Distribution of Galanin Receptor 1Immunoreactivity in the Rat Stomach

and Small Intestine

THOMAS PHAM,1,2,4 STEFANIA GUERRINI,1–4 HELEN WONG,1,2

JOSEPH REEVE, JR,1,2,4AND CATIA STERNINI1–4*

1CURE Digestive Diseases Research Center, Division of Digestive Diseases, University ofCalifornia, Los Angeles, California 90095

2Department of Medicine, University of California, Los Angeles, California 900953Department of Neurobiology, University of California, Los Angeles, California 90095

4Veteran Administrations Greater Los Angeles Healthcare System,Los Angeles, California 90073

ABSTRACTGalanin affects gastrointestinal functions by activating different G protein–coupled

receptors. Here, we identified the sites of expression of the galanin receptor 1 (GAL-R1)subtype in the rat stomach and small intestine by using immunohistochemistry with anantibody raised to the third intracellular loop of rat GAL-R1 (GAL-R1Y225-238) and confocalmicroscopy. Antibody specificity was confirmed by (1) the detection of a band at approxi-mately 70 kDa in Western blot of membranes from GAL-R1 transfected cells, (2) the cellsurface staining of GAL-R1 transfected cells, which was not detected in control cells, and (3)the abolition of Western signal and tissue immunostaining by preadsorbing the antibody withthe peptide used for immunization. GAL-R1 immunoreactivity was localized to the cellsurface of enterochromaffin-like cells, and of myenteric and submucous neurons, and to fibersdistributed to the plexuses, interconnecting strands, muscle layers, vasculature, and mucosa.A dense network of GAL-R1 immunoreactivity was observed in the deep muscular plexus invery close association with interstitial cells of Cajal visualized by c-kit immunostaining. Inthe ileum, 81.6% of GAL-R1 myenteric neurons and 70.7% of GAL-R1 submucosal neuronswere substance P immunoreactive. Vasoactive intestinal polypeptide immunoreactivity wasfound in 48.3% of GAL-R1 submucosal neurons, but not in GAL-R1 myenteric neurons. Thesefindings support the hypothesis that GAL-R1 mediates galanin actions on gastrointestinalmotility and secretion by modulating the release of other neurotransmitters and contributesto galanin-induced inhibition of gastric acid secretion by means of the suppression of endog-enous histamine release. J. Comp. Neurol. 450:292–302, 2002. © 2002 Wiley-Liss, Inc.

Indexing terms: enteric neurons; excitatory neurons; inhibitory neurons; enterochromaffin-like

cells; interstitial cells of Cajal; gastrointestinal motility

Galanin is a neuroendocrine peptide that influences nu-merous functions, including learning, memory, nocicep-tion, feeding behavior, and the release and action of sev-eral neurotransmitters such as acetylcholine, noradrenaline,and serotonin (Kask et al., 1995, 1997). In the gastroin-testinal tract, galanin is expressed in enteric neurons andfibers distributed to all the layers of the gut (Ekblad et al.,1985b) and it exerts a multitude of biological effects,mainly acting as an inhibitory neuromodulator (Rattan,1991). Galanin is involved in the regulation of both secre-tion and contractility. It inhibits acid gastric secretion andregulates the release of numerous peptides, including glu-cagon, insulin, somatostatin, and pancreatic polypeptide

(Bauer et al., 1989). Galanin induces both excitatory andinhibitory effects on the gastrointestinal motility (Fox et

Grant sponsor: National Institutes of Health; Grant number: DK57037;Grant number: DK35740; Grant number: DK41301.

*Correspondence to: Catia Sternini, CURE, Digestive Diseases ResearchCenter, Bldg. 115, Room 224, VAGLAHS, 11301 Wilshire Blvd., Los Ange-les, CA 90073. E-mail: csternin@ucla.edu

Received 9 July 2001; Revised 5 February 2002; Accepted 8 May 2002DOI 10.1002/cne.10311Published online the week of July 15, 2002 in Wiley InterScience (www.

interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 450:292–302 (2002)

© 2002 WILEY-LISS, INC.

al., 1986, 1988; Bauer et al., 1989; Brown et al., 1990;Chakder and Rattan, 1991). For instance, in the rat stom-ach, galanin causes an initial, short-lasting inhibitory ef-fect on the gastric motility followed by an excitatoryphase, perhaps by activating different galanin receptorsubtypes (Guerrini et al., unpublished observations).

Galanin triggers cellular responses by acting on specificG-protein coupled receptors, which in turn influence in-tracellular effectors. Three G protein–coupled receptorsfor galanin have been cloned, named GAL-R1, GAL-R2,and GAL-R3, and pharmacologic studies strongly suggestthe existence of additional unidentified galanin receptors(Floren et al., 2000). Galanin receptors are highly con-served among species, and share a low percentage of ho-mology within the same species (Wang et al., 1997a, b).GAL-R1, the first to be identified and characterized, con-sists of 349 amino acids in humans (346 in rats). In hu-mans, this receptor shares 42% of amino acid sequencehomology with GAL-R2 and 38% with GAL-R3 (Parker etal., 1995). In situ hybridization studies showed the pres-ence of GAL-R1 mRNA in the brain and the spinal cordand in the peripheral nervous system (Parker et al., 1995;Burgevin et al., 1995; Waters and Krause, 2000); further-more, this receptor has also been identified by Northernblot analysis and reverse transcriptase-polymerase chainreaction in human Bowes melanoma cells (Habert-Ortoliet al., 1994), insulinoma cells (Parker et al., 1995), as wellas in the human mucosa epithelial cells of the gastroin-testinal tract (Lorimer and Benya, 1996). GAL-R1 is cou-pled to a Gi/Go-protein, and it is pertussis toxin sensitive.Receptor activation leads to inhibition of adenylate cy-clase with subsequent decrease of cAMP production(Lagny-Pourmir et al., 1989; Habert-Ortoli et al., 1994;Burgevin et al., 1995) and to activation of mitogen-activated protein kinases, which are serine/threonine ki-nases acting on transcription factors and ultimately in-volved in cell proliferation and growth (Wang et al., 1998).

