J. Physiol. (Paris) 94 (2000) 259–264© 2000 Elsevier Science Ltd. Published by Editions scientifiques et medicales Elsevier SAS. All rights reservedPII: S0928-4257(00)00212-6/REV

Localization of five somatostatin receptors in the rat central nervous systemusing subtype-specific antibodies

Stefan Schulz, Manuela Handel, Matthias Schreff, Harald Schmidt, Volker Hollt*

Department of Pharmacology and Toxicology, Otto-6on-Guericke Uni6ersity, 39120 Magdeburg, Germany

Received 21 October 1999; accepted 1 March 2000

Abstract – The cloning of five members of the somatostatin receptor family, sst1-sst5, as well as two isoforms of the somatostatinreceptor 2, sst2A and sst2B, enabled us to generate specific anti-peptide antisera against unique sequences in the carboxyl-terminal tailof each somatostatin receptor subtype. We used these antibodies in multicolor immunofluorescent studies aimed to examine theregional and subcellular distribution of somatostatin receptors in adult rat brain. Several findings are notable: The cloned sst1 receptoris primarily localized to axons, and therefore most likely functions in a presynaptic manner. The cloned sst2 receptor isoforms exhibitstrikingly different distributions, however, both sst2A and sst2B are confined to the plasma membrane of neuronal somata anddendrites, and therefore most likely function in a postsynaptic manner. The cloned sst3 receptor appears to be excluded from ‘classical’pre- or postsynaptic sites but is selectively targeted to neuronal cilia. The cloned sst4 receptor is preferentially distributed to distaldendrites, and therefore most likely functions postsynaptically. The cloned sst5 receptor was not detectable in the adult rat brain,however, prominent sst5 expression was found in the pituitary. Furthermore, sst1-containing axons either co-contained somatostatinor were closely apposed by somatostatin-positive terminals in a regional-specific manner. Neuronal somata and dendrites containingeither sst2A, sst2B or sst4 were found to exist in close proximity, although not necessarily synaptically linked, to somatostatin-positiveterminals. Together, in the central nervous system the effects of somatostatin are mediated by several different receptor proteins whichare distributed with considerable regional overlap. However, there appears to be a high degree of specialization among somatostatinreceptor subtypes with regard to their subcellular targeting. This subtype-selective targeting may be the underlying principal oforganization that allows somatostatinergic modulation of neuronal activity via both pre- and postsynaptic mechanisms. © 2000Elsevier Science Ltd. Published by Editions scientifiques et medicales Elsevier SAS

somatostatin / somatostatin receptor subtypes / antibodies / immunocytochemistry / targeting

1. Introduction

The cyclic tetradecapeptide somatostatin iswidely expressed throughout the body and is animportant regulator of endocrine and brain func-tions. Two biological active forms have been iden-tified in mammals, somatostatin-14 (SS-14) andthe amino-terminally extended somatostatin-28 [2].Recently, a somatostatin-like peptide, cortistatin,with a high degree of homology but a more re-stricted distribution has been isolated [3]. In thecentral nervous system, somatostatin acts as neu-rotransmitter and neuromodulator to regulate neu-ronal firing, and plays a role in the modulation ofcomplex behaviors such as motor activity andcognition (reviewed in [6]). At the level of thespinal cord, locally applied SS-14 inhibits firing of

nociceptive dorsal horn and dorsal root ganglianeurons (reviewed in [6]).

SS-14 mediates its diverse physiological actionsthrough a family of G protein coupled receptorscontaining seven transmembrane domains. Fivegenes encoding distinct somatostatin receptor sub-types, termed sst1-sst5, have so far been cloned inhumans and other species (reviewed in [1, 10]). Inaddition, the carboxy-terminal tail of the sst2 re-ceptor has been shown to undergo alternativesplicing yielding two isoforms, sst2A and sst2B. Anumber of studies has addressed the distributionof the mRNA for the five somatostatin receptorsubtypes (reviewed in [1, 10]). However, little in-formation is available about the cellular localiza-tion of the receptor proteins in the central nervoussystem. Although earlier autoradiographic bindingstudies have examined the overall distribution ofsomatostatin binding sites in mammalian brain,none of the somatostatin receptor ligands availableis sufficiently selective to allow definite discrimina-tion between the various receptor subtypes (re-viewed in [1, 10]).

Elucidation of the cellular and subcellular local-ization of the various somatostatin receptor sub-

Abbre6iations: ir, immunoreactive; Li, like immunoreactivity;MAP-2, microtubule-associated protein 2; SS-14, somatostatin-14; sst, somatostatin receptor.* Correspondence and reprints

E-mail address: volker.hoellt@medizin.uni-magdeburg.de (V.Hollt).

