Immunohistochemical localization of the somatostatin receptor subtype 2 (sst2) in the central...

Immunohistochemical Localization of theSomatostatin Receptor Subtype 2 (sst2)

in the Central Nervous System of theGolden Hamster (Mesocricetus auratus)

LONE HELBOE,1* ANDERS HAY-SCHMIDT,2 CARSTEN E. STIDSEN,3

AND MORTEN MØLLER1

1Department of Medical Anatomy, Panum Institute, University of Copenhagen,2200 Copenhagen, Denmark

2Institute of Anatomy and Physiology, The Royal Veterinary University,1870 Frederiksberg, Denmark

3Department of Molecular Pharmacology, Novo Nordisk A/S, 2760 Maløv, Denmark

ABSTRACTThe 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 haveinvestigated the distribution of one of these receptors, the sst2 subtype, in the brain and spinalcord of the golden hamster (Mesocricetus auratus). Immunohistochemistry was carried out byusing polyclonal antibodies raised against the C-terminal part of the human sst2 receptor. sst2immunoreactivity was found in the forebrain, brainstem, cerebellum, and spinal cord. In theforebrain, strong immunoreactivity was observed in the deep layers of the neocortex as well asin the endopiriform cortex, claustrum, and basolateral amygdaloid nucleus. Immunoreactiv-ity was also found in the CA1 area of the hippocampus and in the subiculum. In thediencephalon, staining was observed in the periventricular area, the dorsomedial and arcuatenuclei of the hypothalamus, and the medial habenular nucleus. Other areas such as thethalamus, striatum, and globus pallidus were almost devoid of staining. In the brainstem,strong immunoreactivity was observed in the locus coeruleus and the parabrachial nucleus. Inaddition, 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, immunoreac-tive cells were widely distributed in the anterior pituitary. The localization of the sst2 receptorin many brain regions suggests that this receptor subtype is involved in different neuromodu-latory actions of somatostatin such as somatosensory, motor, memory, and neuroendocrinefunctions. J. Comp. Neurol. 405:247–261, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: neuropeptide; cortex; hypothalamus; brainstem; neuroanatomy

Based on its ability to inhibit the release of growthhormone, somatostatin was isolated from ovine hypotha-lamic extracts in 1973 (Brazeau et al.). The 14-amino acidcyclic peptide somatostatin-14 (SS-14) and its amino-terminal extended form, somatostatin-28 (SS-28), consti-tute the two bioactive forms of somatostatin. Somatostatinis widely distributed in the mammalian brain and iscontained in short interneurons as well as projectingneuronal pathways (Johansson et al., 1984). In addition toits involvement in hormone secretion, this peptide hasbeen shown to act as a neurotransmitter and neuromodula-tor of central effects such as cognitive function and forma-tion of memory (Matsuoka et al., 1994; Dournaud et al.,1996a), pain (Helmchen et al., 1995; Bereiter, 1997), as

well as feeding behaviour (Danguir, 1988; Feifel andVaccarino, 1990; Cummings, 1997). Furthermore, a changein the level of somatostatin in certain brain areas has beencoupled to brain disorders, such as Alzheimer’s (Davies etal., 1980) and Huntington’s diseases (Nemeroff et al.,1983).

Grant sponsor: Biotechnology Center for Cellular Communication; Grantsponsor: Lundbeck Foundation.

*Correspondence to: Lone Helboe, Department of Medical Anatomy,section B, The Panum Institute, Blegdamsvej 3, DK 2200 N Copenhagen.E-mail: [email protected]

Received 4 February 1998; Revised 29 September 1998; Accepted 8October 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 405:247–261 (1999)

r 1999 WILEY-LISS, INC.

In recent years, five somatostatin receptor subtypes,designated sst1 through sst5, have been identified bymolecular cloning (Schindler et al., 1996). sst2 has beencloned from human (Yamada et al., 1992) and rat (Kluxenet al., 1992). In mouse, two splice variant forms of sst2 werefound, sst2a and the 23-amino acid residue shorter formsst2b, which differ in 15 amino acids at the C-terminus(Vanetti et al., 1992). On the basis of radioligand-bindingstudies, somatostatin receptors were originally dividedinto two classes: SRIF1 (corresponding to the clonedreceptors sst2, sst3, and sst5), which bind somatostatinanalogues such as octreotide with high affinity, and SRIF2(corresponding to sst1 and sst4), showing little affinity foroctreotide (see Bruns et al., 1995).

Previously, the distribution of somatostatin receptorshas been investigated by autoradiographic binding studieswith radioligands (Reubi and Maurer, 1985; Katayama etal., 1990; Holloway et al., 1996) and by in situ hybridiza-tion (Breder et al., 1992; Perez et al., 1994; Senaris et al.,1994; Beaudet et al., 1995; Harrington et al., 1995). Thesestudies revealed a widespread expression of somatostatin-binding sites and receptor mRNA throughout rodent andhuman brain.

Only recently have specific antibodies been developedagainst receptor subtypes and used in mapping the cellu-lar localization of somatostatin receptors. Two recentpapers describe the distribution of the sst2a receptor in ratbrain (Dournaud et al., 1996b; Schindler et al., 1997). As aconsequence of the availability of still more subtype-selective analogues, the localization of somatostatin recep-tors in different physiological systems becomes increas-ingly important. However, there is still only sparseinformation about the roles of individual receptor subtypesin the central nervous system. We have recently raisedrabbit polyclonal antibodies against the five human somato-statin receptors and characterized their specificity byimmunoblotting (Helboe et al., 1997). In this study, we lookin detail at the localization of one of these receptors,subtype sst2, in the brain and spinal cord of the goldenhamster by using an antiserum directed against thecarboxy-terminal part of the sst2 receptor. The goldenhamster is widely used as an animal model in the study ofcircadian rhythms and has been used in several investiga-tions in our laboratory of the anatomy of the circadianrhythm generating centres of the central nervous system(Møller and Korf, 1987; Cozzi and Møller, 1988; Leander etal., 1998).

