immunocytochemical localization of somatostatin receptor sst2a in the rat spinal cord and dorsal...

European Journal of Neuroscience, Vol. 10, pp. 3700–3708, 1998 © European Neuroscience Association

Immunocytochemical localization of somatostatin receptorsst2A in the rat spinal cord and dorsal root ganglia

Stefan Schulz, Matthias Schreff, Harald Schmidt, Manuela Handel, Ryszard Przewlocki1 andVolker HolltDepartment of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Germany1Department of Pharmacology, Polish Academy of Sciences, Cracow, Poland

Keywords: analgesia, antibodies, dorsal root ganglia, immunocytochemistry, octreotide, somatostatin receptor, somatostatin,spinal cord

Abstract

Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, andlocally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the present study, we haveraised polyclonal antibodies that specifically detect the somatostatin receptor sst2A and used these antisera forimmunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In thesuperficial layers of the dorsal horn, sst2A-like immunoreactivity (Li) formed a dense network consisting ofneuronal perikarya and dendrites which were often closely apposed by, but not co-contained within,somatostatin-14-immunoreactive nerve fibres and terminals. sst2A-Li was resistant to dorsal rhizotomy and didnot colocalize with either substance P or calcitonin gene-related peptide suggesting that sst2A-Li was not locatedto primary afferents, but rather confined to second-order spinal neurons. The position of sst2A-Li perikarya anddendrites in the dorsal horn appeared to be similar to those containing µ-opioid receptor-Li; however, doublelabelling experiments revealed no instances of coexistence of these two receptors. sst2A-Li was also observed inthe dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinctfrom those expressing somatostatin-14 or δ-opioid receptors. Thus, the present results not only provide amorphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poisedto modulate nociceptive transmission by distinct anatomical systems.

Introduction

Somatostatin was originally discovered as a hypothalamic neuroendo-crine hormone, which potently inhibits the secretion of growthhormone from the anterior pituitary gland (Brazeauet al., 1973). Twobiological active forms have been identified in mammals, somatostatin-14 (SS-14) and the amino-terminally extended somatostatin-28 (SS-28). SS-14 has a widespread distribution throughout the centralnervous system where it acts as neurotransmitter and neuromodulatorpredominantly in an inhibitory fashion (Ho¨kfelt et al., 1974;Johannsonet al., 1984; Esclapez & Houser, 1995). Recently, asomatostatin-like peptide, cortistatin, with a high degree of homologybut more restricted distribution has been isolated (DeLeceaet al.,1996). At the level of the spinal cord, locally applied SS-14 inhibitsfiring of nociceptive dorsal horn and dorsal root ganglia (DRG)neurons (Havliceket al., 1977; Randic & Miletic, 1978; Muraseet al., 1982; Sandkuhleret al., 1990; Chapman & Dickenson, 1992;Taddeseet al., 1995; Bereiteret al., 1996). Intrathecal administrationof the long-acting somatostatin analogue, octreotide (SMS 201–995),provides pain relief in patients (Sicuteriet al., 1984; Pennet al.,1992; Mollenholtet al., 1994).

SS-14 mediates its physiological effects through a family of Gprotein coupled receptors containing seven transmembrane domains

Correspondence: Volker Hollt. E-mail: [email protected]

Received 11 March 1998, revised 24 June 1998, accepted 13 July 1998

(Hoyer et al., 1995). Five genes encoding distinct somatostatinreceptor subtypes, termed sst1–sst5, have so far been cloned in humansand other species (Brunoet al., 1992; Kluxenet al., 1992; Meyerhofet al., 1992; O’Carollet al., 1992; Yamadaet al., 1992; Yasudaet al.,1992). In addition, the carboxy-terminal tail of the mouse sst2 receptorhas been shown to undergo alternative splicing yielding two isoforms,sst2A and sst2B (Vanetti et al., 1992, 1993). A number of studies hasaddressed the distribution of the mRNA for the five somatostatinreceptor subtypes (Brederet al., 1992; Wulfsenet al., 1993; Konget al., 1994; Perezet al., 1994; Senariset al., 1994). However, littleinformation is available about the cellular localization of the receptorproteins in the central nervous system. Although earlier autoradio-graphic binding studies have examined the overall distribution ofsomatostatin binding sites in mammalian brain, none of the somato-statin receptor ligands available is sufficiently selective to allowdefinite discrimination between the various receptor subtypes(Epelbaumet al., 1982; Uhlet al., 1985; Martinet al., 1991; Schoeffteret al., 1995).