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Table I. Amino acid sequences of COOH-terminal regions ofsomatostatin receptors. Amino acid sequences for peptidescorresponding to COOH-terminal regions of the somatostatinreceptors that were used to immunize rabbits for polyclonalantibody production. Please note that there is a high degree ofhomology between the mouse and rat somatostatin receptors.Thus, all of these antibodies stain effectively both mouse andrat brain tissue.

EpitopeReceptor Antibody

ESGGVFRNGTCASRISTLrsst1 (374–391)a 56056291ETQRTLLNGDLQTSIrsst2A (355–369)

ADNSKTGEEDTMAWVrsst2B (329–343) 55747986rsst3 (417–428) TAGDKASTLSHL6002CQQEPVQAEPGCKQVPFTKTTTFmsst4 (362–384)b

QATLPTRSCEANGLMQTSRIrsst5 (344–363) 6003

a rsst, rat somatostatin receptor.b msst, mouse somatostatin receptor.

somatostatin receptors [7, 13–17]. This review isintended to summarize our light- and electron-mi-croscopic analysis of the cellular and subcellularlocalization of somatostatin receptors in the ratcentral nervous system.

2. Generation and characterization of somatostatinreceptor subtype-specific antibodies

In an effort to localize somatostatin receptorproteins in rat brain, we generated anti-peptideantibodies to the carboxyl-terminal regions of eachof the somatostatin receptor subtypes. The identityof the peptides used for immunizations is given intable I. Peptides were coupled via an amino-termi-nally added cysteine to keyhole limpet hemo-cyanin, and conjugates were then injected intogroups of two to three rabbits for polyclonal anti-body production. After extensive characterizationthe antisera given in table I were found to specifi-cally recognize their targeted receptor and not tocrossreact with other proteins present in braintissue [7, 13–17]. Antibody specificity was ascer-tained by the following criteria: First, in im-munodot-blot assays the anti-sst antisera

types would provide important insights into so-matostatinergic transmission. Therefore, much at-tention has been directed towards the localizationof somatostatin receptors in the central nervoussystem by immunocytochemical techniques [4, 5, 8,9, 11, 12, 18]. During the last years we havesuccessfully generated antibodies to all subtypes of

Figure 1. Immunofluorescent confocal images of rat brain and spinal cord sections stained with anti-sst1 antiserum {5605}. A, Incoronal forebrain sections, dense sst1-Li is seen in the eminentia mediana. B and C, in the medulla and spinal cord, sst1-Li is mostprominent in the spinal trigeminal tract and the superficial layers of the spinal cord. Moderately dense sst1-Li is also seen in theventral areas of the medulla and spinal cord. D, in virtually all regions, sst1-Li is targeted to nerve fibres and terminals that weremorphologically similar to varicose axons. 3V, third ventricle; ME, eminentia mediana; SP5, spinal trigeminal tract. Scale bars: A500 mm; B, C 1 000 mm; D 25 mm.

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Figure 2. Immunocytochemical distribution of sst2A, sst2B and somatostatin in the rat spinal cord. Coronal (A–C) or parasaggital(D–F) spinal cord sections were stained with either anti-sst2A antiserum {6291}, anti-sst2B antiserum {5574} or a mouse monoclonalanti-SS-14 antibody {K121}. Please note, (I) in the spinal cord sst2A-Li and sst2B-Li show strikingly different distributions. Whilesst2A-Li is prominent in the superficial layers of the dorsal horn (2A), sst2B-Li is seen throughout the spinal grey matter (2B). (II)Both sst2A-Li and sst2B-Li are primarily targeted to the somatodendritic compartment of spinal cord neurons (2D, E). (III) SS-14shows a complementary distribution with most dense SS-14-Li in the superficial layers of the dorsal horn (2F) and moderately denseSS-14-Li in the ventral areas of the spinal grey matter. Scale bars: A–D 250 mm; D–F 50 mm.

selectively detected their cognate peptides but notthe peptides corresponding to the carboxy-termi-nal region of other ssts. Second, immunocyto-chemical staining of stably transfected HEK 293cells revealed that the anti-sst antisera selectivelystained cells expressing their targeted receptor butdid not stain wild-type cells or cells transfectedwith other somatostatin receptors. Third, in West-ern blots the antisera detected a band of theappropriate molecular weight in stably transfectedHEK 293 cells as well as in brain tissue. Fourth,the antibodies revealed unique staining patterns intissue sections. This immunostaining was com-pletely abolished after preincubation of the anti-bodies with homologous but not with heterologouspeptides. Finally, the carboxy-terminal peptidesare likely to have served as somatostatin receptorspecific immunogen since these peptides werefound to have minimal homologies to other pep-tide sequences when aligned to current entries inthe EMBL databases using BLASTp or FASTa.Thus, these antisera were affinity purified againsttheir immunizing peptides and used for subsequentstudies.