MATERIALS AND METHODS

Immunoblot

Adult male golden hamsters (Mesocricetus auratus) wereanaesthetized by intraperitoneal injection of tribrometha-nol (400 mg/kg) and killed by decapitation. A brain areacorresponding to the ventrolateral cortical area around therhinal sulcus was dissected out and homogenized in 50 mMTris base, 1 mM EGTA, 5 mM MgCl2, pH 7.4, supple-mented with proteinase inhibitors bacitracin 200 µg/ml,leupeptin 2 µg/ml, phenylmethylsulphonyl fluoride 100µg/ml. The homogenate was pelleted and rehomogenizedin the same buffer. Twenty-five micrograms of membraneprotein was reduced with dithiothreitol, electrophoresedon 10% sodium dodecyl sulphate (SDS)-polyacrylamidegel, and blotted onto a nitrocellulose membrane. The blotwas saturated with 5% dry milk in TBS-T (50 mM Tris-

HCl, 150 mM NaCl, pH 7.5, containing 0.1% Tween 20).The blot was incubated for 1 hour at room temperaturewith antiserum directed against the C-terminus of thehuman sst2 receptor at a dilution of 1:500 (for details, seeHelboe et al., 1997).After washing, horseradish peroxidase-conjugated swine anti-rabbit IgG (DAKO, Copenhagen,Denmark) at 1:2,000 was applied to the membrane andincubated for 1 hour. Immunoreactive bands were detectedby using enhanced chemiluminescence (Amersham, LittleChalfont, UK).

Tissue preparation

Adult male golden hamsters (M. auratus) weighingapproximately 100 g were anaesthetized by intraperito-neal injection of tribromethanol (400 mg/kg) and fixed byvascular perfusion with 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4) for 15 minutes. The brains andspinal cords were removed, postfixed overnight in thesame fixative, and changed to 0.1 M phosphate-bufferedsaline (PBS). The specimens were cryoprotected in 30%sucrose in PBS and were sectioned into 40-µm-thickcoronal cryosections and transferred to PBS, or into 20-µmsagittal or horizontal cryosections on gelatinized glassslides. Coronal 40-µm-thick cryosections were preparedfrom the spinal cord at cervical, thoracic, lumbal, andsacral levels. Pituitaries were cut into 20-µm cryosectionsand placed on gelatinized glass slides.

All experiments with animals were performed in accor-dance with Principles of Laboratory Animal Care (NIHpubl. no. 86–23, revised 1985) as well as in accordancewith Danish national laws.

Immunohistochemistry

Reactions on coronal brain sections and spinal cordswere performed free-floating. Pituitary, sagittal, or horizon-tal brain sections were incubated on glass slides. Speci-mens from a total of six animals were tested. Endogenousperoxidase activity was quenched by incubating the sec-tions in 1% H2O2 in PBS for 15 minutes. This was followedby a 20-minute preincubation with 5% normal swineserum and 1% bovine serum albumin (BSA) in PBS/0.3%Triton X-100. The sections were incubated overnight at4°C with anti-sst2 antiserum diluted 1:5,000 in PBS con-taining 1% BSA and 0.3% Triton X-100. They were thenwashed for 3 3 10 minutes in washing buffer (PBS, 0.25%BSA, 0.1% Triton X-100) followed by 1 hour of incubationwith biotinylated swine anti-rabbit immunoglobulins(DAKO) diluted 1:500 in washing buffer. After washing for3 3 10 minutes, the sections were incubated for 20minutes with the blocking buffer provided with the kit(Biotinyl-tyramide amplification system, DuPont NEN,Boston, MA), followed by 45 minutes of incubation withhorseradish peroxidase-conjugated streptavidin-biotin com-plex (streptABC) (DAKO). The sections were washed,incubated with biotinyl-tyramide at 1:50 for 10 minutes(DuPont NEN), washed again, and finally incubated for afurther 45 minutes with streptABC. Immunoreactivitywas visualized by incubation in a solution of 0.05% diami-nobenzidine and 0.01% H2O2 in 0.05 M Tris buffer, pH 7.6.The sections were mounted on glass slides by using 0.5%gelatine in distilled water, air dried, and coverslipped withDepex.

For absorption controls, the diluted antiserum waspreabsorbed with 50 µg fusion protein/ml overnight at 4°Cbefore incubation of the sections.

248 L. HELBOE ET AL.

RESULTS

Immunoblots

In immunoblots, we detected a smeared band centeredaround an apparent molecular weight of 72 kDa in mem-branes prepared from a brain area corresponding to theventrolateral cortex (Fig. 1). This band was not present inthe absorption control lane. In addition, we detectedseveral sharp bands which could not be absorbed out.

Immunohistochemistry

By using the specific antiserum directed against the sst2receptor, immunoreactivity was observed in several areasof the central nervous system (Table 1). This immunostain-ing was abolished when the antiserum was absorbed withthe fusion protein used for immunization of the rabbits(Fig. 2P,Q).

In many regions (such as the the cortex, hippocampus,and amygdala), the immunolabelling was mainly observed

in fibres and only to a minor extent also in perikarya. Inmost cases it was not possible to determine whetherimmunostained fibre-like structures corresponded to den-drites or axons. Where possible, the type of staining hasbeen indicated; otherwise the term ‘‘fibre’’ or ‘‘process’’ hasbeen applied.

The anatomy of the hamster brain and the anatomicalnomenclature were in accordance with the atlas of thehamster brain by Collins (1969).

Areas of sst2 immunoreactivity

Telencephalon. Rhinencephalon. In the olfactorybulb, immunoreactivity was observed in fibre-like struc-tures in the glomerular layer. This was detected primarilyin the periphery of glomeruli, but some varicose fibreswere also seen to enter the glomeruli. There was amoderately dense immunoreactive staining of the mitral

Fig. 1. Immunoblot showing the sst2 receptor in the ventrolateralcortical area of hamster brain. Protein from membrane preparations(25 µg/lane) was separated by 10% SDS-PAGE, transferred to anitrocellulose membrane, and reacted with the anti-sst2 antiserumdiluted 1:500. A specific broad band of an apparent molecular weightbetween 63 and 80 kDa (average 72 kDa) was immunoreactive for sst2(lane A). The absorption control is shown in lane B. Several sharpbands were detected which could not be absorbed out. Molecular sizemarkers are indicated in kDa.

