietal cortex. Moderate to low somatostatin receptor densities were present in the mesencephalic periaqueductal gray, dorsal raphe, thalamic paraventricular nucleus, interpeduncular nucleus, pineal gland, dorsal tegmental, dorsolateral tegmental and parabrachial nuclei, nucleus of the solitary tract. The distribution of somatostatin binding sites generally correlates with the data obtained on slides dipped in photoemulsion which provided better resolution and more precise localization. In most of the labeled areas, 125I-Tyr0-DTrp8-somatostatin receptor binding was distributed between both neuropil and perikarya. Perikarya bearing 125I-Tyr0-DTrp8-somatostatin receptors were observed in areas which did not display detectable binding sites on film such as the preoptic-anterior hypothalamic complex and arcuate nucleus and in the locus coeruleus. In conclusion, the distribution of 125I-Tyr0-DTrp8-somatostatin binding sites in sheep brain is very reminiscent of other mammals being closer to the human than to rodents.

The regional brain distribution of 125I-somatostatin (SRIH) binding sites was determined by quantitative radioautography in neonatally monosodium glutamate (MSG) treated adult male rats, a procedure which selectively destroys most neurons of the arcuate nucleus. Neonatal MSG treatment did not modify the extrahypothalamic distribution of 125I-SRIH-binding sites. In contrast, the number of 125I-SRIH-labeled cells in the ventrolateral part of the arcuate nucleus was strongly reduced in MSG-treated animals. The effect was selective for the anterior part of the arcuate nucleus and was not found in its posterior part or in the cells located more dorsally, beneath the ependymal zone of the periventricular nucleus. Intracerebroventricular SRIH injections, which increased growth hormone levels in control rats, were totally ineffective in MSG-treated animals. In contrast, the prolactin levels were equally stimulated by intracerebroventricular injections in control and MSG-treated animals. These results demonstrate that extrahypothalamic SRIH-binding sites are not located on neurons originating in the anterior arcuate nucleus neurons. In addition, 125I-SRIH-labeled cells in the ventrolateral part of the arcuate nucleus are necessary for the paradoxical stimulation of growth hormone secretion induced by intracerebroventricular SRIH injection, but do not seem to be essential for the increased prolactin secretion observed under these conditions.

PURPOSE. In the mouse model of oxygen-induced retinopathy (OIR) somatostatin (SRIF) acting at the SRIF receptor subtype 2 (sst2) inhibits angiogenic responses to hypoxia through a downregulation of vascular endothelial growth factor. Information on the sites where SRIF-sst2 interactions take place is lacking, and downstream effectors mediating SRIF-sst2 antiangiogenic actions are unknown. METHODS. In the OIR model, retinal expression of SRIF was evaluated with RT-PCR and RIA. The bindings of [125I]LTT-SRIF-28 and [125I]Tyr3-octreotide were measured in coronal sections of the eye. With Western blot we evaluated the levels of sst2A as well as the expression and the activity of the Signal Transducer and Activator of Transcription (STAT)3. The analysis of STAT3 was performed in hypoxic mice treated with the sst2 agonist octreotide or with the sst2 antagonist D-Tyr8 cyanamid 154806 (CYN). Retinal localization of sst2A was assessed by single and double immunohistochemistry with an endothelial cell marker. RESULTS. In the hypoxic retina, both SRIF and sst2 levels as well as [125I]Tyr3-octreotide binding were downregulated. In addition, sst2A immunostaining was decreased in the neuroretina, but was increased in capillaries. Hypoxia increased both expression and activity of STAT3. This increase was inhibited by octreotide, while was strengthened by CYN. CONCLUSIONS. These data suggest that i. sst2 expressed by capillaries may be responsible of the antiangiogenic effects of SRIF and ii. downstream effectors in this action include the transcription factor STAT3. These results support the possibility of using sst2- selective ligands in the treatment of proliferative retinopathies and indicate STAT3 as an additional target for novel therapeutic approach. L'articolo è disponibile sul sito dell'editore http://www.arvo.org/eweb/StartPage.aspx?Site=arvo2

International audience; 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.

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.

Somatostatin (SRIF) receptors (sst) are present on normal pancreatic endocrine β-cells. However, the use of SRIF analogs in the scintigraphic imaging of insulinomas and in the medical management of these tumors seems to be restricted to a subgroup of patients. The aim of this study was to determine the prevalence of sst expression in vitro and characterize sst subtype binding in insulinomas and its correlation with in vivo sst receptor scintigraphy (SRS). In vitro studies were performed on 27 insulinomas from 25 patients: 22 with benign and three with malignant tumors. Semiquantitative RT-PCR of sst mRNAs was performed for 20 of these insulinomas. Sst2 and sst5 were expressed in 70%, sst1 in 50%, and sst3 and sst4 subtypes only in 15–20% of the tumors. 125I-Tyr0DTrp8SRIF14 binding was assessed by quantitative autoradiography in 18 insulinomas, and competition experiments were performed with SRIF14 and L797–591, L779–976, L796–778, L803–087, L817–818, selective agonists of the five sst subtypes, and BIM23244, a selective agonist of sst2 and sst5. Significant specific binding was observed in 72% of the insulinomas. Displacement experiments with ligands of higher affinity for each of the sst receptors revealed significant binding with the sst2 and sst5 ligands in 72%, sst3 in 44%, sst1 in 44%, and sst4 in 28% of cases. All insulinomas displaying sst2 binding were also sst5 sensitive. However, the ratio of sst5/sst2 displacement was variable and only equal to that for SRIF14 in experiments with the sst2/sst5 agonist BIM23244. SRS was performed 10 times in nine patients; it detected 60% of the tumors, including metastases of a malignant insulinoma. All the tumors detected by SRS displayed high levels of 125I-Tyr0DTrp8SRIF14 binding. The mechanisms underlying the loss of expression of sst2/sst5 in a third of insulinomas remains to be determined, but this loss of expression may be involved in β-cell dysfunction.

