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Differential distribution of substance P binding sites in guinea-pig sympathetic ganglia
Journal of the Autonomic Nervous System 69 Ž1998. 103–114
Differential distribution of substance P binding sites in guinea-pig sympathetic ganglia J.P. Messenger ) , I.L. Gibbins Department of Anatomy and Histology, and Centre for Neuroscience, Flinders UniÕersity of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia Received 3 October 1997; revised 25 November 1997; accepted 28 November 1997
Abstract We have used a combination of autoradiographic and immunohistochemical techniques to investigate the distribution of binding sites for substance P in relation to the distribution of substance P-immunoreactive nerve fibres and specific functional populations of neurons in the sympathetic ganglia of guinea-pigs. There was considerable heterogeneity in the density of binding sites for Bolton Hunter labelled 125 I y substance P ŽBHSP.. Binding sites were more dense in the prevertebral ganglia, such as the coeliac and inferior mesenteric ganglia, than in the paravertebral ganglia, such as the superior cervical or lumbar chain ganglia. The binding sites tended to be clumped within the ganglia. Within the prevertebral ganglia, they were associated predominantly with neurons projecting to the enteric plexuses. Many of these neurons contained somatostatin immunoreactivity. In the lumbar sympathetic chain ganglia, there was a weak association of binding sites with neurons containing immunoreactivity to vasoactive intestinal peptide. Overall, the density of binding sites matched the density of nerve fibres containing immunoreactivity to substance P in different ganglia. However, within particular ganglia, there was little, if any, correlation between the distribution of binding sites and nerve fibres containing substance P. Most of the binding sites in the ganglia had the pharmacological characteristics of NK 1 receptors. Our results show that there is considerable heterogeneity in the expression of NK 1 receptors in the sympathetic ganglia of guinea-pigs. However, given the relatively poor spatial correlation between the distribution of binding sites and potential sites of substance P release from intraganglionic nerve fibres, we suggest that substance P may diffuse for relatively large distances through the ganglia, with actions only on those neurons selectively expressing NK 1 receptors. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Substance P; Neurokinin receptor; Sympathetic ganglia; Sensory; Preganglionic; Autoradiography
1. Introduction Immunoreactivity to substance P ŽSP-IR. is widespread in peripheral neurons of common laboratory mammals. Nerve cell bodies with SP-IR occur in dorsal root ganglia, cranial and pelvic parasympathetic ganglia, sympathetic and enteric ganglia ŽCosta et al., 1981; Gibbins, 1990, 1992; Gibbins et al., 1987; Hokfelt et al., 1975; Ju et al., ¨ 1987; Lindh et al., 1989.. Usually, SP-IR is expressed together with other neuropeptides in specific subpopulations of neurons projecting to well-defined peripheral target tissues ŽGibbins, 1990, 1992; Gibbins et al., 1987; Lindh et al., 1989.. Many autonomic ganglia also contain ) Corresponding author. Department of Anatomy and Histology, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5042, Australia. Tel.: q61 8 82044261; fax: q61 8 82770085; e-mail: [email protected]
