Application of 89Zr-DFO*-immuno-PET to assess improved target engagement of a bispecific anti-amyloid-ß monoclonal antibody.

PURPOSE: The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders. METHODS: Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-ß plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease. RESULTS: A 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice. CONCLUSION: 89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aß plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.

Distribution of serotonin receptor 5-HT6 mRNA in rat neuronal subpopulations: A double in situ hybridization study.

The 5-HT6 receptor (5-HT6R) is almost exclusively expressed in the brain and has emerged as a promising target for cognitive disorders, including Alzheimer's disease. In the present study, we have determined the cell types on which the 5-HT6R is expressed by colocalizing 5-HT6R mRNA with that of a range of neuronal and interneuronal markers in the rat brain. Here, we show that 5-HT6R mRNA was expressed at high levels in medium spiny neurons in caudate putamen and in nucleus accumbens, as well as in the olfactory tubercle. Striatal 5-HT6R mRNA was colocalized with both dopamine D1 and D2 receptor mRNA. 5-HT6R mRNA was moderately expressed in the hippocampus and throughout cortical regions in glutamatergic neurons coexpressing vGluT1. A subset of GAD67-positive GABAergic interneurons (approximately 15%) expressed 5-HT6R mRNA in the cortex and hippocampus, the majority of which belonged to the 5-HT3a receptor (5-HT3aR)-expressing subpopulation. In contrast, 5-HT6R mRNA was only expressed to a minor extent in the parvalbumin and somatostatin subpopulations. A subset of calbindin- and calretinin-positive GABAergic interneurons expressed 5-HT6R mRNA while only a very minor fraction of VIP or NPY interneurons in forebrain structures expressed 5-HT6R mRNA. Serotonergic, dopaminergic or cholinergic neurons did not express 5-HT6R mRNA. These data indicate that the 5-HT6R is located on GABAergic and glutamatergic principal neurons, and on a subset of interneurons mainly belonging to the 5-HT3aR subgroup suggesting that the 5-HT6R is positioned to regulate the balance between excitatory and inhibitory signaling in the brain. These data provide new insights into the mechanisms of 5-HT6R signaling.

Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation.

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimers disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis.

The immunohistochemical localisation of somatostatin receptors 1, 2, 3, and 5 in acoustic neuromas.

AIMS: Acoustic neuroma is a benign tumour, which develops through an overproliferation of Schwann cells along the vestibular nerve. Somatostatin is a naturally occurring peptide, which exerts antiproliferative and antiangiogenic effects via five membrane bound receptor subtypes. The aim of this study was to determine whether somatostatin receptor subtypes (SSTRs) 1, 2, 3, and 5 are present in acoustic neuromas. METHODS: The expression of SSTRs 1, 2, 3, and 5 was studied in both the Schwann cells and blood vessels of eight acoustic neuroma specimens, by means of immunohistochemistry using novel rabbit polyclonal antibodies raised against human SSTR 1, 2, and 5 subtype specific peptides, and a commercial anti-SSTR3 antibody. RESULTS: SSTR2 was the most prevalent subtype in Schwann cells (seven of eight), with intermediate expression of SSTR3 (six of eight), and lower expression of SSTRs 1 and 5 (four of eight and five of eight, respectively). There was ubiquitous vascular expression of SSTR2, with no evidence of SSTR 1, 3, or 5 expression in blood vessels. CONCLUSION: SSTRs 1, 2, 3, and 5 are differentially expressed in acoustic neuromas. Somatostatin analogues may have a therapeutic role in the management of this rare and challenging condition.

Comparison of somatostatin receptor expression in human gliomas and medulloblastomas.

The expression of the five somatostatin receptor subtypes, sst1-5 was compared on tissue containing glial tumours (glioblastomas or oligodendrogliomas), medulloblastomas, and on normal human cortex. By semiquantitative reverse 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, sst1 and sst3 being mostly expressed. By 125I-Tyr0DTrp8 somatostatin-14 or 125I-Leu8DTrp22 Tyr25 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. sst2 immunocytochemistry demonstrated immunostaining of neuronal cells and neuropile; sst2 and sst3 immunostaining was identified on glioblastoma proliferating vessels endothelial cells and on medulloblastomas tumoral cells. Faint sst2 immunostaining among glial tumoral cells was due to microglia, while glioma cells did not significantly stain. In summary, medulloblastoma tumoral cells express sst2/sst3 receptors at a high level while glioma cells do not. In gliomas, sst expression is restricted to endothelial cells on proliferating vessels (displaying both sst2 and sst3 receptors), including parenchyma and reactive microglia (only sst2). The differential expression of sst2/sst3 receptors on gliomas and medulloblastomas has implications for the therapy of these tumours.