3. Distribution and targeting of somatostatinreceptor subtypes in the rat central nervous system

3.1. The sst1 receptor is located primarilypresynaptic

sst1 Receptor-like immunoreactivity (Li) showedan overall discrete distribution in the central ner-vous system with prominent immunostaining inthe main olfactory bulb, nucleus accumbens,globus pallidus, ventral pallidum, medial habe-nula, lateral septum, amygdala, zona incerta, hy-pothalamus, eminentia mediana (figure 1A),substantia nigra, interpeduncular nucleus, peri-aquaeductal grey, fascial nucleus, granular layer ofthe cerebellar cortex, nucleus of the solitary tract,spinal trigeminal tract, superficial layers of thespinal cord, dorsal areas of the medulla and spinalcord (figure 1B, C) as well as the pituitary gland[17]. In most brain regions, sst1-Li was primarilyconfined to fibres and terminals that were morpho-logically similar to varicose axons, suggesting apresynaptic role of this receptor (figure 1D). Duallabeling experiments revealed that in many brain

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regions sst1-Li was often closely apposed by SS-14-immunoreactive (ir) fibres and varicosities while inother regions SS-14-Li was co-contained withinsst1-positive fibres and terminals [17]. These findingssuggest that the sst1 receptor is predominantlytargeted to the presynaptic compartment and,hence, in a position to modulate the release of eithersomatostatin itself or other neurotransmitters.

3.2. The sst2 receptor isoforms exhibit strikinglydifferent distributions

The carboxyl-terminal splice variants of the sst2

receptor are widely distributed throughout theadult rat brain. However, sst2A and sst2B appear tobe expressed by different populations of neurons.We have examined the distribution and targetingof these receptors at the level of the spinal cord[15, 16]. Prominent sst2A-Li was contained in adense network within the superficial dorsal horn atall spinal levels (figure 2A). This network consistedmostly of plasmalemma of neuronal perikarya anddendrites. sst2A-Li was also present in the interme-diolateral cell column. In parasaggital sections ofthe lamina II, sst2A-Li was present on small roundcell bodies with a star-shaped dendritic formation(figure 2D) [15]. sst2B-Li exhibited a strikingly dif-ferent staining pattern with prominent stainingthroughout the spinal grey matter (figure 2B).sst2B-Li was confined to the plasma membrane ofrelatively large neuronal perikarya and their proxi-mal dendrites. Interestingly, immunoreactive sst2B

receptors appeared to be not evenly distributedalong the plasma membrane of these neurons butrather in a patch-like manner. This was particu-larly striking on ventral horn motoneurons (figure2E) [16]. SS-14-Li formed a dense plexus in thesuperficial layers of the dorsal horn, the lateralspinal nucleus, the dorsal grey commisure and theintermediolateral cell column (figure 2C). In addi-tion, a moderately dense plexus of SS-14-Li wasobserved throughout the deeper layers of thespinal grey matter. Most SS-14-Li appeared innerve fibres that were morphologically similar tovaricose axons (figure 2F). In preparations of thespinal cord dually stained for SS-14-Li and eithersst2A- or sst2B-Li, somatostatin receptor-containingplasmalemma were often closely apposed by, butnot co-contained within, SS-14-positive nervefibres and terminals suggesting that both sst2A andsst2B may function in a postsynaptic manner [15,16].

3.3. The sst3 receptor is selecti6ely targeted toneuronal cilia

We observed somatostatin receptor sst3-Li inmany brain regions including the cerebral cortex,hippocampus, hypothalamus, amygdala and cere-bellum (figure 3) [5]. In all of these regions (exceptfor the cerebellar cortex), sst3-like immunoreactiv-ity was selectively targeted to 4 to 8 mm-longrod-shaped profiles which did not co-localize withaxonal or dendritic markers [7]. One immunoreac-tive profile was always associated with one neu-ronal cell body. This staining pattern was resistentto colchicine treatment and showed a closely over-lapping distribution with sst3 mRNA suggestingthat the receptor protein is not transported overlong distances. Electron microscopic analysis re-vealed that sst3-like immunoreactivity is localizedto the plasma membrane of neuronal cilia whichextended into an intercellular pocket and showed a9 + 0 filament pattern in their basal body andproximal segments [7]. Thus, the somatostatin re-ceptor sst3 demonstrates a unique example of a Gprotein-coupled receptor not localized to ‘classical’pre- or postsynaptic sites but selectively targeted to