In the guinea pig intestine, galanin inhibits the electri-cally induced contractile response mediated by either ace-tylcholine or substance P (Fox et al., 1986; Muramatsuand Yanaihara, 1988; Bauer et al., 1989; Akehira et al.,1995) and this inhibitory effect is reversed by pertussistoxin, suggesting the involvement of a Gi/Go protein–coupled receptor. Because galanin acts as an inhibitoryneuromodulator on cholinergic and tachykinergic trans-mission through a pertussis toxin sensitive mechanism, itcan be hypothesized that the inhibitory GAL-R1 mediatesthis galanin effect. The elucidation of the sites of cellularexpression of galanin receptors in the gastrointestinaltract represents a fundamental step to understand thefunction and mode of actions of galanin. The aims of thepresent study were to (1) determine the distribution ofGAL-R1 in the rat stomach and small intestine by meansof immunohistochemistry with a polyclonal antiserum di-rected to the third intracellular loop of rat GAL-R1, and(2) characterize the cell types expressing GAL-R1 by usingdouble-labeling immunofluorescence approaches with an-tibodies to substance P (marker for myenteric excitatorymotor neurons) and vasoactive intestinal polypeptide (amarker for descending myenteric neurons) and antibodiesthat visualize enterochromaffin (ECL)-like cells and inter-stitial cells of Cajal.

MATERIALS AND METHODS

Antibody preparation

A polyclonal antibody was raised in New Zealand Whitefemale rabbits against the third intracellular loop of ratGAL-R1 (GAL-R1Y225-238; antiserum 96202). The antibodywas raised against the following sequence (dYH-KKLKNMSKKSEA), chosen because the same sequencewas not expressed by GAL-R2 or GAL-R3. The peptidewas synthesized on an Advanced Chemtech Peptide Syn-thesizer (model 396; Louisville, KY) and was purified byreverse-phase, high-pressure liquid chromatography. Thecomposition of the peptide was verified by mass spectros-copy, and its purity was evaluated by high performancecapillary electrophoresis. Fractions with greater than 90%purity were pooled and lyophilized. The detailed method-ological procedures for the production, screening, and pu-rification of the antibody have been published previously(Sternini et al., 1997). The antibody was screened byenzyme-linked immunosorbent assay and Western blotanalysis as previously described (Sternini et al., 1997).

Western blotting

KNRK cells transfected with GAL-R1 cDNA were lysedin Laemmli buffer. Aliquots (20 mg total protein/lane)were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) on a 4–15% polyacryl-amide gel (Bio-Rad, Hercules, CA) and then transferred tonitrocellulose. After incubation with blocking solution (3%nonfat dry milk in 0.1 M phosphate buffered saline, pH 7.4[PBS], 0.1% Tween 20) for 1 hour at room temperature,membranes were incubated with the receptor antiserumat a final dilution of 1:3,000 overnight at room tempera-ture, then washed and incubated with goat anti-rabbitimmunoglobulin G (IgG) conjugated to peroxidase at1:7,500 dilution for 1 hour at room temperature. Immuno-reactive bands were detected by using an Amersham ECLdetection kit (Amersham, Arlington Heights, IL), accordingto the manufacturer’s protocol. As a control, the receptorantiserum was preadsorbed with 1 �M of GAL- R1Y225-238peptide for 1 hour at 37°C and used in place of the receptorantibody. As additional control, the nontransfected KNRKcells were used together with GAL-R1–expressing KNKRcells.

Immunostaining of HEK293 cells

HEK293 cells transfected with cDNA encoding forGAL-R1 were grown on glass coverslips in Dulbecco’s mod-ified Eagle medium (Life Technologies, Grand Island, NY),supplemented with 10% fetal bovine serum (Life Technol-ogies), Hepes buffer (25 mM, pH 7.55; Life Technologies),and gentamycin (20 �g/ml; Life Technologies) at 37°C in ahumidified incubator containing 5% CO2. Cells werewashed in PBS and then fixed in 4% paraformaldehyde for10 minutes at room temperature. Cells were then perme-abilized with 0.1% Triton-X in PBS for 15 minutes at roomtemperature, washed twice with PBS, incubated with 10%normal goat serum in PBS for 30 minutes, and then incu-bated with GAL-R1 antiserum (1:2,000 dilution) for 60minutes. Subsequently, cells were washed twice in PBSand incubated with goat anti-rabbit IgG conjugated withfluorescein isothiocyanate (FITC, 1:100 dilution; JacksonImmunolabs, West Grove, PA) for 2 hours at room tem-perature. Finally, coverslips were washed in PBS twiceand mounted with glycerol on slides. As a control, non-

293GAL-R1 IN THE RAT GI TRACT

transfected HEK293 cells were simultaneously processed.Cells were analyzed by using a Zeiss Laser Scanning mi-croscope 410 with a krypton/argon laser and attached to aZeiss Axiovert 100 microscope with a PlanApo (Carl Zeiss,Thornwood, NY) 100� 1.4 na objective. Images were col-lected at a magnification zoom of 1 (z-axis of 0.7 �m).Images were prepared and labeled by using Adobe Photo-shop v. 5.0.1 (Adobe Systems, Mountain View, CA).