Localisation of somatostatin and somatostatin receptors in benign and malignant ovarian tumours.

Somatostatin has been identified as having anti-proliferative, anti-angiogenic and pro-apoptotic actions in many tumour systems, and these effects are mediated through a family of five transmembrane G-protein coupled SRIF receptors. Ovarian cancer is the commonest gynaecological malignancy in the UK and maintenance therapy is urgently required. Native somatostatin expression and its receptors sst(1,2,3 and 5) were studied with immunohistochemistry in 63 malignant and 35 benign ovarian tumours of various histological types. Fifty-seven out of 63 (90%) of malignant and 26/35 (74%) benign tumours expressed somatostatin. Receptors sst(1,2,3 and 5) were expressed variably in epithelial, vascular and stromal compartments for both benign and malignant tumours. Somatostatin was found to correlate significantly with stromal sst(1) (P=0.008), epithelial sst(1) (P<0.001), stromal sst(2) (P=0.019), vascular sst(2) (P=0.026), epithelial sst(3) (P=0.026), stromal sst(5) (P=0.013) and vascular sst(5) (P=0.038). Increased expression of native somatostatin correlating with somatostatin receptors in malignant ovarian tumours raises the possibility that either synthetic somatostatin antagonists or receptor agonists may have therapeutic potential.

Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis.

Somatostatin receptors (SSTRs) have been extensively mapped in human tumors by means of autoradiography, reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). We analyzed the SSTR type 1-5 expression by means of RT-PCR and/or IHC in a series of 81 functioning and non-functioning gastroenteropancreatic (GEP) endocrine tumors and related normal tissues. Moreover, we compared the results with clinical, pathological and hormonal features. Forty-six cases (13 intestinal and 33 pancreatic) were studied for SSTR 1-5 expression using RT-PCR, IHC with antibodies to SSTR types 2, 3, 5 and ISH for SSTR2 mRNA. The vast majority of tumors expressed SSTR types 1, 2, 3 and 5, while SSTR4 was detected in a small minority. Due to the good correlation between RT-PCR and IHC data on SSTR types 2, 3, and 5, thirty-five additional GEP endocrine tumors were studied with IHC alone. Pancreatic insulinomas had an heterogeneous SSTR expression, while 100% of somatostatinomas expressed SSTR5 and 100% gastrinomas and glucagonomas expressed SSTR2. Pre-operative biopsy material showed an overlapping immunoreactivity with that of surgical specimens, suggesting that the SSTR status can be detected in the diagnostic work-up. It is concluded that SSTRs 1-5 are heterogeneously expressed in GEP endocrine tumors and that IHC is a reliable tool to detect SSTR types 2, 3 and 5 in surgical and biopsy specimens.

Somatostatin receptor 2 expression in the human endometrium through the menstrual cycle.

OBJECTIVE: Somatostatin mediates its many inhibitory functions through five G-protein-coupled receptors (sstr1-5); however, it is not known whether somatostatin or its receptors are present in the endometrium. DESIGN: We have used immunohistochemistry on formalin-fixed paraffin-embedded sections of normal human endometrium from the menstrual (n = 6), proliferative (n = 15) and secretory (n = 10) stages of the endometrial cycle to determine the pattern of expression of somatostatin receptor (sstr) subtype 2. In addition, we have used quantitative polymerase chain reaction (PCR) to determine the level of expression of the sstr2 mRNA in 17 samples of normal human endometrium. PATIENTS: Endometrial tissue had been removed from patients undergoing dilation and curettage (D&C) for menorrhagia and had been determined to be normal histologically. MEASUREMENTS: Immunostaining in the epithelium, endothelium and the stroma of the endometrial sections was characterized and was scored positive or negative. The PCR results were analysed using the software provided to standardize the expression of sstr2 against that of constitutively expressed beta-glucoronidase in the same sample. A final percentage value of the level of sstr2 expression was then determined. RESULTS: sstr2 was expressed variably throughout all the stages of the menstrual cycle in the epithelium, the endothelium and the stroma. In particular, the position of sstr2 expression varied in the epithelial cells surrounding the endometrial glands from being basal or diffuse in the proliferative and secretory phase to being lumenal in the menstrual stage. Quantitative PCR showed that 15 of 17 samples expressed sstr2 mRNA and the level of expression between individual samples varied dramatically. CONCLUSIONS: These data show that sstr2 is present in the endometrium and its location seems to vary through the menstrual cycle.