Figure 3. Immunofluorescent confocal images of rat brainsections stained with anti-sst3 antiserum {7986}. A and C, incoronal forebrain sections sst3-Li is seen in many regionsincluding the cerebral cortex (A) and the ventromedial hypo-thalamic nucleus (C) but not in the caudate putamen. B, invirtually all of these brain regions, sst3-Li is selectively local-ized on small, rod-shaped profiles which represent neuronalcilia. C and D, sst3-ir profiles appear to be more denselypacked in many (e.g. ventromedial hypothalamic nucleus) butnot all in brain regions (e.g. eminentia mediana) where highlevels of SS-14-Li occur. 3V, third ventricle; CPu, caudateputamen; ME, eminentia mediana; VMH, ventromedial hypo-thalamic nucleus. Scale bars: A, C, D 1 000 mm; B 50 mm.

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Figure 4. Immunofluorescent confocal micrographs showing the regional and subcellular localization of sst4-Li in rat hippocampus.Coronal rat brain sections were immunofluorescently stained with anti-sst4 antiserum {6002}. A and B, sst4-Li is enriched in thehippocampal formation with high levels found in the Ammon’s horn and the hilar region of the dentate gyrus. C, sst4-Li is primarilytargeted to distal dendrites of CA1 pyramidal cells. DG, dentate gyrus; SO, stratum oriens; SP, stratum pyramidale, SR, stratum,radiatum. Scale bars: A, B 250 mm; C 50 mm.

neuronal cilia. The presence of the sst3 receptor onneuronal cilia suggests that these presumably non-motile cilia may not merely represent developmen-tal remnants but rather function as chemicalsensors of the immediate milieu [7].

3.4. The sst4 receptor is located predominantlypostsynaptic

sst4-Li was most prominent in many forebrainregions including the cerebral cortex, hippocam-pus, striatum, amygdala and hypothalamus (figure4) [13]. sst4-expressing neurons seem to target thisreceptor to their somatodendritic domain as evi-denced by its frequent co-localization with MAP-2.Analysis at the electron microscopic level revealedan exclusive postsynaptic sst4 localization at den-dritic shafts, symmetrical and in some instancesasymmetrical synapses [13]. In most regions, sst4-immunoreactive dendrites were closely apposed by,but not co-contained within, somatostatin-14-con-taining fibres and terminals. With one notableexception being the hilus of the dentate gyruswhere sst4-Li appeared to decorate processes ofsomatostatin-14-positive interneurons [13]. Thesefindings suggest that the sst4 receptor protein ispredominantly targeted to the somatodendritic do-

main where it most likely functions postsynapti-cally.

3.5. The sst5 receptor is prominent in the pituitary

Finally, we also examined the distribution ofsst5, however, failed to detect immunoreactive sst5

receptors in rat central nervous system. Neverthe-less, under the same condition robust sst5 expres-sion was seen in the anterior lobe of the pituitarygland (figure 5).

Figure 5. Immunofluorescent confocal images of rat pituitarystained with anti-sst5 antiserum {6003}. A, Approximately30 % of cells in the anterior lobe of the pituitary gland stainedpositive for sst5-Li. B, Corresponding adsorption control.Please note, sst5-Li was not detected in adult rat brain. Scalebar: A, B 100 mm.

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4. Conclusion

In the central nervous system, the effects ofsomatostatin are mediated by several different re-ceptor proteins. Many of the various somatostatinreceptor subtypes show greatly overlapping distri-butions. However, there appears to be a highdegree of specialization among somatostatin recep-tor subtypes with regard to their subcellular target-ing. While sst2A, sst2B and sst4 can mediateregional- and cell-specific postsynaptic responses,the sst1 is poised to modulate presynaptic re-sponses. In contrast, the sst3 receptor appears tobe excluded from ‘classical’ pre- or postsynapticsites, and, is selectively targeted to neuronal cilia.Thus, the subtype-selective targeting of somato-statin receptors in the central nervous system maybe the underlying principal of organization thatallows somatostatinergic modulation of neuronalactivity via both pre- and postsynaptic mecha-nisms.

Acknowledgements

We thank Dana Wiborny and Dora Nuß forexcellent technical assistance. This work was sup-ported by grant SCHU 924/4-1 (S.S.) from theDeutsche Forschungsgemeinschaft, grant I/75 172(S.S.) from the Volkswagen-Stiftung, grant 1908A/0025 (S.S.) from the Kultusministerium desLandes Sachsen/Anhalt, grant SFB 426 TPA2(V.H.) from the Deutsche Forschungsgemeinschaftand a grant from the Fonds der Chemischen In-dustrie (V.H.).

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