nerve fibres with SP-IR ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins, 1992; Gibbins et al., 1987; Hokfelt et al., 1977; Matthews and Cuello, 1982, 1984; ¨ Morris and Gibbins, 1987.. Within the sympathetic ganglia of guinea-pigs, SP-IR fibres can arise from two different sources. One population of fibres contains immunoreactivity to calcitonin gene-related peptide ŽCGRP-IR. in addition to SP-IR and almost certainly represents collaterals of capsaicin-sensitive, unmyelinated sensory neurons with cell bodies in the dorsal root ganglia ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins et al., 1987; Matthews and Cuello, 1982, 1984.. The other population of SP-IR fibres lacks CGRP-IR and is most likely to represent the endings of preganglionic neurons that presumably also contain acetylcholine ŽGibbins, 1992; Heym et al., 1990, 1993.. In the prevertebral ganglia, sensory fibres containing both SP-IR and CGRP-IR predominate, while in the lumbar sympathetic chain ganglia, preganglionic SP-IR
0165-1838r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 0 7 - 1
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fibres predominate ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins, 1992; Gibbins et al., 1987; Heym et al., 1990; Matthews and Cuello, 1982, 1984.. As has been seen for other peptide-containing inputs to sympathetic ganglia ŽGibbins, 1992; Lindh et al., 1986; Macrae et al., 1986; Morris and Gibbins, 1992., SP-IR fibres tend to be associated with specific populations of ganglionic neurons ŽGibbins, 1992; Heym et al., 1990.. For example, in the lumbar sympathetic chain ganglia of guinea-pigs, preganglionic fibres containing SP-IR are associated selectively with non-noradrenergic vasodilator neurons that contain immunoreactivity for vasoactive intestinal peptide ŽVIP-IR. and neuropeptide Y ŽNPY-IR. ŽGibbins, 1992.. Exogenously applied substance P generally has excitatory effects on sympathetic neurons, and there is evidence that it can mediate slow synaptic potentials in the prevertebral sympathetic ganglia ŽDun and Karczmar, 1979; Dun and Minota, 1981; Dun and Jiang, 1982; Konishi and Otsuka, 1985; Konishi et al., 1983, 1992; Kreulen and Peters, 1986; Krier and Szurszewski, 1982; Tsunoo et al., 1982; Zhao et al., 1995, 1996.. In the coeliac ganglia of guinea-pigs, these effects are mediated by NK 1 and NK 3 receptors, which appear to be present on both phasic and tonic neurons ŽZhao et al., 1995, 1996.. Hybridization experiments with synthetic DNA probes specific to each of the tachykinin receptor types indicate that NK 1 , NK 2 and NK 3 tachykinin receptors all are expressed in rat prevertebral ganglia ŽKonishi et al., 1993.. SP binding sites also have been localised autoradiographically in sympathetic ganglia ŽJames and Burnstock, 1988; Mantyh et al., 1992; Niwa et al., 1985.. Nevertheless, although the density of substance P binding sites varies within ganglia, it is not known how this variation relates to the distribution of intraganglionic SP-IR fibres or to particular functional populations of ganglionic neurons. It might be expected that variation in the density of substance P binding sites in sympathetic ganglia would be related to the differential distribution of SP-IR fibres in the ganglia. However, in many parts of the nervous system, there may be, at best, only a loose correlation between the distribution of peptide-containing nerve fibres and the receptors for that peptide ŽZoli and Agnati, 1996.. Therefore, this study aims to identify and map the distribution of substance P binding sites in the sympathetic ganglia of guinea-pigs, using autoradiographic methods, and to correlate the distribution of binding sites with immunohistochemically characterised populations of neurons and SP-IR fibres in these ganglia.