In the present study, we have generated polyclonal antibodiesspecific for sst2A and determined the immunocytochemical localizationof this receptor in the spinal cord and DRG of rats.

Immunolocalization of somatostatin receptor sst2A 3701

Materials and methods

Animals

Adult male Wistar rats (200–230 g;n 5 8) were deeply anaesthetizedwith chloral hydrate and transcardially perfused with Tyrode’s solutionfollowed by a fixative containing 4% paraformaldehyde and 0.2%picric acid in 0.1M phosphate buffer, pH 6.9. Spinal cords and DRGwere rapidly dissected and postfixed in the same fixative for 2 h atroom temperature. Unilateral dorsal rhizotomies were performedat spinal segments L3–S1 in two rats anaesthetized with sodiumpentobarbital. Six days after recovery, these animals were fixed byvascular perfusion and spinal cords were removed as above.

Generation of antipeptide antisera

Polyclonal antisera were generated against the carboxy-terminal tailof sst2A. The identity of the peptide was ETQRTLLNGDLQTSI,which corresponds to residues 355–369 of sst2A. The peptide wascustom-synthesized by Gramsch Laboratories (Schwabhausen,Germany), purified by HPLC and coupled via an amino-terminallyadded cysteine and a SMCC (succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate) linker to keyhole limpet haemocyanin(Lerner et al., 1981). The conjugates 500µg/mL were mixed 1 : 1with Freund’s adjuvant and injected into one group of four guinea-pigs GP1–GP4 and one group of two rabbits 6291 and 6292. Animalswere injected at 4-week intervals, and serum was obtained 2 weeksafter immunizations beginning with the second injection.

Dot–blot analysis

The specificity of the antisera as well as possible cross-reactivitywith other somatostatin receptor subtypes was initially tested in dot-blot assays. Serial dilutions of the unconjugated peptides correspond-ing to the carboxy-terminal sequences of sst1, sst2A, sst2B, sst3, sst4and sst5 were blotted on to nitrocellulose membranes. The identityof the peptides was: ESGGVFRNGTCASRISTL which correspondsto residues 374–391 of the mouse sst1; ETQRTLLNGDLQTSI whichcorresponds to residues 355–369 of the rat/mouse sst2A; ADNSQSG-AEDIIAWV which corresponds to residues 332–346 of the mousesst2B; TAGDKASTLSHL which corresponds to residue 417–428 ofthe rat/mouse sst3; CQQEPACKRVPFTKTTTF which corresponds toresidues 367–384 of the rat sst4; and QATLPTRSCAENGLMQTSRIwhich corresponds to residues 344–363 of the rat sst5 receptor.Membranes were then incubated with the antisera at a dilution of1 : 2000 for 60 min at room temperature (RT). Blots were subsequentlyincubated with biotinylated antirabbit antibodies (1 : 5000) andavidin–biotin complex (both from Vector, Burlingame, CA, USA)and developed with 3,39-diaminobenzidine (DAB). For subsequentanalysis of antibody specificity either these crude antisera were usedor selected antisera (GP3; 6291) were affinity purified using theimmunizing peptide bound to Sulfo-Link coupling gel (Pierce,Rockford, LI, USA) according to the manufacturer’s instructions.

Western blot analysis

Membranes were prepared from several brain regions including spinalcord, hippocampus, cortex and cerebellum, and glycoproteins werepartially purified using wheat germ lectin agarose (WGA, Vector)essentially as described by Hunyadyet al. (1997). Briefly, tissue waslysed in homogenization buffer (5 mM EDTA, 3 mM EGTA, 250 mM

sucrose, 10 mM Tris–HCl, pH 7.6 containing 1 mM phenylmethylsul-phonylfluoride, 1µM pepstatin, 10µg/mL leupeptin and 2µg/mLaprotinin). The homogenate was spun at 500g for 5 min at 4 °C toremove unbroken cells and nuclei. Membranes were then pelleted at

© 1998 European Neuroscience Association,European Journal of Neuroscience, 10, 3700–3708