Leptin receptor immunoreactivity is present in ascending serotonergic and catecholaminergic neurons of the rat.

Using double-labelling immunohistochemistry we have studied the localisation of leptin receptor proteins including both long and short forms and their possible presence in serotonergic (5-HT) and catecholaminergic neurons in the rat brain. Leptin receptor immunoreactivity was found to be widely distributed in the central nervous system including cortical areas, amygdala, several hypothalamic and thalamic nuclei, the raphe system, pontine nuclei, locus coeruleus, parabrachial nucleus, tractus solitarus and the medullary reticular formation. Serotonergic cell groups were identified by 5-HT immunocytochemistry and classified according to standard nomenclature. High degrees of co-existence of leptin receptor immunoreactivity with serotonin in the raphe system were observed in B1, B5, B6, B7, B8 and B9. In B3 and B2 less than 50% of the 5-HT cells colocalised leptin receptor immunoreactivity. Brainstem and diencephalic (catecholaminergic) neurons were identified by tyrosine hydroxylase immunocytochemistry and classified according to standard nomenclature. Within the periventricular hypothalamic dopaminergic nuclei A14 and A12, the metencephalic noradrenergic A6, A7, A2, A1, and the adrenergic C3, C2 and C1 cell groups, nearly all tyrosine hydroxylase-positive cells colocalised with leptin receptor immunoreactivity. In contrast, co-existence of tyrosine hydroxylase and leptin receptor immunoreactivities in the dopaminergic A13, A11, A10, A9 and A8 cell was practically non-existent. Thus leptin, the adipose tissue-derived ligand of the leptin receptor, may in some brain areas directly influence serotonergic, dopaminergic, adrenergic and noradrenergic inputs to the periventricular and medial hypothalamic nuclei.

In vivo internalization of the somatostatin sst2A receptor in rat brain: evidence for translocation of cell-surface receptors into the endosomal recycling pathway.

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.

Localization of somatostatin receptors at the light and electron microscopial level by using antibodies raised against fusion proteins.

Somatostatin mediates its multiple biological effects via specific plasma membrane receptors belonging to the family of G-protein coupled receptors with seven putative membrane-spanning domains. Five somatostatin receptor subtypes (sst1-sst5) have been cloned in human, mouse, and rat. We have raised specific antibodies against the five human somatostatin receptors by using the fusion protein technique. DNA sequences encoding C-terminal parts of the somatostatin receptors were inserted into a pGEX-2T plasmid vector. E. coli bacteria were transformed with the recombinant plasmid and fusion proteins were expressed and purified using the glutathione S-transferase Gene Fusion System. The fusion proteins were emulsified with Freund's complete adjuvant and polyclonal antibodies were raised in rabbits. The antisera were tested for specificity in Western blot analysis of membrane preparations from cell lines expressing the receptors and in membrane preparations of brain tissues. The receptors were visualized at the light microscopical level in paraformaldehyde fixed tissue sections by use of biotin labelled secondary antibodies as well as by amplification with biotinylated tyramide. The final step in the immunohistochemical visualization of the receptors was done by both peroxidase labelled streptavidin/biotin and different fluorophores. At the electron microscopical level, some of the receptors could be visualized in tissues fixed with a combination of paraformaldehyde and low concentrations of glutaraldehyde. In the hamster brain, sst2 receptors labelling was observed in both neuronal processes and perikarya. The staining was present in neo-, and allocortical areas of the forebrain, the hypothalamus, brain stem, and spinal cord. In the rat and human, sst1 receptor was shown to be an auto receptor on somatostatinergic neurons located in the hypothalamus. In the retina both sst1 and sst2 receptors were present. sst1 receptors were confined to amacrine cells, few ganglionic cells, and Müller cell-end feet. sst2 receptors were more widespread than the sst1 receptors. sst2-immunoreactivity was present in dopaminergic amacrine cells, the Müller cell-end feet, and in the inner segments of the cone photoreceptors. Thus, the availability of subtype specific antibodies against the five somatostatin receptors makes it possible to identify the receptors involved in the multiple somatostatinergic system in the body.