TABLE 1. Distribution of sst2-immunoreactivity in Hamster CNS1

Area Level Area Level

Telencephalon HypothalamusRhinencephalon Periventricular area 11

Olfactory bulb, gl. layer 11 Dorsomed. hypothalamic n. 11Anterior olfactory n. 1 Paraventricular n. 2

Septum Arcuate n. 111Lateral septal n. 11 Suprachiasmatic n. 2

Neocortex Supramammilary n. 11Layer I 2 Preoptic area 2Layer II–III (1) BrainstemLayer V–VI 111 Superior colliculus 11

Allocortex Barrington n. 11Piriform cortex 1 Locus coeruleus 111Endopiriform n. 111 Parabrachial n. 111CA1 111 Central grey 11CA2 (1) N. of the solitary tract 1(1)CA3 (1) Spinal trigeminal n. 111Dentate gyrus (1) Circumventricular organsSubiculum 111 Subfornical organ 111

Amygdala Median eminence 2Lateral amygdaloid n. 1 Area postrema 11Basolateral amygdaloid n. 111 Cerebellum (vermis, flocculus)

Basal ganglia Molecular layer 1Striatum 2 Granular layer 2Globus pallidus 2 Medulla spinalisClaustrum 111 Layer I and II 111

Diencephalon Anterior pituitary 11Thalamus

Medial habenular n. 111Intergeniculate leaflet 11

1Relative levels of immunoreactivity: 2, not detectable; 1, weak; 11, moderate; 111,intense.

Fig. 2. (Overleaf) A–O: A series of coronal sections from the rostral(A) to the caudal (O) parts of the adult hamster brain showing thedistribution of sst2 immunoreactivity. Several well-defined regions inthe brain, especially neocortical and allocortical areas, showed distinctimmunoreactive labelling. P–Q: Preabsorption controls, correspond-ing to sections F and L, respectively. 12, hypoglossal nucleus; aca,anterior commissure, anterior part; ACo, anterior cortical amygdaloidnucleus; AHi, amygdalohippocampal area; AOP, anterior olfactorynucleus, posterior part; AP, area postrema; APir, amygdalopiriformtransition area; Arc, arcuate hypothalamic nucleus; BL, basolateralamygdaloid nucleus; CG, central grey: CGM, central grey, medial part;Cl, claustrum; CnF, cuneiform nucleus; cp, cerebral peduncle, basalpart; CPu, caudate putamen; Cu, cuneate nucleus; DEn, dorsalendopiriform cortex; DLG, dorsal lateral geniculate nucleus; DM,dorsomedial hypothalamic nucleus; DR, dorsal raphe nucleus; f,fornix; fr, fasciculus retroflexus; GP, globus pallidus; Gr, gracilenucleus; hbc, habenular commissure; ic, internal capsule; IC, inferiorcolliculus; IGL, intergeniculate leaflet; IO, inferior olive; IP, interpedun-cular nucleus; La, lateral amygdaloid nucleus; LC, locus coeruleus;LHb, lateral habenular nucleus; lo, lateral olfactory tract; LPB, lateralparabrachial nucleus; LRT, lateral reticular nucleus; LSD, lateral

septal nucleus, dorsal part; LSV, lateral septal nucleus, ventral part;Ma, mammillary nuclei; mcp, middle cerebellar peduncle; Me5, mesen-cephalic trigeminal nucleus; MeA, medial amygdaloid nucleus; MG,medial geniculate nucleus; MHb, medial habenular nucleus; ml,medial lemniscus; MnR, median raphe nucleus; Mo5, motor trigemi-nal nucleus; MPB, medial parabrachial nucleus; mt, mammillotha-lamic tract; opt, optic tract; pc, posterior commissure; Pe, periventricu-lar hypothalamic nucleus; Pir, piriform cortex, PMCo, posteromedialcortical amygdaloid nucleus; Pr5, principal sensory trigeminal nucleus;PVA, paraventricular thalamic nucleus, anterior part; py, pyramidaltract; pyx, pyramidal decussation; R, red nucleus; Rt, reticular tha-lamic nucleus; s5, sensory root of the trigeminal nerve; SCh, suprachi-asmatic nucleus; scp, superior cerebral peduncle; SFO, subfornicalorgan; sm, stria medullaris; SNR, substantia nigra; SO, supraopticnucleus; Sol, nucleus of the solitary tract; sp5, spinal trigeminal tract;Sp5C, spinal trigeminal nucleus, caudal part; SubB, subbrachialnucleus; SuG, superficial grey layer of the superior colliculus; VLG,ventral lateral geniculate nucleus; VMH, ventromedial hypothalamicnucleus; xscp, decussation of the superior cerebellar peduncle. Scalebar 5 1 mm.

sst2 IMMUNOREACTIVITY IN HAMSTER CNS 249

Figure 2

Figure 2 (Continued)


cell bodies. A moderate immunoreactivity was also de-tected in the anterior olfactory nuclei.

Septum. A moderate dense immunoreactivity was ob-served in the lateral septal nucleus, in which both peri-karya and fibres could be recognized (Fig. 2C).

Neocortical areas. An intense immunoreactive label-ling of fibres was present in the cingulate cortex (Fig. 2C–F).This staining was primarily located in layers 5 and 6.Immunoreactive processes from layer 5 were seen toradiate into layers 2 and 3. However, in the more caudalpart of the cingulate cortex, immunoreactivity was absentin layer 5 (Fig. 2F).

In the motor and sensory cortical areas, a moderateimmunoreactivity was observed in layer 5. Furthermore,in caudal areas of the inferior somatosensory cortex, closeto the piriform cortex, immunoreactive medium-sizedmultipolar neurons were present in layer 5 and 6 (Figs.2C–H, 3C). Immunoreactive fibres (probably axons) wereseen to leave the cortical layer 6 and projected into theexternal capsula. Immunoreactive fibres were not ob-served in corpus callosum.