Tissue preparation

Care and handling of the animals were approved by theAnimal Research Committee of the Veteran Administra-tions Greater Los Angeles Healthcare System in accor-dance with all NIH guidelines. Adult Sprague-Dawley180- to 300-g rats of either sex were anesthetized withintraperitoneal sodium pentobarbital (Nembutal, 50 mg/kg; Abbott Laboratories, Chicago, IL). For cryostat sec-tions, rats were perfused with 50 ml of 0.1 M phosphatebuffer, pH 7.4 (PB), followed by 500 ml of 4% paraformal-dehyde in PB. Segments of the stomach and small intes-tine were collected and rinsed, post-fixed for 1 hour atroom temperature, rinsed thoroughly in PBS, and thenstored in 25% sucrose in PBS at 4°C. Tissues were cut at12 �m thickness by using a cryostat perpendicularly to thelumen and collected onto gelatin-coated slides. For whole-mount preparations, animals were injected intraperitone-ally with colchicine (5 mg/kg; Sigma Chemical, St. Louis,MO) 18–24 hours before tissue collection. Specimens ofthe ileum were collected, thoroughly washed in saline,opened along the mesenteric region, stretched and pinnedonto a waxed plate (mucosa down), and fixed in 4% para-formaldehyde in 0.1 M PB for 2 hours at room tempera-ture. Tissues were then stored in 25% sucrose in 0.1 M PBwith 0.1 g/L of sodium azide at 4°C and sectioned flat byusing a sliding microtome from the serosa through themucosa at 25 �m thickness (Sternini et al., 1995, 1997).

Immunohistochemistry

Cryostat sections and whole-mount preparations wereprocessed by the immunofluorescence method for singleand double labeling as previously described (Sternini etal., 1995, 1997). For single-labeling, tissues were washedin PB, incubated in 10% normal goat serum for 1 hour atroom temperature to minimize the background, incubatedin GAL-R1 antiserum (1:2,000–1:3,000), 18–24 hours forcryostat sections, 72 hours for whole-mounts at 4°C, fol-lowed by a 2-hour incubation in affinity purified goatanti-rabbit IgG conjugated with FITC at 1:100 dilution,washed and, if free floating, mounted on slides, and thencover-slipped. For double-label immunofluorescence, cryo-stat sections of the stomach were incubated in a mixture ofrabbit GAL-R1 antiserum and mouse monoclonal antibodyto either calbindin (1:2,000) or to histidine decarboxylase,the histamine-forming enzyme (HDC; 1:2,000) (Table 1),

both markers for enterochromaffin-like (ECL) cells, over-night at 4°C. Whole-mounts of the ileum were incubatedin a mixture of rabbit GAL-R1 antiserum and mousemonoclonal vasoactive intestinal polypeptide (VIP) anti-body (VIP55; 1:1,500 dilution), or guinea pig substance Pantiserum (SP7K; 1:1,000 dilution) (Table 1) for 3 days at4°C, followed by a 2-hour incubation at room temperaturewith a mixture of affinity-purified donkey anti-rabbit IgGcoupled with FITC (1:100; Jackson Immunolabs) and don-key anti-mouse or anti-guinea pig IgG conjugated withrhodamine red-X (Red-X; 1:300–1:500; Jackson Immuno-labs). Tissues were mounted and cover-slipped with 90%glycerol and 2% potassium iodide in 0.1 M PB. Donkeyanti-rabbit antibody was absorbed against mouse andguinea pig serum, respectively, and donkey anti-guineapig and anti-mouse IgGs were preadsorbed against rabbitserum to minimize cross-reaction of the secondary anti-body with the inappropriate antigen-antibody complex.Primary and secondary antibodies were diluted in 0.5%Triton X-100 in 0.1 M PB.

Specificity controls included preadsorption of the GAL-R1, SP, and VIP antibodies with the appropriate peptides(10 �M) for 16–24 hours at 4°C. SP and VIP antibodieshave been previously validated for immunohistochemicaluse in gut tissue (Sternini and Anderson, 1992; Wong etal., 1996). Tissues were examined with confocal micros-copy and images were prepared with Adobe Photoshop asstated above. All confocal images shown here are singleoptical sections. Three colchicine-treated rats were usedfor cell counting. For each animal, 10 ganglia were ran-domly selected, the number of myenteric and submucousneurons expressing GAL-R1, SP, or VIP were counted,and the mean percentage of GAL-R1 neurons containingSP or VIP was calculated.

RESULTS

Western blotting

The GAL-R1 antiserum 96202 recognized a broad bandat approximately 70 kDa in membranes obtained fromKNRK cells transfected with cDNA for GAL-R1 (Fig. 1A).When the same membranes where incubated with theantiserum preadsorbed with the peptide, no immunoreac-tive band was detected (Fig. 1B). Similarly, GAL-R1 im-munoreactivity was not detected in membranes of non–receptor-expressing KNRK cells (not shown).