20 000g for 30 min at 4 °C. Membranes were then dissolved in lysisbuffer (150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM HEPES,pH 7.4 containing 4 mg/mL dodecyl-beta-maltoside and proteinaseinhibitors as described above) and incubated with 150µL WGA-beads for 90 min at 4 °C. Beads were washed five times in lysisbuffer, and adsorbed glycoproteins were eluted with sodium dodecylsulphate (SDS) sample buffer for 60 min at 37 °C. Either crudemembrane proteins (100µg/lane) or WGA-extracts purified from200µg membrane proteins were subjected to 8% SDS–polyacrylamidegel electrophoresis (SDS–PAGE) and immunoblotted on to nitrocellu-lose. Blots were incubated with antisera GP3 or 6291 either crude ata dilution of 1 : 20 000 or after affinity purification at a concentrationof 1 µg/mL overnight at 4 °C. Blots were developed using peroxidase-conjugated secondary antibodies (1 : 5000) and enhanced chemi-luminescence (Amersham, Braunschweig, Germany). For adsorptioncontrols, antisera were preincubated with 10µg/mL of their cognatepeptide for 2 h at RT.

Immunocytochemistry

Human embryonic kidney HEK-293 cells stably transfected witheither sst2A or sst2B were grown on coverslips overnight and fixedwith 4% paraformaldehyde and 0.2% picric acid in 0.1M phosphatebuffer, pH 6.9 for 1 h at RT. Cells were washed several times inTPBS (in mM: Tris, 10; phosphate buffer, 10; NaCl, 137; thimerosal,0.05%; pH 7.4) and preincubated with TPBS containing 0.3% TritonX-100 and 3% NGS for 1 h at RT. Cells were then incubated eitherwith anti-sst2A (GP3 or 6291) or anti-sst2B (9990) antibodies at adilution of 1 : 5000 in TPBS containing 0.3% Triton X-100 and 1%NGS at 4 °C overnight. Anti-sst2B antibodies were raised in rabbitsagainst the following peptide ADNSQSGAEDIIAWV, which corres-ponds to residues 332–346 of the mouse sst2B (Schulzet al. unpub-lished observation). For homologous and heterologous adsorptioncontrols, antisera were preincubated with 10µg/mL of peptidescorresponding to the carboxy-terminal tail of either sst2A or sst2B.Bound primary antibody was detected with biotinylated secondaryantibodies (1 : 1000, Vector) followed by cyanine 3.18 (Cy3)-conjug-ated streptavidin (1 : 400, Amersham). Cells were then dehydrated,cleared in xylol and permanently mounted in DPX (Fluka, Neu-Ulm,Germany).

Free-floating sections (40µm) were washed in TPBS placed inmethanol containing 0.3% H2O2 for 30 min and incubated in 3%normal goat serum (NGS) in TPBS with 0.3% Triton X-100 for 1 h.Tissue sections were then incubated with primary antisera for 72 hin TPBS with 0.3% Triton X-100 and 1% NGS at RT. The followingantisera were used: guinea-pig anti-sst2A (GP3, 1 : 15 000), rabbit anti-sst2A (6291, 1 : 30 000) and rabbit anti-SS-14 (generously provided byDr G. Sperk, 1 : 30 000, Sperk & Widmann, 1984). Affinity-purifiedpreparations of antisera GP3 and 6291 were used in concentrationsranging from 0.1 to 1µg/mL. After washing, sections were processedusing the biotin amplification procedure as previously described,immunofluorescently labelled with streptavidin-Cy3, and mounted onto chrome alum gelatin-subbed glass slides (Adams, 1992; Schulzet al., 1996, 1998).

Thaw-mounted cryostat sections (14µm) were processed usingindirect immunofluorescence methods. Primary antibodies (GP3 and6291) were applied at a dilution of 1 : 500 in TPBS with 0.3% TritonX-100 and 1% NGS overnight at RT. After rinsing in TPBS, sectionswere incubated with Cy3-labelled secondary antibodies (1 : 50,Jackson ImmunoResearch, West Grove, PA) for 1 h at RT. For duallabelling experiments, sections were incubated with a mixture ofprimary antibodies, which were subsequently detected with species-specific secondary antibodies labelled with Cy3 and Cy5. The