Somatostatin and somatostatin receptors in the pig pineal gland during postnatal development: an immunocytochemical study.

An immunohistochemical study of the pineal gland of the domestic pig was carried out using rabbit antisera raised against synthetic peptide fragments corresponding to different amino acid sequences of the prosomatostatin, the somatostatin-14, and the somatostatin-28 molecule. The study was supplemented by immunohistochemical staining with rabbit antisera raised against five subtypes of somatostatin receptors. The pineal glands were taken from the newborn, 21-day-old and 7-month-old pigs. Immunoreactive nerve fibers and cells were observed in the pineal gland with all the antisera against somatostatin and prosomatostatin. The nerve fibers were located throughout the pineal gland-in the capsule, connective septa, and parenchyma-with the highest density in proximo-ventral part of the gland. The somatostatin positive fibers were also found in the habenular and posterior commissurae areas. Somatostatin-immunoreactive cell bodies were observed mostly in the central part of the gland. These results point to the existence of two somatostatin sources in the pig pineal gland: 1) nerve fibers, probably of central origin; and 2) cells that may represent intrapineal neurons or specialised pinealocytes. A clear difference in the immunoreactivity between newborn, 21-day-old, and 7-month-old pigs was found. Generally, the density of nerve fibers was lower in adult than young animals. The number of the cells also decreased with age. By using the antisera against the five somatostatin receptors, only sst3 - receptor immunoreactivity could be detected. The receptor-immunoreactivity was confined to varicose and smooth fibers and some cells. The sst(3)-receptor positive structures were localised in all parts of the gland and their number was higher in younger pigs.

Involvement of the Sst1 somatostatin receptor subtype in the intrahypothalamic neuronal network regulating growth hormone secretion: an in vitro and in vivo antisense study.

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.

Immunohistochemical localization of somatostatin receptor subtypes sst1 and sst2 in the rat retina.

PURPOSE: To investigate the distribution of somatostatin receptor subtypes sst1 and sst2 in the rat retina by immunohistochemistry and to characterize further the neurotransmitters of the sst1- and sst2-immunoreactive cells. METHODS: Polyclonal antibodies raised against sst1 and sst2 receptors were applied to 12-microm cryostat sections of rat retinas fixed in paraformaldehyde. Further, immunofluorescence double labeling was performed for the sst1 and sst2 receptors with somatostatin, tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD). RESULTS: Immunoreactivity for sst1 was present in somatostatinergic amacrine cells located in the inner nuclear layer (INL) and in displaced amacrine cells in the ganglion cell layer of the retina. Also, a small number of ganglion cells were sst1 immunoreactive. Immunoreactivity for sst2 was observed in many medium-sized amacrine cells in the middle part of the INL, with a central process projecting to the sublaminae of the inner plexiform layer. Furthermore, sst2 immunoreactivity was found in large amacrine cells of the INL. These cells also contained TH. Inner segments of cone receptors were stained with the sst2 antiserum. Immunostaining for sst2, and to a minor extent for sst1, was found in Müller cell fibers. None of the somatostatin receptors colocalized with GAD. CONCLUSIONS: These findings suggest that the sst1 receptor may function as an autoreceptor on retinal somatostatinergic cells. The presence of sst2 receptors on the TH-immunoreactive amacrine cells indicates an influence of somatostatin on the secretion of dopamine in rat retina.

Immunohistochemical localization of the somatostatin receptor subtype 2 (sst2) in the central nervous system of the golden hamster (Mesocricetus auratus).