2. Materials and methods 2.1. Tissue preparation Male and female guinea-pigs weighing 200–400 g were used. The superior cervical ganglia, stellate ganglia, lum-
bar sympathetic chain ganglia, coeliac ganglion and inferior mesenteric ganglion were dissected from guinea-pigs that had been stunned by a blow to the head and bled via the carotid arteries. Ganglia were cleared of fat and connective tissue, placed in cryomoulds containing OCT ŽTissue-Tek, Miles, Elkhart, IN, USA., and rapidly frozen in isopentane cooled in liquid nitrogen. The tissue was sectioned at 10–12 m m on a cryostat at y208C. Sections were thaw mounted to slides coated with polyethylenimine ŽSigma, Castle Hill, NSW, Australia. and allowed to air dry. 2.2. Autoradiography Ganglia to be analysed both autoradiographically and immunohistochemically were processed in the following way ŽBurcher and Buck, 1986; Hudson, 1993; Wharton et al., 1993.. Alternate sections were dried onto different slides. Alternate slides were processed for autoradiographic localization of substance P binding sites or for immunohistochemical analysis Žsee Section 2.3.. Slides for autoradiography were pre-incubated in Tris–HCl-buffer Ž0.05 M, pH 7.4. containing 0.02% bovine serum albumin ŽBSA. for 2 = 15 min. This solution was removed and replaced with incubation solution ŽTris-buffer containing 0.02% BSA, 2 m grml chymostatin, 4 m grml leupeptin, 40 m grml bacitracin and 3 mM MgCl 2 . also containing Bolton Hunter-labelled 125 I y substance P ŽBHSP. for 2 h. Slides were washed for 6 = 90 s in ice cold Tris-buffer0.02% BSA followed by one 90-s wash in distilled water. Sections were air dried and fixed in paraformaldehyde vapour, at 608C for 90 min. Slides were then taken up through graded alcohols to xylene, and down through graded alcohols to distilled water. The slides were dried again and dipped in radiation sensitive emulsion ŽLM-1, Amersham Australia, Sydney, NSW., and left for 7–14 days in the dark at 48C, before developing with Kodak D19 developing solution ŽKodak ŽAustralasia., Coberg, Victoria.. The developed slides were coverslipped with DPX mountant ŽBDH, Poole, UK.. In all experiments, a control slide was incubated in solution that did not contain BHSP. Non-specific binding was determined on sections incubated in parallel by adding excess unlabelled substance P Ž10y6 M. to the incubation solution. The specific binding of BHSP was determined in a saturation binding assay, in each of the ganglia Ž n s 2, in duplicate.. Specific binding is defined as the total binding minus the non-specific binding. Total binding is that binding achieved in the presence of radioligand alone, while non-specific binding is the binding seen in the presence of radioligand plus excess unlabelled ligand. The optimum binding concentration was that dose of the radioligand that gave the highest percentage of specific binding. Several time points were tested for each concentration, giving the optimal incubation period. For all ganglia, the optimal
J.P. Messenger, I.L. Gibbinsr Journal of the Autonomic NerÕous System 69 (1998) 103–114
binding concentration was 0.1 nM, the optimal incubation period was 120 min, and these parameters were used routinely. The aim of the first series of experiments was to determine the density of BHSP binding sites in each of the ganglia. To do this, the specific binding of BHSP was determined as described above. Sections that had been incubated with radioligand were wiped from the slides with moistened filter paper after the final wash in distilled water. The wiped sections and filter paper were placed in glass tubes and gamma counts per minute were recorded by a Riastar gamma counter ŽCanberra-Packard, Mt. Wa-
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verly, Australia.. Alternate sections, not incubated in radioligand, were fixed in 4% formaldehyde, and stained with cresyl violet. The stained sections were drawn with a camera lucida attachment on an Olympus BH2 microscope and digitised so that their area could be measured with NIH Image running on a Macintosh 8500 computer. This allowed us to derive an estimate of specific binding as gamma counts per squared millimeter for each of the ganglia Ž n s 3, done in duplicate.. The aim of a further series of experiments was to determine the type of tachykinin receptor to which BHSP was binding. Sections of ganglia were incubated in radiola-
Fig. 1. Distribution of substance P-IR fibres in guinea-pig sympathetic ganglia. Ža. Sparse SP-IR fibres in the superior cervical ganglion ŽSCG.. Žb. SP-IR fibres in the lumbar sympathetic chain ganglia ŽLCG. mostly form dense pericellular baskets around unlabelled neurons. Žc,d. A high density of SP-IR fibres is found in the coeliac ganglion ŽCG, c., and, in the inferior mesenteric ganglion ŽIMG, d.. Že,f. Coexistence of SP-IR and CGRP-IR in most fibres in the CG. Žg,h. In the LCG, however, SP-IR fibres forming baskets around neurons do not contain CGRP-IR. Scale bar: a–d, 50 m m; e–h, 20 m m.