3702 S. Schulzet al.

following antibodies were included in dual labelling experiments:rabbit anti-µ-opioid receptor (MOR1, 9998, 1 : 500) and rabbit anti-δ-opioid receptor (DOR1, 5055, 1 : 1000) both of which have beencharacterized extensively (Schulzet al., 1998), mouse anti-SS-14(K121, 1 : 500, Biomeda, Foster City, CA, USA), rat antisubstanceP (SP, NC1/34, 1 : 1000, Accurate Chemicals, Westbury, NY, USA),mouse anticalcitonin gene-related peptide (CGRP, 1 : 400, Biotrend,Koln, Germany) and mouse antiparvalbumin (PA-235; 1 : 1000,Sigma, Deisenhofen, Germany). Sections were then dehydrated

FIG. 1. Immunodot-blot analysis of the specificity of anti-sst2A antisera. Serialdilutions (0–2000 ng) of the peptides corresponding to the carboxy-terminalregions of sst1, sst2A, sst2B, sst3, sst4 and sst5 were blotted on to nitrocellulosemembranes and incubated with anti-sst2A (GP3). Blots were subsequentlyincubated with biotinylated secondary antibodies and ABC complex anddeveloped with DAB.

FIG. 2. Characterization of anti-sst2A antisera using stably transfected HEK-293 cells. Immunofluorescent labelling and confocal imaging of wild-type HEK-293cells (A,B) and HEK-293 cells transfected with a construct coding for sst2A (C,D,G,I) or sst2B (E,F,H,J) using the anti-sst2A antiserum GP3 (A,C,E,G,I) and theanti-sst2B antiserum 9990 (B,D,F,H,J). Homologous absorption controls with 10µg/mL the cognate peptides (G,H). Heterologous absorption controls with10 µg/mL of the non-cognate peptides (I,J). Scale bar5 15 µm.


through several concentrations of alcohol, cleared in xylol andcoverslipped with DPX. For immunocytochemical controls, theprimary antibody was either omitted, replaced by preimmune sera oradsorbed with several concentrations ranging from 1 to 10µg/mL ofhomologous or heterologous peptides for 2 h at RT.

Specimens were examined using a Leica TCS-NT laser scanningconfocal microscope equipped with a krypton/argon laser. Cy3 wasimaged with 568 nm excitation and 570–630 nm bandpass emissionfilters, and Cy5 was imaged with 647 nm excitation and 665 nmlongpass emission filters.

Results

Characterization of antisera

Specificity of the antisera was monitored using immunodot-blotanalysis. After four boost injections three guinea-pig anti-sst2A antiseraand one rabbit anti-sst2A developed a titre against their immunizingpeptides. As shown in Fig. 1, the anti-sst2A antiserum GP3 detectedquantities as low as 25 ng of their cognate peptide but not the peptidescorresponding to other somatostatin receptor subtypes. The anti-sst2Aantiserum 6291 gave similar results (data not shown).

These antisera were further characterized using immunofluorescentstaining of stably transfected HEK-293 cells. When either wild-type,sst2A- or sst2B-transfected HEK-293 cells were stained with the anti-sst2A antiserum GP3, prominent immunofluorescence localized at thelevel of the plasma membrane was only seen in HEK-293 cellsbearing the somatostatin receptor sst2A (Fig. 2A,C,E). This stainingpattern was completely blocked by preincubation of the antiserumwith homologous but not heterologous peptides (Fig. 2G,I). Similarresults were obtained with the anti-sst2A antiserum 6291 (data notshown). When either wild-type, sst2A- or sst2B-transfected HEK-293cells were stained with the anti-sst2B antiserum 9990, prominentimmunofluorescence localized at the level of the plasma membranewas only seen in HEK-293 cells transfected with the mouse somato-statin receptor sst2B (Fig. 2B,D,F). This staining was also completelyblocked by preincubation of the antiserum with homologous but not


heterologous peptides (Fig. 2H,J) showing that these cells were indeedexpressing the mouse somatostatin receptor sst2B.

Next, the antisera were tested for possible cross-reactivity withother proteins present in brain tissue. When membrane preparationsfrom rat brain were subjected to SDS–PAGE and Western blotting,the antiserum GP3 detected a broad band of 80 kDa and a smallerband at 55 kDa in the spinal cord, hippocampus and cortex but notin the cerebellum (Fig. 3, left panel). The same bands were detectedwhen either crude membrane preparations or WGA-purified membraneproteins were used. These bands were no longer detected when theantiserum was preincubated with its cognate peptide (Fig. 3, rightpanel). The anti-sst2A antiserum 6291 gave similar results (not shown).