Allocortical areas. Throughout the dorsal endopiri-form nucleus, fibres in the superficial layer were stronglylabelled, whereas the staining in the deeper layers wasweak (Fig. 2A–H). Moderate immunoreactivity was con-tained in fibres of layer 2 of the piriform cortex (Fig. 2).

In the entorhinal cortex, an intense immunoreactivitywas present in layers 5 and 6 (Fig. 3B). Immunoreactivefibres radiated from the deep layers of the entorhinalcortex into superficial layers (moderate labelling), with aprominent radiating pattern in layer 3.

In the hippocampal area CA1, there was a strong label-ling of radiating structures in stratum radiatum, probablythe dendrites of the pyramidal cells (Figs. 2F–I, 3A).However, the perikarya in the pyramidal cell layer wereunstained. In stratum oriens, a high number of fibre-likestructures were also stained. The medial part of CA1showed the most intensive labelling. However, in themedialmost part of CA1 at the level of habenula, noimmunostaining was present. The staining of CA1 weak-ened when the CA2 area was approached. The perikarya ofthe granular layer were unstained. The dentate gyrusexhibited only very weak or no staining. A strong stainingof dendrites was detected in the subiculum (Fig. 3B). Thearea corresponding mainly to presubiculum was un-stained, whereas an area probably including parasubicu-lum and lamina dissecans of the entorhinal cortex showeda moderate labelling of fibres.

Amygdala and stria terminalis. In the basolateralamygdaloid nucleus, an intense immunoreactive stainingof fibres was detected (Fig. 2F,G), whereas the lateralamygdaloid nucleus exhibited only a weak staining. Noimmunostaining was observed in the central nucleus.

Thick immunoreactive varicose fibres (probably axons)were detected in stria terminalis.

Basal ganglia. A very strong immunoreactivity wasobserved in fibres throughout the entire claustrum(Fig. 2A–E). On the other hand, virtually no immunoreac-tivity was observed in the striatum or globus pallidus.

Diencephalon. Thalamus. An intense labelling waspresent in the medial part of the medial habenular nucleus(Figs. 2F, 3E) with a clear immunoreactivity of bothperikarya and the processes emerging from them.

Moderate to strong labelling was detected in the interge-niculate leaflet of the lateral geniculate nucleus (IGL)located to varicose fibres (Figs. 2H, 4A). Rostrally, the IGLforms a band interposed between the dorsal and ventrallateral geniculate nucleus, whereas caudally, the IGLtakes up a more ventral position. The immunoreactivitywas observed in both rostral and caudal parts of the IGL.

A small terminal field exhibiting immunoreactivity wasobserved in coronal sections at the level of the posteriorcommissure (Fig. 2H). The immunostained field was lo-cated in the intralaminar area of the thalamus.

Hypothalamus. Immunoreactive perikarya and fibreswere observed throughout the hypothalamic periventricu-lar nucleus (Figs. 2D, 5D). This periventricular layer oflabelled neurons extended into the preoptic area.

Immunoreactive fibres were identified in the area corre-sponding to the dorsomedial hypothalamic nucleus(Fig. 2G). In the arcuate nucleus, several intensely stainedperikarya with processes were observed close to the ven-tricular system (Figs. 2F–I, 3D). Stained processes werealso observed to penetrate the ependymal layer of thisregion. A number of processes which might be tanycyteprocesses terminated on the inferior surface of the brain.Staining of cell bodies in the ependymal layer was notobserved.

No specific immunoreactivity was observed in the supra-chiasmatic nucleus, the paraventricular nucleus, and thepreoptic area. Moderate immunoreactivity was detected inthe supramammilary nuclei.

Brainstem. Moderate immunoreactivity was observedin fibres of the superior colliculus (Fig. 2I–J), mostlyconfined to the superficial grey layer.

The nucleus of Barrington and the locus coeruleuscontained strongly labelled processes and perikarya(Figs. 2L, 5A–C). In the rostral part of the parabrachialnucleus, intensely immunoreactive fibres were located inthe following subnuclei (adapted from Fulwiler and Saper,1984): internal lateral, superior lateral, extreme lateral,ventral lateral, and the most ventral part of the medialsubnucleus. Sparse immunoreactive fibres were also ob-served transversing the ventrolateral part of the superiorcerebellar peduncle. Further caudally at the level of thelocus coeruleus, immunoreactivity was mainly located inthe ventral and central lateral parabrachial nucleus and inthe waist of the superior cerebellar peduncle. Most cau-dally, immunostaining was located in the dorsal part of themedial subnucleus and in the waist of the superior cerebel-lar peduncle. In addition to immunoreactive fibres, a fewperikarya were also labelled mainly in the ventral lateralsubnucleus. The external lateral and the Kolliker-Fusesubnuclei appeared to be without immunostained struc-tures.

In the caudal part of the central grey at the level of theinferior colliculus, immunoreactive perikarya were pres-ent close to the cerebral aqueduct. Processes emerged fromthese perikarya in a dorsoventral direction (Fig. 2K). Inthe more lateral part of the central grey, single immunore-active perikarya and many fibres were detected(Figs. 2J,K, 4E). Immunoreactive fibres were present inthe dorsal raphe (Fig. 4E), whereas no immunoreactivitywas observed in the laterodorsal tegmental nucleus. Nospecific labelling was present in either motor neurons of


Fig. 3. A: Photomicrograph of a coronal section showing part of theCA1 region exhibiting strong fibre-like immunoreactivity in thestratum oriens (Or) and radiatum moleculare (Rad), without stainingof the pyramidal cell layer. The very medial part of the CA1 was devoidof staining. B: Horizontal section through the transition zone betweenthe entorhinal cortex and the hippocampus. Note the sharp borderseparating presubiculum (PrS) from the immunoreactive subiculum(S) and an area corresponding to parasubiculum (PaS)/entorhinalcortex (Ent). Processes from the subiculum radiated into the outer

cortical layers. C: Part of the deep layers of the neocortex. Intenselyimmunoreactive neurons (arrows) were present in layers 5 and 6 withprocesses mainly oriented toward the cortical surface. D: Photomicro-graph of part of the arcuate nucleus where many immunoreactiveperikarya (arrows) and fibres were observed. E: Strongly stainedperikarya and processes are seen in the the medial habenular nucleus(MHb) close to the third ventricle (3V). No specific staining waspresent in the lateral habenular nucleus (LHb). Scale bars 50 µm in Cand D, 100 µm in A and E, 200 µm in B.-


the trigeminal, facial, and hypoglossal nuclei or neurons ofthe trapezoid nucleus.