Immunocytochemical localization of GAL-R1in HEK293 cells

The GAL-R1 antiserum 96202 stained the plasma mem-brane of HEK293 cells transfected with GAL-R1 cDNA(Fig. 1C). No staining could be detected in nontransfectedHEK293 cells (Fig. 1D).

Cellular localization of GAL-R1 in the ratstomach and small intestine

In the stomach, GAL-R1 immunoreactivity was localizedto epithelial cells of the oxyntic region of the gastric mucosa,which have the distribution and morphologic appearance ofECL-like cells (Figs. 2A, 3C), and to myenteric neurons andfibers (Figs. 2A,C, 3A,B). GAL-R1 immunoreactivity waspredominantly localized at the cell surface of both neuronsand epithelial cells. GAL-R1–immunoreactive fibers wereabundant within the myenteric plexus (Fig. 3A) and in in-

TABLE 1. Characteristics of Primary Antibodies

Antigen Host Dilution Code, reference

Calbindin Mouse 1:1000 Sigma, St. Louis, (MO)Histidine decarboxylase Mouse 1:50 (Ohning et al., 1998)c-kit (M-14) Goat 1:500 Sc-1494, Santa Cruz

Biotechnology, Inc.,Santa Cruz (CA)

Substance P7K Guinea pig 1:1000 (Sternini et al., 1995)VIP55 Mouse 1:1500 (Wong et al., 1996)

294 T. PHAM ET AL.

terconnecting strands. Bundles of fibers were distributed tothe muscle layers (Figs. 2C, 3A), mucosa (Fig. 2A), andvasculature (not shown). In the circular muscle layer, wherethe deep muscular plexus is located, there was a densenetwork of GAL-R1–immunoreactive fibers, which appearedto be associated with putative interstitial cells of Cajal (Fig.3B). In the mucosa, GAL-R1 processes ran parallel to theepithelial cells, from the base of the mucosa to the lumen(Fig. 2A). Abolition of immunostaining obtained by pread-sorption of the GAL-R1 antiserum 96202 with its homolo-gous peptide used for immunization confirmed the specificityof GAL-R1 immunostaining in tissue (Fig. 2B,D).

In the small intestine, GAL-R1 immunoreactivity was ob-served in both myenteric and submucous neurons (Fig. 4A,B) as well as in fibers. GAL-R1–immunoreactive fibers wereabundant within the plexuses and in interconnectingstrands, as well as in the intestinal wall. As in the stomach,there was a dense network of GAL-R1–immunoreactive fi-bers in the deep muscular plexus in close association tointerstitial cells of Cajal, visualized by c-kit immunostaining(see below). GAL-R1–immunoreactive fibers were also ob-served in the intestinal mucosa, where they ran up to thetips of the villi (not shown). GAL-R1 immunoreactivity wasnot detected in smooth muscle cells of the rat stomach andsmall intestine.

Double-labeling experiments

In the gastric mucosa, epithelial cells expressingGAL-R1 were immunoreactive for either calbindin (Fig. 5),a marker for ECL cells (Buffa et al., 1989; De Giorgio etal., 1996) or HDC, the enzyme responsible for histaminesynthesis (Ohning et al., 1998). GAL-R1 immunoreactivitywas mainly concentrated at the cell surface, whereas cal-bindin (Fig. 5) or HDC immunoreactivity was detectedinside the cytoplasm. HDC-positive cells showed a broadrange of shapes, from small oval to larger irregular shapes

(Ohning et al., 1998). GAL-R1 was found to colocalizemainly with smaller HDC-immunoreactive cells (notshown).

Double-labeling experiments using a c-kit antibody, amarker for interstitial cells of Cajal (Vanderwinden et al.,2000), showed that these cells in the deep muscular plexuswere closely wrapped by dense networks of GAL-R1-immunoreactive fibers. GAL-R1-immunoreactivity did notappear to be expressed by the interstitial cells of Cajal asindicated by confocal microscopy analysis (Fig. 5), eventhough a final determination would require electron mi-croscopy.

A relative quantification and characterization of thetypes of enteric neurons bearing the GAL-R1 was con-ducted in the ileum. In the myenteric plexus, 4.7 neuronsper ganglion were GAL-R1-immunoreactive, with a max-imum of 10 per ganglion (total number of GAL-R1 cellscounted 147). In the submucosal plexus, 3.6 neurons perganglion were GAL-R1-immunoreactive, with a maximumof 7 per ganglion (total number of GAL-R1 cells counted226). The vast majority of GAL-R1 myenteric neurons wasimmunoreactive for SP, a marker for excitatory, cholin-ergic myenteric neurons (Fig. 6).

By contrast, immunoreactivity for VIP, a marker fordescending myenteric neurons, was not detected in anyGAL-R1 myenteric neurons (data not shown). In the sub-mucous plexus, GAL-R1 immunoreactivity appeared tocolocalize with both SP (Fig. 7) and VIP immunoreactivity(Fig. 8).