In addition, when spinal cord sections were immunocytochemicallystained with these antisera, immunostaining was dose-dependentlyabolished by preabsorption of the antisera with increasing concentra-tions of homologous but not of heterologous peptides (1–10µg/mL)(Fig. 4A,B).

Immunocytochemical distribution of sst2A and SS-14 in thespinal cord

The sst2A antisera GP3 and 6291 yielded an identical staining patternwith prominent sst2A-Li contained in a dense network within thesuperficial dorsal horn at all spinal levels (Fig. 4A). This networkconsisted mostly of plasmalemma of neuronal perikarya and dendrites(Figs 4C and 5A). In parasaggital sections of the lamina II outer zone(II o), densely packed sst2A-immunoreactive dendrites and perikaryaextending their processes in rostrocaudal orientation could be seen(Fig. 5B). sst2A-Li was also present in the intermediolateral cellcolumn (not shown). In parasaggital sections of the lamina II inner

FIG. 3. Western blot analysis of sst2A-immunorectivity in rat brain. Membranepreparations from the spinal cord, hippocampus, cortex and cerebellum wereseparated on a 8% SDS–polyacrylamide gel and blotted on to nitrocellulose.Membranes were then incubated with anti-sst2A antiserum GP3 at a dilutionof 1 : 20 000 in the absence (–) or presence (1) of the peptide antigen(10 µg/mL). Blots were developed using enhanced chemiluminescence.Ordinate, migration of protein molecular weight markers (Mr 3 10–3).


zone (IIi) sst2A-Li was present on small round cell bodies with a star-shaped dendritic formation (Fig. 4C). In preparations of the dorsalhorn dually stained for sst2A- and SS-14-Li, somatostatin receptorsst2A-positive plasmalemma appear in many cases to be apposed by,but not co-contained within, SS-14-positive nerve fibres and terminals(Fig. 5A,A9). The position of sst2A-Li perikarya and dendrites in thedorsal horn appeared to be similar to those containing MOR1-Li;however, two colour, double immunostaining revealed no instancesof coexistence of these two receptors (Fig. 5B,B9). In order todetermine whether sst2A-Li is located on the terminals of dorsal rootganglion neurons or second-order spinal neurons, we double labelledtissue sections with antibodies to either SP or CGRP, both of whichare synthesized in DRG neurons. No evidence of coexistence ofsst2A-Li and either SP- (Fig. 5C,C9) or CGRP-containing fibres wasfound (not shown).

SS-14-Li formed a dense plexus in the superficial layers of thedorsal horn, the lateral spinal nucleus, the dorsal grey commissureand the intermediolateral cell column (Fig. 4D). In addition, amoderately dense plexus of SS-14-Li was observed throughout thedeeper layers of the spinal grey matter (Fig. 4D,E). Most SS-14-Liappeared in nerve fibres that were morphologically similar to varicoseaxons (Figs 4F and 5A9).

Immunocytochemical localization of sst2A in the dorsal rootganglia

In the DRG, a subset of medium diameter neurons was stained withanti-sst2A. In these neurons most prominent sst2A-Li was seen in thesomatic plasmalemma (Fig. 6D,E). In dual-stained preparations, itwas evident that SS-14-Li was also contained in neurons medium insize but never co-contained with sst2A-Li (Fig. 6D,D9). In theseneurons SS-14-Li was seen in the perinuclear cytoplasm as well asin vesicle-like structures within the axoplasm presumably giving riseto SS-14-containing primary afferents (Fig. 6B,D19). DOR1-Li wasfound in small diameter neurons. In these cells, a punctuate stainingwas seen in the cytoplasm and axoplasm possibly representing vesiclestransporting the receptor (Fig. 6C,E9). In preparation dually stainedwith anti-sst2A and anti-DOR1 antisera, no colocalization of the tworeceptors was found (Fig. 6E,E9). Interestingly, SS-14-Li and DOR1-Li but not sst2A-Li was present on fibres in the dorsal root entry zone.For SS-14 or DOR1 immunofluorescently labelled fibres (presumablyprimary afferents) were also seen in the pia (Fig. 6A–C). In addition,sst2A-Li was resistant to dorsal rhizotomy (not shown). These findingssuggest that sst2A-Li did not occur on primary afferent fibres.