A moderate labelling of fibres was detected in thenucleus of the solitary tract (Fig. 2M–N). In general, the

pontine and medullary reticular formation were devoid ofimmunoreactivity.

Strongly immunoreactive fibres and a few perikaryawere present in the outer layers (lamina 1 and 2) of the

Fig. 4. A: sst2 immunoreactivity was observed in varicose fibres ofthe intergeniculate leaflet, whereas no staining was seen in eitherdorsal (DLG) or ventral geniculate nuclei (VLG). B: Intense immunore-activity was detected in coarse fibres of the subfornical organ. C: In ahorizontal section through the area postrema, many intensely stainedperikarya and fibres were observed. D: A coronal section through thecerebellar cortex in the flocculus showing immunoreactive stainingradiating into the molecular (Mol) layer (arrows). The stained struc-

tures resembled Bergmann glia. Purkinje cells (arrowheads) wereunstained. No immunoreactive structures were observed in the granu-lar layer (Gran). E: Coronal section of the ventrolateral area of thecentral grey. We observed many immunoreactive perikarya (arrows)as well as nerve fibres, especially in the medial and ventrolateral area.Immunoreactive fibres were present also in the dorsal raphe (DR). Aq,aqueduct. Scale bars 5 50 µm in A and D, 100 µm in B, C, and E.


Fig. 5. A–C: Coronal sections of the dorsolateral part of themesencephalon with the superior cerebellar peduncle (scp) sur-rounded by parabrachial nuclei. In the more rostral section (A),immunoreactive fibres were observed in the lateral parabrachialnucleus (LPB). In this section, immunoreactive perikarya and fibreswere also seen in the Barrington nucleus (Bar). The outline ofBarrington nucleu is marked by arrowheads. In the medial section (B),immunostained fibres were present in lateral as well as medial (MPB)parabrachial nuclei. Note that immunoreactive fibres transverse thesuperior cerebellar peduncle and seem to connect the two parabrachialnuclei. In this section, locus coeruleus (LC; marked by arrowheads) ispresent and exhibits sst2-positive perikarya and processes. In the

caudal part of this area (C), immunoreactivity was observed only inthe medial parabrachial nucleus and the locus coeruleus (marked byarrowheads). Only very weak immunoreactive staining was seen inthe mesencephalic sensory trigeminal nucleus (Me5). D: In thehypothalamic periventricular area, immunostained perikarya (ar-rows) and varicose fibres were observed. E: Sagittal section with atangentially cut ependymal layer (E) of the third ventricle showedimmunoreactive ependyma cells. Furthermore, in the subependymallayer immunoreactive varicose nerve fibres (arrows) were seen.F: High-power photomicrograph showing a small area of the anteriorpituitary, where many immunoreactive cells (arrows) were present.Scale bars 5 25 µm in F, 50 µm in D and E, 200 µm in A–C.


spinal trigeminal nucleus (Fig. 2N–O). Some positivefibres were also seen to radiate into the trigeminal tract.

Ependyma and circumventricular organs. Ependy-mal cells exhibited immunostaining mostly confined to thecell membrane (Fig. 5E). Some immunoreactive nervefibres were seen to extend from the brain into the ependy-mal layer. This staining was most prominent in theependyma of the third ventricle and was observed only to alesser extent in the fourth and lateral ventricles.

Strongly stained immunoreactive coarse fibres withlarge varicosities were present in the subfornical organ(Figs. 2D, 4B). No immunostained perikarya were detectedin this area. In the area postrema, a strong immunoreactiv-ity was found in intrinsic perikarya and fibres (Figs.2M, 4C). No immunostaining was observed in the medianeminence.

Cerebellum. Immunoreactive radiating processes witha punctate appearance were observed in the molecularlayer of the cerebellar cortex, whereas no specific stainingwas observed in Purkinje cells or in the granular layer(Figs. 2L, 4D). The immunolabelling was prominent in thelateral ventral part of the cerebellar hemisphere (espe-cially in the flocculus and paraflocculus) and in the cortexcovering the superior layer of the vermis. When comparedwith immunostaining with the glial cell marker glialfibrillary acidic protein (GFAP), the sst2 immunoreactivityappeared glia-like. However, the receptor antibody re-sulted in a more punctate type of staining than that ofGFAP. Radiating punctate processes were sometimes ob-served to emerge from perikarya-like structures whichmight represent the cell bodies of Bergmann glia. Thisstaining was eliminated in absorption controls.

Medulla spinalis. At all levels of the spinal cord,immunoreactivity was observed in varicose fibres in layersI and II of the dorsal horn (Fig. 6).

Pituitary. In the anterior pituitary, many immunore-active cells were observed throughout (Fig. 5F). The propor-tion of immunolabelled cells was estimated to be 20–40%;there was a tendency for most cells to be labelled in theperipheral parts of the anterior lobe.

DISCUSSION

Western blot analysis

In membranes prepared from hamster ventrolateralcortical area, a broad protein band was specifically de-tected with the anti-sst2 antiserum. This smeared bandwas centered around an apparent molecular weight of72 kDa. A similar broad migration pattern was alsoobserved in rat brain (Dournaud et al., 1996b; Schindler etal., 1997) and is characteristic of glycosylated proteins.The molecular size of the rat sst2a receptor calculated fromthe primary sequence is 41 kDa, indicating that thehamster sst2 receptor is also subjected to post-transla-tional glycosylation. In extracts from rat cortex, the molecu-lar weight of the sst2a receptor was previously estimated atan average of 72 kDa (Dournaud et al., 1996b) or between80 and 85 kDa (Schindler et al., 1997). The size of thehamster sst2 receptor corresponds well to that of the rat,thus supporting the view that our antiserum raised againstthe human sst2 receptor cross-reacts with the hamster sst2receptor.