Approximately 81.6% of GAL-R1 myenteric neurons(120 of 147 GAL-R1 myenteric neurons counted) and70.7% of GAL-R1 submucous neurons (75 of 106 GAL-R1submucous neurons counted) were immunoreactive for SP(Table 2). Conversely, 57.9% of SP-immunoreactive myen-teric cells and 52.8% of SP-immunoreactive submucosalcell bodies expressed GAL-R1. In the submucous plexus,

Fig. 1. Western blot of membranes from KNRK cells transfectedwith galanin receptor 1 (GAL-R1) cDNA incubated with the GAL-R1antiserum 96202 (A) and incubated with the antiserum preadsorbedwith the homologous peptide (B). The 96202 antiserum labels a broadband at around 70 kDa in KNRKs membranes, whereas a comparableimmunoreactive band is lacking when the antiserum is preadsorbedwith the peptide. Molecular size markers in kilodaltons are shown on

the left. C,D: Confocal images of HEK293 cells transfected with cDNAencoding for GAL-R1 (C) and non–receptor-expressing HEK293 cells(D) incubated with the GAL-R1 antiserum. Note the immunostainingconfined to the cell surface in transfected cells (arrows) and the lack ofimmunoreactivity in control nontransfected cells. Scale bar � 20 �min D (applies to C,D).

295GAL-R1 IN THE RAT GI TRACT

VIP immunoreactivity was observed in 48.3% of GAL-R1neurons (58 of 120 GAL-R1 submucous neurons counted),whereas 61.1% of VIP submucosal cell bodies displayedGAL-R1 immunoreactivity (Table 2). Preadsorption of theGAL-R1 antibody with SP or VIP did not modify GAL-R1immunostaining. Similarly, preadsorption with an excessof SP or VIP prevented SP or VIP staining, respectively,whereas an excess of GAL-R1 peptide did not affect the SPor VIP staining, but it quenched the GAL-R1 staining.Controls for double-labeling confirmed that the GAL-R1antiserum and the SP or VIP antibody do not cross-reactwhen mixed and that the secondary antisera bind only toappropriate antigen-antibody complexes.

DISCUSSION

We have developed an antibody specific for the GAL-R1subtype and have shown that this receptor is widely ex-pressed in the rat stomach and small intestine. GAL-R1 islocalized to neuronal structures of the stomach and intes-tine and to endocrine cells of the stomach, indicating thatthe galanin effects mediated by this receptor involve theactivation of both neuronal and nonneuronal (endocrine)pathways.

Specificity of the antiserum

Several lines of evidence indicate the specificity of the96202 antiserum for GAL-R1. First, the antibody detecteda band of protein of approximately 70 kDa in Westernblots of KNRK cells transfected with GAL-R1, which isconsistent with the molecular mass of a glycosylated re-ceptor, and not in membranes from nonreceptor express-ing KNRK cells, indicating that the band specifically cor-responds to GAL-R1. A further confirmation of thespecificity of the 96202 antiserum for GAL-R1 is shown bythe immunostaining of HEK293 cells. HEK293 cells bear-ing GAL-R1 showed a diffuse surface staining, which waslacking in nontransfected HEK293 cells. Finally, the abo-lition of staining in the Western blot and gastrointestinaltissue by preadsorbing the antiserum with the homolo-gous peptide used for immunization provides further evi-dence to the specificity of the antiserum.

Sites of expression of GAL-R1immunoreactivity and functional

implications

Enteric neurons and fibers represent the predominantsites of expression of GAL-R1 throughout the stomach andsmall intestine. Indeed, GAL-R1 is expressed by function-

Fig. 2. Galanin receptor 1 (GAL-R1) immunoreactivity in the mu-cosa (A) and in the myenteric plexus and muscle layers (C) of the ratstomach. Fibers immunoreactive for GAL-R1 in the gastric mucosarun parallel to the epithelial cells from the base (which is on the leftof the image) to the lumen (A; arrows). GAL-R1 immunoreactivity isexpressed by epithelial cells, mainly on the cell surface (A; arrow-heads point to some examples). In the myenteric plexus, GAL-R1immunoreactivity is located on myenteric neurons (C; arrows) and in

fiber bundles within the plexus. Fibers immunoreactive for GAL-R1are located in the deep muscular plexus (C; arrowhead). Incubationwith GAL-R1 96202 preadsorbed with homologous peptide results inlack of immunostaining in the mucosa (B) and in the myenteric plexus(D) of the stomach. Cryostat sections: lm, longitudinal muscle; mp,myenteric plexus; cm, circular muscle. Scale bars � 50 �m in B(applies to A,B), 100 �m in D (applies to C,D).

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ally distinct populations of enteric neurons that send fi-bers to a variety of structures in these regions of thegastrointestinal tract, including the enteric plexuses, mu-cosa, muscle, and vasculature. The distribution of GAL-R1

neurons and their sites of innervation are in accord withthe galanin distribution (Parker et al., 1995; Burgevin etal., 1995; Gustafson et al., 1996; Ekblad et al., 1985b) andprovide the morphologic basis for many sites of action ofgalanin involving this receptor. Activation of GAL-R1 onenteric neurons by endogenously released galanin mightcontrol gastrointestinal motility and secretion indirectlyby modulating the release of other transmitters/modulators. Furthermore, the presence of GAL-R1 on neu-ronal cell surface and processes distributed to the gut wallsuggests pre- and postsynaptic actions of galanin bymeans of this receptor and supports the concept thatGAL-R1 is transported to axon terminals.