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belled substance P, with a tachykinin receptor agonist or antagonist. In these experiments, the BHSP binding also was measured by gamma counting. The sections were incubated in radioligand as described above, with or without the agonist or antagonist, and the sections were wiped from the slides, after washing, for gamma counting. The peptide agonists ŽRegoli et al., 1988. and non-peptide antagonists ŽEdmonds-Alt et al., 1992, 1993, 1994. used were, respectively: NK 1 receptor, Sar 9 MetŽO 2 .11 -SP and SR140333; NK 2 receptor, Nle 10 -NKA Ž4–10 . and SR48968; NK 3 receptor, senktide and SR142801. Each compound was used in concentrations ranging from 10y6 M to 10y9 M, on sections from each of the ganglia Ž n s 3, done in duplicate.. Results were analysed statistically with multivariate repeated measures analysis of variance, using the GLM procedure of SAS on a personal computer ŽRelease 6.04; SAS Institute, Cary, NC, USA.. The small size of the sympathetic ganglia was a limiting factor in the design of these experiments. It was not possible to obtain reliably more than 36 sections 10-m m thick from a single ganglion. Therefore, only a limited number of slides could be used for receptor characterization experiments. For each ganglion, four doses of each ligand were selected, plus a slide for total binding and a slide for non-specific binding. 2.3. Immunohistochemistry The aim of one series of experiments was to determine the chemical coding of the postganglionic neurons over which autoradiographic grains were localized. In the prevertebral ganglia, neurons can be differentiated by their immunoreactivity for somatostatin ŽSOM-IR. or NPY: neurons with NPY-IR tend to be located more laterally in the ganglia and project to blood vessels; neurons with SOM-IR or which lack both NPY-IR and SOM-IR tend to be located more medially and project to the enteric plexuses ŽCosta and Furness, 1984; Lindh et al., 1986; Macrae et al., 1986.. In the paravertebral ganglia, VIP-IR was used to localize neurons that are surrounded preferentially by SP-IR fibres ŽGibbins, 1992.. It was not possible to determine the relationship between clumps of BHSP binding sites and the distribution of SP-IR fibres directly, since the SP antiserum did not produce specific labelling on the freshly cut and post-fixed tissue used for autoradiography. However, double labelling immunohistochemistry for SP and CGRP was performed on freshly fixed tissue that was not processed for autoradiography. Sections to be immunohistochemically analysed were fixed in phosphate buffered 0.2% picric acid and 4% formaldehyde for 1 h then cleared with dimethylsulphoxide ŽDMSO. for 15 min and washed in phosphate buffered saline ŽPBS. for 15 min. Whole ganglia were fixed overnight and then cleared in ethanol followed by DMSO, and again in ethanol. Ganglia were embedded in polyethylene glycol, and sections were cut with a rotary
microtome. All sections were incubated in 10% normal donkey serum for 30 min, and then incubated in primary antiserum containing 10% normal donkey serum for 48 h. Primary antisera used were raised against VIP Žraised in rabbit; Incstar; code 9032039., SOM Žraised in mouse; source J.C. Brown; code SOMA 08., NPY Žraised in rabbit; source C. Maccarone and B. Jarrott; code JrM 264 ŽMorris et al., 1986.. substance P Žraised in rat; Sera Lab; code B8E035. and CGRP Žraised in goat; Arnel Laboratories, code F1124459.. Secondary antisera used were Cy3 or DTAF conjugated anti-rabbit, anti-rat, anti-sheep or anti-mouse IgG, all raised in donkey ŽJackson ImmunoResearch Laboratories, West Grove, PA, USA.. These antibodies have been used extensively in our laboratory and show no inappropriate cross-reactivity ŽGibbins, 1992; Gibbins and Matthew, 1996.. 2.4. Microscopy and digital imaging Autoradiographic grains were visualized using an Olympus BX-50 or BH-2 microscope, with either bright field or dark field optics. Immunofluorescent labelling on the sections adjacent to the autoradiographically labelled sections was analysed on the BX-50 microscope. Images were captured with a Sony CCD camera ŽModel SSCM370CE. and a Scion LG-3 frame grabber in a Macintosh Quadra 850AV computer, using NIH Image software. Digital enhancement and image analysis was performed on a Macintosh 8500 computer using NIH Image and Adobe Photoshop software ŽAdobe Systems, Mountain View, CA, USA.. Grey scale images were enhanced in Photoshop by altering brightness and contrast to appropriate levels for publication. Images of radiolabelled whole ganglia were thresholded and converting to binary format in NIH Image before assembling into montages using Powerpoint ŽMicrosoft..