Immunohistochemical distribution of sst2

immunoreactivity

Strong immunoreactivity for sst2 was detected in severalwell-confined forebrain areas as well as in some motor andsensory brainstem nuclei, certain cerebellar cortical areas,and the dorsal horn of the spinal cord. The pattern ofstaining was mostly diffuse and fibre-like. However, dis-tinct staining of perikarya could also be observed in someareas.

In the forebrain, immunoreactivity was found in bothneocortical and allocortical areas and some areas of thehypothalamus but was lacking in most areas of the thala-mus, striatum, and globus pallidus.

Comparison of sst2 localizationwith previous studies

The immunostaining observed in this study of thehamster mainly agrees with the distributional pattern ofsst2 observed in the rat brain by immunohistochemistry(Dournaud et al., 1996b; Schindler et al., 1997) and byautoradiography using radioligands (Uhl et al., 1985;Reubi and Maurer, 1985; Katayama et al., 1990; Martin et

Fig. 6. Section of the spinal cord at the cervical level. sst2 immuno-reactivity was observed in layers I and II of the dorsal horn. The weakstaining of motor neurons in the ventral horn was not specific. Thesame pattern of immunostaining was also observed at thoracic,lumbar, and sacral levels of the spinal cord. CC, central canal. Scalebar 5 200 µm.


al., 1991; Schoeffter et al., 1995; Holloway et al., 1996).Also, extensive in situ hybridization studies have beenconducted for the sst2 receptor in rat brain (Perez et al.,1994; Senaris et al., 1994). The areas with major signalsfor sst2 mRNA (e.g., deep layers of neocortex, claustrum,endopiriform nucleus, hippocampus, basolateral amyg-dala, medial habenula, locus coeruleus) correlate with thelocalization of the sst2 receptor protein. This could indicatethat most of the sst2 immunoreactivity in these regions islocated postsynaptically on dendrites or alternatively onpresynaptic axon terminals of small interneurons.

There are, however, some apparent differences betweenthe immunolabelling in the rat and the hamster indicativeof species-specific localizations of the sst2 receptor in somebrain regions. In the hamster hippocampal area CA1, sst2immunoreactivity in stratum radiatum could be localizedto the dendrites of the pyramidal cells. Staining in thestratum oriens might be due to the presence of receptorson the axons of the pyramidal cells or processes frominterneurons. However, sst2-immunoreactive staining wasnot detected on the pyramidal cell bodies. This contrastswith the finding by Dournaud et al. (1996b) in the rat,where the most prominent staining was observed in thepyramidal cell layer including proximal dendrites. AlsoSchindler et al. (1997) detected some labelling in the ratpyramidal cell bodies. Moreover, in the two studies of therat there was a moderate, diffuse labelling of the dentategyrus. This was not observed in the hamster. However, wealso see diffuse staining of the dentate gyrus and somelabelling of pyramidal cell bodies in the CA1/CA2 areawhen applying our sst2 antiserum to rat brain (unpub-lished observations). This points to a true difference indistribution of this receptor between the two rodent spe-cies.

In all immunohistochemical studies, an intense label-ling of fibres was seen in the basolateral amygdaloidnucleus. However, whereas Dournaud et al. (1996b) and toa minor extent also Schindler et al. (1997) reported thepresence of immunoreactive perikarya in the central amyg-daloid nucleus of the rat, we detected no labelling of thisnucleus in the hamster. In the rat central amygaloidnucelus, we detected a moderate staining (unpublishedobservations).

In contrast to the observations in the rat, in which fibrestaining was mainly reported, the hamster arcuate nucleuscontained densely stained perikarya as well as fibres. Alsoin the hypothalamus, strongly stained perikarya andfibres were present in the periventricular area of thehamster brain. This was not observed in the rat.

The intense labelling of perikarya in the outer layers ofthe neocortex observed by Dournaud et al. (1996b) was notobserved in the hamster, nor did we see such a labelling inthe rat brain. This difference in labelling pattern mayreflect a preferential recognition of membrane-associatedreceptors with our antibody as opposed to receptors orreceptor fragments present in the cytoplasm of neurons.

Functional implications of the localizationof somatostatin receptors

In some of the areas exhibiting immunoreactivity, a rolefor somatostatin and its receptors has previously beenindicated.

Regulation of growth hormone secretion. In theanterior pituitary gland, we detected many immunoreac-tive cells. The sst2 receptor is most likely responsible for

mediating the inhibitory effect of somatostatin on growthhormone secretion in rat (Raynor et al., 1993) and inhuman, where sst5 is also believed to be involved (Shimonet al., 1997). Kumar et al. (1997) recently reported thatimmunoreactivity for all five somatostatin receptors waspresent in the rat anterior pituitary. Approximately half ofthe somatotrophes expressed the sst2 receptor.

We showed that sst2 immunoreactivity was present innerve fibres and perikarya in the hamster arcuate nucleus.Also in the rat, the sst2a receptor was detected in fibres inthis structure (Dournaud et al., 1996b; Schindler et al.,1997). In the arcuate nucleus, somatostatin-containingfibres have been shown to innervate growth hormone-releasing hormone (GHRH)-containing neurons (Daikokuet al., 1988; Liposits et al., 1988; Horvath et al., 1989).Furthermore, double localization studies have revealedthat somatostatin-binding sites (McCarthy et al., 1992;Bertherat et al., 1992) and sst1 and sst2 mRNA (Tannen-baum et al., 1998) were present in a subpopulation ofGHRH-containing neurons in the arcuate nucleus. Somato-statin has been suggested to inhibit the secretion ofGHRH, thereby indirectly inhibiting the release of growthhormone from the anterior pituitary gland. Thus, it isconceivable that somatostatin and GHRH interact at thehypothalamic level to regulate growth hormone secretion,with sst2 as a possible candidate for mediating this effect.