In the enteric nervous system, neurons can be differen-tiated on the basis of their morphology, projections, andcombination of transmitters that they synthesize and re-lease. Enteric neurons can be categorized into severalfunctional classes, including primary afferent neurons,interneurons, excitatory and inhibitory motor neurons,secretomotor, and vasomotor neurons. For instance, SP isa well-established neurotransmitter for excitatory motorneurons that project orally to the muscle (Furness et al.,1984, 1992), for ascending interneurons, and for intrinsicprimary afferent neurons (Furness et al., 1998). The find-ing that as many as 81.6% of GAL-R1 myenteric neuronsand 70.7% of GAL-R1 submucosal neurons contain SP,and that GAL-R1/SP neurons represent a substantial pro-portion (�50%) of SP enteric neurons supports the possi-bility that GAL-R1 might be expressed by ascending exci-tatory neurons as well as intrinsic primary afferentneurons. Because SP neurons are often cholinergic, we candeduce that GAL-R1 is expressed by cholinergic neurons.Indeed, we have observed colocalization of GAL-R1 andvesicular acetylcholine transporter (VAChT), a cholinergicmarker, in enteric neurons of the rat stomach (Guerrini etal., unpublished data) and of the rat ileum in a pilot,unpublished study. However, a systematic analysis ofGAL-R1 and VAChT colocalization was not conducted inthis investigation nor did we attempt quantification.There is a large body of evidence indicating that galanin isresponsible for both excitatory and inhibitory actions ongut motility. Galanin acts either directly on the muscula-ture (Ekblad et al., 1985a; Botella et al., 1995; Gu et al.,1995), or indirectly by either suppressing the nicotinicsynaptic transmission (Tamura et al., 1987) or by modu-lating the release of excitatory neurotransmitters. Forinstance, previous studies have shown that, in the guineapig intestine, galanin inhibits the contractile response toelectrical stimulation mediated by endogenous substanceP and acetylcholine and that this neuromodulatory effectseems to be presynaptic (Ekblad et al., 1985a; Akehira etal., 1995). Furthermore, Bauer et al. (1989) have demon-strated that galanin strongly inhibits the postprandialgastrointestinal motility, significantly delaying the gastricemptying and increasing the mouth-to-cecum transit. Ga-lanin is abundantly distributed in the gastrointestinaltract, being expressed in myenteric and submucous neu-rons, and in numerous fibers innervating the gut wall atall levels (Ekblad et al., 1985b). Therefore, one of themechanisms through which galanin might influence thegastrointestinal motility could be indirect inhibition ofacetylcholine and substance P release from excitatory mo-tor neurons by activating GAL-R1. The observation thatthe inhibitory effect of galanin on stimulated acetylcholinerelease is reversed by pertussis toxin indicates that a

Fig. 3. Galanin receptor 1 (GAL-R1) immunoreactivity on neuronsand fiber bundles of the myenteric plexus of the rat stomach (A), infibers in the deep muscular plexus (B), and on the surface of epithelialcells in the secretory region of the gastric mucosa (C). Cryostat sec-tions; dm, deep muscular plexus. Scale bar � 50 �m in C (applies toA–C).

297GAL-R1 IN THE RAT GI TRACT

Gi/Go-protein–coupled receptor is involved in mediatingthis action, thus providing further evidence for this hy-pothesis, because GAL-R1 is an inhibitory G-protein-coupled receptor.

Our findings support the hypothesis that GAL-R1 isprimarily involved in the prejunctional neuromodulationexerted by galanin on the ascending excitatory reflex ofthe intestinal peristalsis by depressing substance P andacetylcholine release in response to mechanical stimula-

tion. Galanin’s effect on the ascending excitatory reflexcould be the result of activation of GAL-R1 on excitatoryascending neurons and on primary afferent neurons. Thelack of colocalization of GAL-R1 and VIP in myentericneurons of the small intestine provides further evidencefor a prominent inhibitory role played by GAL-R1 on ex-citatory neurons in the galanin-induced effects on gastro-intestinal motility. An additional pathway through whichgalanin might affect gastrointestinal motility could in-

Fig. 4. Confocal images (single optical sections) of whole-mount preparations showing galanin recep-tor 1 (GAL-R1) immunoreactivity on myenteric (A; arrows point to representative examples of GAL-R1stained neurons) and submucous (B; arrows) neurons of the rat ileum. Fiber bundles are observed in theinterconnecting strands leaving the plexuses. Scale bars � 20 �m in A,B.

Fig. 5. Double-label immunofluorescence of cryostat sections ofthe stomach. In the top panels, confocal images showing galaninreceptor 1 (GAL-R1) immunoreactivity (green) on the cell surface(arrow), calbindin (Calb.) immunoreactivity (red) mainly inside thecytoplasm (asterisk), and simultaneous visualization of GAL-R1(green on the surface) and calbindin (red in the cytoplasm) inenterochromaffin-like cells. In the bottom panels, GAL-R1 immuno-

reactivity (green) in dense networks of fibers (arrows) in the innerpart of the circular muscle layer of the rat ileum (whole-mount prep-aration), which surround non-neuronal structures identified as inter-stitial cells of Cajal (red) as revealed by c-Kit immunostaining.GAL-R1 immunoreactivity appears to be confined to fibers and doesnot seem to be located on interstitial cells of Cajal surface. Scalebars � 10 �m in top panels, 25 �m in bottom panels.