Discussion

In the present study, we have raised antisera in rabbits and guinea-pigs that specifically react with somatostatin receptor sst2A. Severallines of evidence suggest that these antibodies selectively detect theirtargeted receptor. First, in immunodot-blot assays the anti-sst2Aantisera specifically detected their cognate peptides but not thepeptides corresponding to the carboxy-terminal region of othersomatostatin receptor subtypes. Second, immunocytochemical stainingof transfected HEK-293 cells revealed that the anti-sst2A antiseraselectively stained cells expressing the sst2A receptor but did not stainwild-type cells or cells transfected with the sst2B receptor. Third, inWestern blots the antisera detected a band of the appropriate molecularweight (80 kDa) in brain regions known to contain dense somatostatinreceptors but not in the cerebellum where neither somatostatin bindingsites nor sst2 mRNA has been detected. This staining was neutralizedby preincubation of the antisera with their cognate peptide. Fourth,


both antisera stained tissue sections. The staining pattern of theantisera GP3 and 6291 was identical and completely abolished afterpreincubation with the peptides (10µg/mL) used for immunizations.In addition, the overall distribution of sst2A-Li in rat brain was inexcellent agreement to that previously reported by Schindleret al.(1997). Finally, the carboxy-terminal peptide is likely to have servedas sst2A-specific immunogen as this peptide was found to havehomologies no greater than 58% to other peptide sequences whenaligned to current entries in the EMBL databases using BLASTpor FASTa.

FIG. 4. Immunocytochemical distribution of sst2A and SS-14 in the spinal cord. (A) Coronal spinal cord section stained with anti-sst2A antiserum GP3.(B) Corresponding adsorption control. Anti-sst2A antiserum was preincubated with 10µg/mL of its cognate peptide. (C) Parasaggital section of lamina II innerzone (IIi) of the spinal cord stained with anti-sst2A. (D) Coronal spinal cord section stained with rabbit anti-SS-14. (E) Higher magnification of D. (F) Parasaggitalsection of lamina II outer zone (IIo) of the spinal cord stained with anti-SS-14. Note, in the spinal cord sst2A-Li is enriched in the superficial layers of the dorsalhorn. SS-14-Li shows a largely overlapping distribution in that it is most dense in the superficial dorsal horn. While sst2A-Li appears to be predominantlytargeted to the somatodendritic compartment, SS-14-Li is predominantly contained in fibres and varicosities. Scale bars: A, B, D5 500µm; C, E, F5 50 µm.


Previous autoradiographic binding studies could not unequivocallyidentify the cellular location of somatostatin receptors in the spinalcord (Uhl et al., 1985; Hollowayet al., 1996). Using iodinated SS-28 or iodinated BIM-23027, a sst2-selective ligand, moderate bindingwas seen in the superficial layers of the dorsal horn, and low tomodest binding was seen in the deeper layers of the spinal cord. Thissuggests that the majority of binding sites in the substantia gelatinosa(lamina II) may represent sst2A. Thus, the distribution of sst2A-Li isin good agreement with the distribution of somatostatin receptor sst2-binding sites in the spinal cord. The light microscopic analysis


FIG. 5. Immunofluorescent confocal images of spinal cord sections after double labelling with guinea-pig anti-sst2A and rabbit anti-SS-14 (A and A9), guineapig anti-sst2A and rabbit anti-MOR1 (B and B9) or guinea-pig anti-sst2A and rat anti-SP (C and C9). A-A9, in coronal dorsal horn sections sst2A-Li is seen inthe plasmalemma of neuronal perikarya and dendrites which are in many cases closely opposed, but not co-contained within, SS-14-Li fibres and terminals. Band B9 in parasaggital dorsal horn sections sst2A-Li appears to be in a similar position but not coexistent with MOR1-Li. C and C9 in parasaggital dorsal hornsections no colocalization of sst2A-Li and SP-Li is seen. Scale bar5 25 µm.