Memory and learning—hippocampus. Ahigh concen-tration of somatostatin is known to be present in thehippocampal areas. Somatostatinergic cell bodies (most ofthem interneurons) have been found mainly in the stra-tum oriens and pyramidale of CA1, all strata of CA3, thehilus of the dentate area, and subicular regions (Roberts etal., 1984; Sloviter and Nilaver, 1987). A region of somato-statinergic fibres is located in the CA1 molecular layer.These fibres may originate in the contralateral CA1 or inthe entorhinal region (Roberts et al., 1984). In accordancewith this, we found immunoreactivity mainly in the stra-tum oriens and stratum radiatum of CA1 and in thesubiculum. No immuno staining of the pyramidal cell layerwas observed.

The functional role of somatostatin in the hippocampushas been indicated by several lines of evidence. Cyste-amine, a depletor of somatostatin, was shown to impair theretention of memory, and exogenous somatostatin re-versed amnesia associated with cholinergic deficits (Mat-suoka et al., 1994). It has also been suggested thatsomatostatinergic interneurons in the frontal cortex par-ticipate in learning behaviour since a decline in somatosta-tin mRNA levels in this area correlates with an impair-ment in memory performance (Dournaud et al., 1996a).

A recent study has shown that somatostatin regulatesneurotransmission at excitatory synapses in cultured ratglutamatergic hippocampal neurons (Boehm and Betz,1997). This action is most likely mediated by presynapticsomatostatin receptors, probably of the sst2 subtype. Itthus indicates an axonal location of sst2 receptors. Further-more, somatostatin has been demonstrated to inhibithigh-voltage-activated Ca21 channels in fresh cultures ofrat hippocampal CA1 neurons (Ishibashi and Akaike,1995) possibly resulting in a reduced release of neurotrans-mitters in the hippocampus.

sst2 receptors in the dentate gyrus have been suggestedto reduce the susceptibility to convulsions in kainic acid-treated rats (Perez et al., 1995). Immunoreactivity wasmedium to strong in the rat dentate gyrus (Dournaud et


al., 1996b; Schindler et al., 1997), thus supporting thistheory. However, we failed to observe the same pattern inthe hamster dentate gyrus, where virtually no immunore-activity was detected, possibly indicating a species differ-ence concerning this function.

Circadian systems. sst2-immunoreactive fibres wereobserved in the intergeniculate leaflet (IGL) of the lateralgeniculate body. This is in accordance with previous recep-tor autoradiographic studies showing the presence ofsomatostatin receptors in this area (Bodenant et al., 1991).

The IGL is a part of the circadian rhythm-generatingsystem, consisting of the retina, the suprachiasmaticnucleus, the IGL, and the retinal projections to the supra-chiasmatic nucleus and to the IGL (Moore and Card, 1994).The IGL projects back to the suprachiasmatic nucleus viathe geniculohypothalamic tract (Morin et al., 1992) throughwhich pathway neurons of the IGL are able to phase shiftthe suprachiasmatic nucleus (Rusak et al., 1989)—theendogenous clock of the brain (Moore, 1992).

The sst2-immunoreactive fibres of the IGL might bedendrites emerging from perikarya that did not exhibitsst2 immunoreactivity. However, the sst2 might also be anautoreceptor located on somatostatinergic axons project-ing to the IGL. Thus, somatostatin-containing neurons arelocated in the ganglion cell layer of the retina (Larsen etal., 1990), and somatostatin-immunoreactive fibres havebeen shown in the IGL (Laemle and Feldman, 1985).

Somatostatin-containing neurons innervating the IGLmight also be located outside the retina. Thus, somatosta-tinergic neurons are also present in the superior colliculus(Taber-Pierce et al., 1985), and projections from the super-ficial grey layer of the superior colliculus to the IGL havebeen shown in the hamster in our laboratory by retrogradetracing (Vrang et al., personal communication).

Somatostatin is present in the suprachiasmatic nucleusand has been shown to exhibit a circadian rhythm with lowlevels during the dark phase (Shinohara et al., 1991). Nosst2 immunoreactivity was found in this nucleus, but wehave previously detected sst1-immunolabelled nerve fibresin the rat supachiasmatic nucleus (Helboe et al., 1998).

Pain. Analgesic effects have been attributed to somato-statin in clinical trials (Mollenholt et al., 1994; Paice et al.,1996), and it was shown that somatostatin injected intonucleus raphe magnus or central grey of rats leads todescending inhibition of spinal nociceptive transmission(Helmchen et al., 1995).

In some of the central areas known to take part innociceptive pathways, we detected strong immunoreactivesignals, thus indicating a role of the sst2 receptor innociception. We found an intense immunoreactive stainingof layers I and II (substantia gelatinosa) in the dorsal hornof the spinal cord, which has been shown to contain a highconcentration of immunoreactive somatostatin (Finley etal., 1981; Johansson et al., 1984). Previously, receptorscorresponding to sst2 have been detected in substantiagelatinosa of rats by autoradiography (Reubi and Maurer,1985; Schoeffter et al., 1995; Holloway et al., 1996) and byimmunohistochemistry (Schindler et al., 1997). Excitatoryand inhibitory interneurons are present in the substantiagelatinosa where they are known to modulate or processthe nociceptive response. Somatostatin is present in inhibi-tory interneurons (Willis et al., 1995).

In the caudal spinal trigeminal subnucleus, which isinvolved in nociception from cranial inputs, we detectedintense immunoreactivity in the outer layers.Also, somato-

statin has been shown to be present in these laminae I andII (Alvarez and Priestly, 1990). In a recent study, somato-statin has been shown to reduce the number of Fos-positive neurons at the subnucleus interpolaris/caudalistransition in the spinal trigeminal nucleus in response tochemical irritant stimulation of the corneal surface (Bere-iter, 1997), indicative of nociceptor activity.

The central grey (CG) is a major site for ascending paintransmission as well as descending inhibition of dorsalhorn nociceptive neurons (Behbehani, 1995). Somatostatinimmunoreactivity has been detected in rat mainly in themore lateral aspects of CG extending into a ventrolateralarea (Johansson et al., 1984). We observed immunoreac-tive perikarya and fibres in lateral areas of the CG and anintense immunoreactivity in the area immediately sur-rounding the aqueduct with immunoreactive perikaryaand fibres spreading in a parallel dorsoventral direction incaudal CG.