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volve interstitial cells of Cajal, which are surrounded by adense network of GAL-R1-immunoreactive fibers. An im-portant role in the control of the gut motility has beenattributed to these cells, a subpopulation of which is lo-cated in the inner part of the circular muscle layer in strictassociation with the deep muscular plexus and smoothmuscle cells of the circular muscle layer itself (Gabella,1974). Interstitial cells of Cajal might act as pace-makers

and have been implicated in signaling between nerves andmuscle (Sanders, 1996). Given the high level of coexpres-sion of GAL-R1 and SP in myenteric neurons, it is reason-able to speculate that at least some of the GAL-R1 fibersclosely apposed to interstitial cells of Cajal are tachykin-ergic. These cells express the SP preferred receptor neu-rokinin 1 receptor (Sternini et al., 1995). Therefore, acti-vation of GAL-R1 by galanin might result in inhibition of

Fig. 6. Confocal images (single optical sections) of a whole-mountpreparation of the rat ileum showing colocalization of galanin receptor1 (GAL-R1) immunoreactivity (on the cell surface) (A) with substanceP (SP) immunoreactivity (in the cytoplasm) (B) in the myentericplexus. Arrows point to neurons bearing GAL-R1 at the cell surface

and containing SP in the cytoplasm, whereas the arrowheads point toa neuron containing SP in the cytoplasm (B) that lacks GAL-R1immunoreactivity. Arrowhead in A points to a GAL-R1–immunoreactive fiber bundle in close vicinity to a SP�/GAL-R1- neu-ron. Scale bar � 50�m.

Fig. 7. Confocal images (single optical sections) of a whole-mountpreparation of the rat ileum showing colocalization of galanin receptor1 (GAL-R1) immunoreactivity (on the cell surface) (A) with SP immu-noreactivity (in the cytoplasm) (B) in the submucous plexus. Arrows

point to a neuron bearing GAL-R1 at the cell surface and containingSP in the cytoplasm. Arrowheads point to submucosal neurons con-taining SP but lacking GAL-R1. Scale bars � 50 �m.

299GAL-R1 IN THE RAT GI TRACT

interstitial cells of Cajal activity by means of a reductionof SP release. However, confirmation of possible colocal-ization of GAL-R1 and SP immunoreactivities in fibersrequires electron microscope analysis, and it is beyond thepurpose of the present investigation. Previous studieshave provided evidence for a direct effect of galanin onintestinal smooth muscle cells (Ekblad et al., 1985a; Fox etal., 1986; Muramatsu and Yanaihara, 1988). The absenceof GAL-R1 immunoreactivity on smooth muscle cells sug-gests that this receptor does not mediate the direct myo-genic effect of galanin on gastrointestinal contractility. Adifferent galanin receptor subtype, likely the other gala-nin inhibitory receptor GAL-R3, might be responsible forthe direct galanin action on the gastrointestinal muscula-ture, even though the possibility that GAL-R1 is ex-pressed at a level below detectability with immunohisto-chemical techniques cannot be discounted.

In this study, we also showed that GAL-R1 is localizedto an abundant population of VIP submucous neurons ofthe small intestine. VIP is a well-established neurotrans-mitter for secretomotor neurons (Furness et al., 1992),therefore, the expression of GAL-R1 on VIP-positive neu-rons of the submucous plexus is consistent with the hy-pothesis that galanin influences intestinal electrolyte ex-change and fluid homeostasis by activating GAL-R1.Galanin might also be involved in control of blood flow.Enteric vasodilator neurons have been demonstrated to benoncholinergic (Vanner and Surprenant, 1991), the pri-

mary transmitter being VIP. Galanin might be involved inthe modulation of local vasodilator reflexes in the gut byindirectly acting on VIP/GAL-R1 neurons. Detection ofchanges in the luminal chemistry and water content leadsto activation of primary afferent neurons communicatingwith myenteric and submucous neurons that in turn feed-back to secretomotor neurons located in the submucousplexus. The effects of galanin on the fluid homeostasis inthe gastrointestinal tract have not been extensively inves-tigated. Nevertheless, galanin appears to be involved inthe control of intestinal fluid secretion. On the basis of ourfindings, we propose that galanin might influence gastro-intestinal secretion by indirectly modulating VIP releaseby means of the activation of GAL-R1. Secretomotor re-flexes can occur physiologically due to chemical interac-tions between luminal content and the mucosa but also asa defense mechanism against pathogens or toxins presentin the lumen. Galanin might be involved in this pathway,as suggested by our data, by means of the activation ofGAL-R1. Indeed, a recent study by Matkowskyj et al.(2000) proposed that up-regulation of GAL-R1 might rep-resent a novel mechanism explaining the increased colonicfluid secretion occurring in infectious diarrhea. The au-thors demonstrated that gastrointestinal infection bypathogens such as Salmonella typhimurium and Shigellaflexerii and not by commensal organisms results in in-creased GAL-R1 expression in the human colon and sub-sequent increase in colonic fluid secretion.

Fig. 8. Confocal images (single optical sections) of a whole-mountpreparation of the rat ileum showing colocalization of galanin receptor1 (GAL-R1) immunoreactivity (on the cell surface) (A) with vasoactiveintestinal polypeptide (VIP) immunoreactivity (in the cytoplasm)

(B) in the submucous plexus. Arrows point to neurons bearingGAL-R1 at the cell surface and containing VIP in the cytoplasm.Arrowheads point to a submucosal neuron containing VIP immuno-reactivity but lacking GAL-R1. Scale bars � 50�m.