FIG. 6. Immunofluorescent confocal images of spinal cord sections stained with anti-sst2A, anti-SS-14 or anti-DOR1 (A–C) and DRG preparations double stainedwith guinea-pig anti-sst2A and rabbit anti-SS-14 (D and D9) or guinea-pig anti-sst2A and rabbit anti-DOR1 (E and E9). Note, (1) SS-14- and DOR1- but notsst2A-Li were prominent in the dorsal root entry zone of the dorsal horn and (2) sst2A-Li is most prominent in the somatic plasmalemma of a subpopulation ofmedium size neurons. A different population of medium size neurons contains SS-14-Li in their cytoplasm and axoplasm. DOR1-Li is seen in vesicle-likestructures within the axoplasm and cytoplasm of small diameter neurons. Scale bars: A–C5 250µm; D–E9 5 100µm.



performed in the present study provides the cellular resolutionnecessary to establish that sst2A is primarily targeted to the plasmamembrane of nerve cell bodies and primary dendrites and, hence,may function in a postsynaptic manner, e.g. in the superficial layersof the spinal cord, sst2A-Li was found in plasmalemma of small roundcell bodies and their dendrites. The distribution of SS-14-Li resembledclosely the pattern of sst2A-Li, with the highest density in thesubstantia gelatinosa where in many cases sst2A-positive plasmalemmawere directly apposed by SS-14-positive terminals indicating that SS-14 may be a physiological relevant ligand for sst2A.

Somatostatin suppresses spinal nociceptive responses differentlythan opioids do. Electrophysiological evidence indicates that somato-statin hyperpolarizes spinal dorsal horn neurons and inhibits neuralresponses to noxious thermal or chemical stimulation of cutaneoustissue (Randic & Miletic, 1978; Muraseet al., 1982; Sandkuhleret al., 1990; Chapman & Dickenson, 1992). Interestingly, the potencyof the sst2-selective ligand, octreotide, to affect the neural activity ofdorsal horn neurons greatly exceeds that seen for somatostatin (Maureret al., 1982; Chapman & Dickenson, 1992). These responses are notreversed by addition of naloxone. Octreotide has also been usedclinically to treat certain cancer pain and to alleviate the pain ofclustered headache, a trigeminal-mediated syndrome (Sicuteriet al.,1984; Pennet al., 1992). Taddeseet al. (1995) showed that small(25 µm in diameter) nociceptive DRG neurons preferentially respondto opioids while nociceptive DRG neurons larger in size (45µm indiameter) were inhibited by somatostatin. Taken together, althoughadditional somatostatin receptor subtypes are likely to be involved,the present results not only provide a morphological substrate forspinal octreotide analgesia but also show that somatostatin and opioidsare poised to modulate nociceptive transmission by distinct anatomicalsystems. Whileµ and δ opioid receptors are primarily present onsmall diameter DRG neurons (Dadoet al., 1993; Arvidsonet al.,1995), sst2A-Li was seen on medium size DRG neurons. In thesuperficial layers of the dorsal horn, sst2A-Li appeared to parallelMOR1-Li; however, no instances of colocalization have beenobserved. The presence of both sst2A-Li and high levels of SS-14-Liin the intermediolateral cell column suggests that this receptor mayalso participate in autonomic regulation at the spinal cord level.

In conclusion, we show that the sst2A receptor is in a position tomodulate the processing of sensory information at the level of thespinal cord. The present findings reinforce the notion that somesubtypes of somatostatin receptors may inhibit nociceptive transmis-sion in a manner fundamentally different from opioid receptors.

Acknowledgements

We thank Dana Wiborny and Dora Nu¨β for skilful technical assistance, andDr G. Sperk for kindly providing ant-SS-14 antisera. This work was supportedby a grant from the Fonds der Chemischen Industrie to V.H. and grants(1908 A/0025 to S.S.) from the Kultusministerium des Landes Sachsen/Anhaltand by grant (to V.H.) from the BMBF (Schwerpunkt Neurotraumatologie).

Abbreviations

Cy3 cyanin 3.18CGRP calcitonin gene-related peptideDAB 3,39-diaminobenzidineDOR1 δ opioid receptorDRG dorsal root ganglionLi like immunoreactivityMOR1 µ opioid receptorNGS normal goat serumSDS–PAGE sodium dodecyl sulphate–polyacrylamide gel electrophoresisSS-14 somatostatin-14


sst somatostatin receptorSP substance PRT room temperature

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immunocytochemical localization of somatostatin receptor sst2a in the rat spinal cord and dorsal...

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