Somatostatin receptors on lactotroph cells of the anterior pituitary are positively regulated by estradiol. In the present work, we investigated whether estradiol regulation of somatostatin receptors also occurred in the female rat brain. 125I-Tyr0-DTrp8-somatostatin (125I-SRIF: 780 Ci/mM) was used as a ligand. Female adult rats were ovariectomized and treated or not with estradiol benzoate (20 micrograms/day for 1 or 8 days). In female brains, 125I-SRIF binding, as assessed by film radioautography, was high in the basolateral amygdala, CA1 field and dentate gyrus of the hippocampus and locus coeruleus, moderate in the median habenula and deep layers all through the cortex. Castration or estradiol treatment did not modify 125I-SRIF binding in these regions. By light-microscopic radioautography, a subpopulation of 125I-SRIF-labeled cells was localized in the ventrolateral portion of the arcuate nucleus. Ovariectomy alone did not significantly affect the number and binding density of 125I-SRIF-labeled cells in the arcuate nucleus. However, estradiol treatment in ovariectomized animals significantly increased both parameters. Along the estrus cycle, the number of 125I-SRIF-labeled cells was not significantly modified but 125I-SRIF binding density was significantly higher in proestrus as compared to diestrus I, diestrus II and estrus. These results demonstrate that brain 125I-SRIF binding sites are positively regulated by estradiol only in the arcuate nucleus of the hypothalamus.

In the present work, we have characterized by film radioautography the effects of divalent cations and guanine nucleotide on specific receptor for somatostatin (SRIF) using 125I-TyrO-DTrp8-SRIF14 (125I-ToD8-SRIF) as a ligand. The experiments were performed on coronal 20-microM-thick sections cut at the level of the amygdala, thus allowing to study binding sites in several regions enriched in binding sites (frontal cortex, hippocampus CA1 and dentate gyrus, habenula, basolateral nucleus of the amygdala). In a preliminary set of experiments using brain cortical membranes it was found that 3 mM Mg2+ ions doubled the specific binding of 125I-ToD8-SRIF. However, Mg2+ enhanced equally by a factor of 3 affinities of high- and low-affinity binding sites as evidenced by SMS 201.995 displacement curves without modifying the ratio between high and low affinity sites. In radioautographic studies while SRIF14 and SRIF28 elicited monophasic displacement curves, SMS 201.995 displaced 125I-ToD8-SRIF binding in a biphasic manner in all regions tested but the baso-lateral nucleus of the amygdala. Radioautographic distribution of 125I-ToD8-SRIF binding sites was identical whether the sections were incubated with MgCl2 or with MnCl2 and almost undetectable in the absence of ions. In all structures investigated increasing concentrations of GTP totally inhibited 125I-ToD8-SRIF binding with an IC50 value of 3 microM. In conclusion, our results demonstrate that 125I-ToD8-SRIF-binding sites in brain occur on two different affinity states as assessed by a displacement curve using endogenous ligands and SMS 201.995. According to the comparable effects of divalent cations and GTP, the two subtypes of 125I-ToD8-SRIF-binding sites discriminated by SMS 201.995 are likely to correspond to interconvertible forms of the same receptor coupled to a G protein-transducing system.

Somatostatin (SRIF) actions in the brain and pituitary are mediated by specific receptors. Using radioiodinated ligands it has been possible to characterize the kinetics of specific binding sites in the brain and pituitary, and to determine their cellular localization by autoradiography. At the pituitary level, the inhibition of growth hormone, prolactin and thyrotropin secretions induced by SRIF is mediated through a single binding site which is coupled to the inhibition of adenylate cyclase. In the brain, SRIF receptors are localized on neurons and glial cells and are also coupled to adenylate cyclase inhibition. Two sites are differentiated in the brain with an analogue of somatostatin, SMS 201995. In humans, SRIF-binding sites have been related to a number of pathologies. At the pituitary level, it has been shown that the number of binding sites was negatively correlated to growth hormone levels in acromegaly. Furthermore, SRIF-binding sites were undetectable in a patient which did not respond to SMS 201995 therapy. In the brain, meningiomas and gliomas are rich in SRIF binding sites. This suggests a possible role for SRIF on glia. In neurodegenerative diseases, cortical SRIF concentrations are decreased in Alzheimer's and Parkinson's disease associated with dementia while SRIF-binding sites are only affected in Alzheimer's disease. In conclusion, the physiological role of SRIF in the brain and pituitary can be evaluated by studying the receptors of the peptide. Such studies allow to question the implication of SRIF in endocrine and neuropathologies.