Fig. 2. Density of BHSP binding sites in guinea-pig sympathetic ganglia. There is a significant overall difference in binding density. One-way ANOVA and post-hoc comparisons show Ž). that the stellate ganglion has less binding than all other ganglia and that the IMG has a greater binding density than all other ganglia Ž FŽ4,15. s 4.8, P s 0.01.. SCG, superior cervical ganglion; LCG, lumbar chain ganglia; CG, coeliac ganglion; IMG, inferior mesenteric ganglion.
J.P. Messenger, I.L. Gibbinsr Journal of the Autonomic NerÕous System 69 (1998) 103–114
2.5. Chemicals The peptides Sar 9 MetŽO 2 .11 -SP, Nle10-NKAŽ4–10. and senktide were made by Auspep ŽParkville, Victoria, Australia.. Bolton Hunter 125 I y substance P was made by Amersham ŽNorth Ryde, NSW, Australia.. The peptidase inhibitors chymostatin, leupeptin and bacitracin were obtained from Sigma ŽAustralia.. The tachykinin receptor antagonists were a generous gift of Sanofi ŽSydney, Australia.. They were SR140333, Ž1-2-w3-Ž3,4-dichlorophenyl . -1- Ž 3-isopropoxyphenylacetyl .piperidin-3-yl xethyl4 -4-phenyl-1-azoniabicyclow2.2.2xoctone.; SR48968,
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Ž S .-N-methyl-N w4-acetylamino-4-phenylpiperidino.-2-Ž3,4dichloro phenyl.butylx benzamide and SR142801, ŽŽ S-Ž N .-Ž1-Ž3-Ž1-benzoyl-3-Ž3,4-dichlorophenyl.piperidin3-yl.-4-phenylpiperidin-4-yl.-N-methylacetamide. 3. Results 3.1. Distribution of SP-IR and CGRP-IR fibres in sympathetic ganglia Varicose nerve fibres with SP-IR were found in all sympathetic ganglia analysed, as has been reported previ-
Fig. 3. Distribution of BHSP binding sites Ždarkfield microscopy., NPY-IR neurons and SOM-IR neurons Žimmunofluorescence. in the inferior mesenteric ganglion ŽIMG. and coeliac ganglion ŽCG.. Binding sites are most dense where SOM-IR neurons are present and least dense where NPY-IR neurons occur. Ža,b. Low magnification montages of one lobe of an IMG showing Ža. clustered BHSP binding sites Žarrows. and Žb. corresponding clusters of SOM-IR neurons in the adjacent section Žarrows.. Žc,d. Micrographs showing the negative correlation between Žc. BHSP binding sites and Žd. NPY-IR neurons in the IMG. Že,f. micrographs showing a good positive correlation between Že. clustered BHSP binding sites Žarrow. and Žf. a corresponding group of SOM-IR neurons Žarrow. in the CG. Scale bar: a–b, 200 m m; c–f, 100 m m.