TABLE 2. GAL-R1 Enteric Neurons Expressing Either SP or VIP1

GAL-R1 and SP in the myenteric plexusNo. of GAL-R1 cells � 147 No. of SP cells � 207 No. of GAL-R1/SP cells � 120 % of GAL-R1 cells SP� � 81.6 % of SP cells GAL-R1� � 57.9

GAL-R1 and SP in the submucosal plexusNo. of GAL-R1 cells � 106 No. of SP cells � 142 No. of GAL-R1/SP cells � 75 % of GAL-R1 cells SP� � 70.7 % of SP cells GAL-R1� � 52.8

GAL-R1 and VIP in the submucosal plexusNo. of GAL-R1 cells � 120 No. of VIP cells � 95 No. of GAL-R1/ VIP cells � 58 % of GAL-R1 cells VIP� � 48.3 % VIP cells GAL-R1� � 61.1

1GAL-R1–immunoreactive neurons expressing either SP or VIP in the myenteric and submucosal plexus of the rat ileum. Cell numbers were obtained from randomly selectedganglia from three colchicine-treated rats (10–12 ganglia from each animal). GAL-R1, galanin receptor 1; SP, substance P; VIP, vasoactive intestinal polypeptide.

300 T. PHAM ET AL.

The finding that 48.3% of GAL-R1 submucous neuronscontained VIP immunoreactivity and that 70.7% were im-munoreactive for SP suggests that a portion of submucousGAL-R1 neurons contains both SP and VIP. SP and VIPlabel distinct populations of myenteric neurons in theguinea pig ileum (Costa et al., 1996) and their colocaliza-tion has not been observed in the rat and guinea pig colon(Schultzberg et al., 1980), even though this last studyrelied on the use of consecutive sections and elution-restaining techniques that are not as sensitive as doublelabeling with whole-mount preparations. By contrast, SPand VIP colocalize in a subpopulation of myenteric neu-rons of the guinea pig antrum (Vanden Berghe et al.,1999). The colocalization of SP and VIP in the rat ileumsubmucous plexus has not been reported nor excluded,and it is not surprising because peptide colocalizationdiffers among species and among different regions of thegastrointestinal tract in the same species.

Galanin acts as an inhibitory neuromodulator on thepostprandial release of several substances and hormones,including insulin, glucagon, and glucose (Bauer et al.,1989). Furthermore, galanin inhibits gastric acid secre-tion (Yagci et al., 1990), although the mechanismsthrough which this effect is induced remain to be clarified.The presence of GAL-R1 on ECL-like cells supports thehypothesis that galanin inhibits gastric secretion indi-rectly by blocking the gastrin-induced secretion of hista-mine from ECL-like cells. ECL-like cells actively produceand store histamine, which is an important physiologicalstimulant of acid secretion from the parietal cells, in re-sponse to gastrin-evoked stimulation (Hakanson et al.,1994; Andersson et al., 1998). Furthermore, an indirecteffect by means of the inhibition of acetylcholine releasecan be postulated, because GAL-R1 is expressed by fibersinnervating the gastric mucosa, most of which are likely tobe cholinergic (Guerrini et al., unpublished observations).A direct action of galanin on gastric G-cells suggested bySchepp et al. (1990) cannot be excluded, and it is sup-ported by the presence of galanin-immunoreactive fibersinnervating the gastric mucosa (Sengupta and Goyal,1988). However, if that is the case, another galanin recep-tor subtype is likely to be involved, because GAL-R1 im-munoreactivity could not be identified on G-cells.

In summary, this study demonstrates that GAL-R1 isexpressed by different structures of the stomach and smallintestine, including enteric neurons and ECL-like cells,and that neurons immunoreactive for GAL-R1 innervateseveral different targets. Indeed, this study provides evi-dence that this receptor is expressed by functionally dis-tinct populations of enteric neurons, which are likely toinclude excitatory motor neurons, primary afferent neu-rons, and secretomotor neurons. These findings supportthe hypothesis that galanin’s effects on gastrointestinalmotility and secretion mediated by the GAL-R1 occurthrough the activation of neuronal and non-neuronal (en-docrine) pathways and involve the inhibition ofneurotransmitter/modulator and hormone release. An au-tocrine mechanism of action of galanin cannot be ex-cluded, although further investigations to clarify whetherthe peptide colocalizes in the same structures in whichGAL-R1 is identified are needed. In addition, GAL-R1distribution cannot account for all reported galanin ac-tions on the gastrointestinal system, which are probablymediated by other GAL-Rs. Therefore, further studies areneeded aimed to identify the sites of localization and dis-

tribution of the other known galanin receptor subtypes,and this information will be fundamental to determine thedifferent routes through which galanin exerts its effectson the digestive system.

ACKNOWLEDGMENTS

We thank John H. Walsh (deceased on June 14, 2000)for making available many of the antibodies used in thisstudy, James Krause and Stephen M. Waters for provid-ing the GAL-R1 cDNA, Nicholas Brecha for useful com-ments on the manuscript and many fruitful discussions,and Sean Kohlmeier for helping with the preparation ofthe images.

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