The many actions of somatostatin in the central nervous system are mediated through specific membrane receptors of which five have been cloned. In this study, we have investigated the distribution of one of these receptors, the sst2 subtype, in the brain and spinal cord of the golden hamster (Mesocricetus auratus). Immunohistochemistry was carried out by using polyclonal antibodies raised against the C-terminal part of the human sst2 receptor. sst2 immunoreactivity was found in the forebrain, brainstem, cerebellum, and spinal cord. In the forebrain, strong immunoreactivity was observed in the deep layers of the neocortex as well as in the endopiriform cortex, claustrum, and basolateral amygdaloid nucleus. Immunoreactivity was also found in the CA1 area of the hippocampus and in the subiculum. In the diencephalon, staining was observed in the periventricular area, the dorsomedial and arcuate nuclei of the hypothalamus, and the medial habenular nucleus. Other areas such as the thalamus, striatum, and globus pallidus were almost devoid of staining. In the brainstem, strong immunoreactivity was observed in the locus coeruleus and the parabrachial nucleus. In addition, immunostaining was observed in the cortex of the cerebellum. In the spinal cord, intense immunoreactivity was seen in lamina I and II of the dorsal horn. Finally, immunoreactive cells were widely distributed in the anterior pituitary. The localization of the sst2 receptor in many brain regions suggests that this receptor subtype is involved in different neuromodulatory actions of somatostatin such as somatosensory, motor, memory, and neuroendocrine functions.

Immunohistochemical and cytochemical localization of the somatostatin receptor subtype sst1 in the somatostatinergic parvocellular neuronal system of the rat hypothalamus.

Somatostatin is known to mediate its actions through five G-protein-coupled receptors (sst1-sst5). We have studied the expression of the sst1 receptor in the rat hypothalamus by using a subtype-specific antiserum. In Western blotting, the antiserum reacted specifically with a band with an apparent molecular weight of 80,000 in membranes prepared from hypothalamic tissue. The localization of the sst1 receptor was investigated by immunohistochemistry in hypothalamus sections. Additionally, an immunofluorescent double-labeling was performed for the sst1 receptor and somatostatin. Light microscopy revealed that the sst1 receptor is located in perikarya and nerve fibers in the rostral periventricular area surrounding the third ventricle as well as in nerve fibers projecting from the perikarya to the external layer of the median eminence. In these neuronal structures, sst1 immunoreactivity was found to be colocalized with somatostatin. Furthermore, the location of sst1 receptors was studied by immunoelectron microscopy in the median eminence. In the external layer, receptor immunoreactivity was confined to nerve terminals. Immunoreactive nerve terminals were seen to make synapse-like junctions with other both stained and unstained nerve terminals. Thus, the sst1 receptor is present in the classic somatostatinergic hypothalamic parvocellular system inhibiting hormone secretion from the anterior pituitary gland. These findings indicate that the sst1 receptor may act as an autoreceptor and inhibit the release of somatostatin from periventricular neurons projecting to the median eminence.

Development of selective antibodies against the human somatostatin receptor subtypes sst1-sst5.

Antisera selective for five somatostatin receptor subtypes, human sst1-sst5, were raised in rabbits. C-terminal parts of human sst1-sst5 receptors were expressed as fusion proteins with glutathione S-transferase. Fusion proteins were affinity-purified and used for raising polyclonal antibodies. In Western blot analysis, all five antisera were tested on preparations of mammalian cell lines transfected with human sst1-sst5, respectively. sst1 antiserum reacted with a broad band of 53-72 kDa. A band of 71-95 kDa was detected with the antiserum raised against sst2, 65-85 kDa with sst3 antiserum, 45 kDa with sst4 antiserum and 52-66 kDa with sst5 antiserum. No cross-reactivity could be detected to any of the other four somatostatin receptor subtypes. Enzymatical deglycosylation of the receptors revealed that sst1, sst2, sst5 and possibly sst3 in this system are subjected to N-linked glycosylation, whereas sst4 is not. Two of the antisera (sst2 and sst5) were used for immunohistochemical localization of the receptors. sst2 and sst5 antisera labeled neurons in e.g. the amygdaloid complex, hippocampus, fascia dentata and the neocortex in rat and monkey tissue. This is the first report on antisera against all five somatostatin receptor subtypes and the first immunohistochemical visualization of sst5 receptors in the mammalian brain.