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ously ŽDalsgaard et al., 1983; Gamse et al., 1981; Heym et al., 1990, 1993; Hokfelt et al., 1977; Matthews and Cuello, ¨ 1982, 1984.. The density of the SP-IR fibres in each of the ganglia, and characteristics of their distribution within a ganglion, varied between ganglia ŽFig. 1.. The superior cervical ganglion had only sparse SP-IR fibres which did not appear to target selectively any structures within the ganglion ŽFig. 1a.. These fibres mostly contained CGRP-IR in addition to SP-IR. The stellate ganglion had a low density of SP-IR fibres, some of which formed pericellular baskets around the cell bodies of unlabelled neurons. SP-IR fibres in the lumbar chain ganglia were numerous. They mostly lacked CGRP-IR, and formed frequent baskets of boutons around unlabelled neurons ŽFig. 1b,g,h.. Such baskets of SP-IR boutons are associated selectively with VIP-IR neurons in the lumbar chain ganglia ŽGibbins, 1992.. SP-IR fibres in the coeliac and inferior mesenteric ganglia were densely distributed throughout the ganglia
ŽFig. 1c,d.. They formed obvious baskets less often than in the lumbar chain ganglia, and there was no obvious bias in the distribution of SP-IR fibres in the medial or lateral lobes of the coeliac ganglion. In both prevertebral ganglia, most SP-IR fibres also contained CGRP-IR ŽFig. 1e,f.. 3.2. Substance P binding sites Bolton Hunter 125 I y substance P ŽBHSP. binding was observed in all the sympathetic ganglia we examined. However, the density and distribution pattern of binding sites varied consistently from ganglion to ganglion ŽFig. 2.. 3.2.1. Inferior mesenteric ganglion This ganglion had a highest density of BHSP binding sites of all ganglia analysed ŽFig. 2.. Mean binding density over the entire ganglion was 36.2 " 9.9 cpmrmm2 Žmean
Fig. 4. Distribution of BHSP binding sites and VIP-IR neurons in two examples of lumbar sympathetic chain ganglia. Ža,b. Clusters of BHSP binding sites Žarrows, a. only loosely match with the groups of VIP-IR neurons in the adjacent section Žarrows, b.. Žc,d. A cluster of BHSP binding sites Žc. is associated with a VIP-IR neuron on the adjacent section Žd., but also extend into regions of the ganglion devoid of VIP-IR neurons. Scale bar: a–b, 200 m m; c–d, 100 m m.
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" SEM, n s 3 animals, experiments were done in duplicate.. The binding sites were distributed in large clumps throughout the ganglion ŽFig. 3a.. Subsequent immunohistochemistry for SOM-IR on adjacent sections revealed that the BHSP binding was closely correlated with the distribution of SOM-IR cells ŽFig. 3b.. The areas of sparse BHSP binding matched the distribution of NPY-IR neurons, seen in adjacent sections ŽFig. 3c,d.. Neurons without either SOM-IR or NPY-IR occurred both in areas with high and low density BHSP binding. 3.2.2. Coeliac ganglion The coeliac ganglion consists of two lobes. NPY-IR neurons tend to be located laterally in each lobe, while SOM-IR neurons and neurons lacking NPY-IR and SOMIR tend to be located more medially ŽCosta and Furness, 1984; Keast et al., 1993; Lindh et al., 1986; Mantyh et al., 1992.. In the coeliac ganglion as a whole, there was a high density of BHSP binding sites Ž29.0 " 3.4 cpmrmm2 ; Fig. 2.. The BHSP binding sites were distributed in a patchy manner over large regions of the ganglion. However, there was a distinct polarity across each lobe so that the greatest density of BHSP binding sites was found over the medial region of each lobe. This region corresponds to the part of the ganglion containing predominantly SOM-IR neurons ŽFig. 3e,f.. 3.2.3. Lumbar sympathetic chain ganglia The average density of BHSP binding sites for lumbar chain ganglia was 13.8 " 3.9 cpmrmm2 ŽFig. 2.. BHSP binding sites generally were concentrated into small