The dorsal raphe, locus coeruleus, and parabrachialnuclei are also implicated in pain modulation (Saper, 1995;Willis et al., 1995). In these areas, we detected moderate tostrong immunoreactivity.

Neocortex. Immunoreactivity for the sst2 receptor wasabundantly present in all neocortical areas. The presenceof somatostatin-immunoreactive neurons in the cortex hasbeen known for the last two decades (Hokfelt et al., 1974;Mizukawa et al., 1987). Somatostatin in neocortex issensitive to external stimulation (Nilsson et al., 1995) andhas been associated with the pathogenesis of both schizo-phrenia and Alzheimer’s disease (Gabriel et al., 1996).Somatostatin is confined to nonpyramidal neurons inlayers II, III, V, and VI (Mizukawa et al., 1987). Thesomatostatinergic neurons of the neocortex form an intrin-sic cortical system because undercuts of the cortex do notchange the density of nerve fibres in overlying corticalareas (Morrison et al., 1983).

The sst2 immunoreactivity observed in the neocortex inour study was confined mostly to the deeper layers IV, V,and VI, indicating that the receptor is located more onneuronal cell bodies than the radial dendrites of the cortex.Thus, staining of layer I was always very weak and inlayers V and VI, staining of the outline of small neuronscould actually be observed. Our localization of sst2 immu-noreactivity in the hamster corresponds well with thelocalization of sst2a immunoreactivity in the rat brain(Dournaud et al., 1996b; Schindler et al., 1997), with theexception of the immunolabelled perikarya in layers II andIII observed in the former study. It also corresponds withreceptor autoradiographic studies in which 125I SS-14 wasused as ligand (Katayama et al., 1990; McCarty andPlunkett, 1987). Furthermore, in situ hybridization stud-ies of the rat neocortex have shown cells with mRNAencoding the sst2 in neurons located in layers V and VI(Perez et al., 1994; Senaris et al., 1994).

Locus coeruleus. Somatostatinergic processes havebeen found in locus coeruleus (LC) in the cat (De Leon etal., 1992) and rat (Johansson et al., 1984). These somatosta-tin-immunoreactive processes are said to originate inperiventricular hypothalamic somatostatinergic cells(Palkovits et al., 1982), or from somatostatinergic neuronslocated in the nucleus paragigantocellularis and nucleusprepositus hypoglossi (Aston-Jones et al., 1986), whichrepresent the major input to LC.


SS-14 binds in LC to the noradrenergic cells (Gagne etal., 1990). Octreotide, which has high affinity for sst2,inhibits firing of these neurons, whereas an sst3-selectiveagonist (BIM-23056) has no effect (Chessell et al., 1996).The inhibitory effect on the firing rate of LC neurons seemsto be directly on somatodendritic somatostatin receptorsand not indirectly by the release of noradrenaline (Ches-sell et al., 1996), although somatostatin has been shown torelease noradrenaline from LC neurons (Tsujimoto andTanaka, 1981).

Parabrachium. Somatostatin-immunoreactive fibreshave been localized in the dorsal (including central andsuperior), ventral and medial parabrachial subnuclei, inthe waist of the superior cerebellar peduncle and in theKolliker-Fuse subnucleus, and perikarya have been ob-served in the dorsal parabrachial nucleus (Johansson etal., 1984). This correlates with our localization of sst2immunoreactivity in most of these subnuclei, sparing theKolliker-Fuse nucleus, however.

The somatostatinergic afferents to the parabrachialnucleus originate in several areas and indicate a wide-spread somatostatinergic input to the parabrachium. Pro-jections to the parabrachium derive from the nucleus ofthe solitary tract (Riche et al., 1990), bed nucleus of thestria terminalis, and central amygdala, but the parabra-chial terminal subnuclei of these projections have not beendetermined (Moga and Gray, 1985; Moga et al., 1989).Minor somatostatinergic projections to parabrachiumemerge from the intercalated nuclei of the amygdala andthe lateral part of the central amygdala (Veening et al.,1984), whereas there is probably no input to the parabra-chial nucleus from the hypothalamus (Palkovits et al.,1982; Moga et al., 1990). Functionally, the central amyg-dala somatostatinergic efferent to parabrachium could beinvolved in cardiovascular responses to stress (Moga andGray, 1985). Furthermore, somatostatin injected into theparabrachial nucleus inhibits spontaneous firing of tha-lamic visceral neurons but is not a part of the ascendingvisceral sensory pathway (Saleh and Cechetto, 1993).These somatostatinergic responses may thus be mediatedvia the sst2 receptor found in medial and ventral parabra-chial nuclei.

Cerebellum. In the cerebellum, somatostatin is mainlypresent during prenatal (Inagaki et al., 1982) and earlypostnatal development (Villar et al., 1989), indicating arole for somatostatin in the organization of the developingnervous system. However, in the adult rat, somatostatinimmunoreactivity has been detected in Purkinje cells,Golgi cells, and climbing fibres, especially in the areas ofthe flocculus and paraflocullus and in superior vermallayers (Villar et al., 1989). In this study, we detected aspecific immunolabelling of Bergmann glia-like structuresin the molecular layer in the flocculus and paraflocullusand in superior vermal layers. The presence of the sst2receptor or its mRNA has previously been demonstrated inrat and mouse astrocytes (Feindt et al., 1997; Viollet et al.,1997a), thus indicating that sst2 receptors are indeedexpressed in glia cells.

In previous immunohistochemical studies (Dournaud etal., 1996b; Schindler et al., 1997) no sst2 immunoreactivitywas found in the rat cerebellum, nor were somatostatin-binding sites detected by autoradiography in rat cerebel-lum (Martin et al., 1991; Holloway et al., 1996).

In contrast, however, binding studies using selectivesomatostatin analogues revealed sst2 to be the predomi-

nant receptor in the immature cerebellum (Viollet et al.,1997b), and furthermore, in the adult rat cerebellum a lowbut significant level of sst2 receptor was demonstrated inthe molecular cell layer (Piwko et al., 1995). Thus, therestill seems to be some dispute as to the presence ofsomatostatin receptors in the cerebellum.

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Immunohistochemical localization of the somatostatin receptor subtype 2 (sst2) in the central...

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