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clumps, scattered irregularly throughout the ganglia ŽFig. 4a,b.. Most ganglia had several clumps of grains, but some ganglia had no clumps of BHSP binding sites and other ganglia had a high density of binding sites uniformly over the ganglion. Alternate sections were labelled for VIP-IR and there was an inconsistent association between clumps of BHSP binding sites and VIP-IR cells. Although BHSP binding sites often were localized over VIP-IR cells ŽFig. 4c,d., clumps of BHSP binding sites also were associated with VIP immunonegative cells. Many VIP-IR cells were not associated with BHSP binding sites ŽFig. 4a,b.. 3.2.4. Superior cerÕical ganglion and stellate ganglion The average density of BHSP binding sites was 19.6 " 3.1 cpmrmm2 for the superior cervical ganglion and 7.0 " 2.4 cpmrmm2 for the stellate ganglion ŽFig. 2.. In the superior cervical ganglion, BHSP binding sites were distributed evenly throughout the ganglion without obvious clustering ŽFig. 5a.. In the stellate ganglion, occasional clumps of BHSP binding sites were seen ŽFig. 5b.. Labelling for VIP-IR on sequential sections of the stellate ganglion showed that there was no correlation between the distribution of VIP-IR cells and BHSP binding sites. 3.3. Characterization of BHSP binding sites In a further series of autoradiographic experiments, the effects of the proposed endogenous ligand for the NK 2 receptor, neurokinin A ŽNKA., and the proposed endogenous ligand for the NK 3 receptor, neurokinin B ŽNKB., on BHSP binding were tested. As seen in Fig. 6, an excess
Fig. 5. Distribution of BHSP binding sites in darkfield montages of the superior cervical ganglion ŽSCG, a. and the stellate ganglion Žb.. BHSP binding sites are patchy and relatively sparse. Scale bar: 500 m m.
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Fig. 6. BHSP binding in the coeliac ganglion ŽCG., inferior mesenteric ganglion ŽIMG. and the lumbar chain ganglia ŽLCG., and the effect on BHSP binding of adding excess unlabelled SP, NKA or NKB to the incubation solution. In all ganglia, 1 m M SP completely inhibited BHSP binding Žsecond column of micrographs.. However, an excess of NKA or NKB did not affect BHSP binding in any ganglia Žthird column of micrographs.. Scale bar: 500 m m.
Ž10y7 M. of unlabelled NKA or NKB in the incubation medium did not inhibit BHSP binding in any ganglia, suggesting that BHSP is binding predominantly to NK 1 receptors. In order to test this conclusion further, we examined the ability of selective agonists and antagonists for NK 1 , NK 2 and NK 3 receptors to inhibit BHSP binding, as determined by gamma counting on sections wiped from slides. In each of the ganglia analysed, unlabelled substance P Ž1 nM. in
the incubation medium completely inhibited BHSP binding. The selective NK 1 peptide agonist, Sar 9 MetŽO 2 .11 -SP Ž1 nM., also abolished BHSP binding in all ganglia, as did the NK 1 selective non-peptide antagonist SR140333 Ž1 nM.. These results confirm that most of the BHSP binding occurred at NK 1 receptors. Consistent with the preceding results, selective NK 2 and NK 3 agonists and antagonists only inhibited BHSP binding significantly at the highest concentrations tested
J.P. Messenger, I.L. Gibbinsr Journal of the Autonomic NerÕous System 69 (1998) 103–114
Ž10y6 M. if at all. For example, in the inferior mesenteric ganglion, BHSP binding was partially inhibited by the NK 3 receptor agonist, senktide Ž45% inhibition at 10y7 M; FŽ1,2. s 47.7, P - 0.05., and the NK 3 receptor antagonist, SR142801 Ž39% inhibition at 10y7 M; FŽ1,2. s 25.4, P - 0.05.. In the stellate ganglion, the non-peptide NK 2 antagonist partially inhibited BHSP binding Ž61% inhibition at 10y7 M; FŽ1,2. s 278.4, P - 0.05., although the NK 2 agonist, Nle10 -NKAŽ4–10. had little or no effect on binding. Similarly, the non-peptide NK 3 antagonist, but not the peptide agonist, partially inhibited BHSP binding in the superior cervical ganglion Ž68% inhibition at 10y7 M; FŽ1,2. s 28.4, P - 0.05.. As a well-characterised control tissue, we examined BHSP binding in the urinary bladder, where the tachykinin binding sites have been described previously as representing predominantly NK 1 receptors ŽBurcher and Buck, 1986.. The presence of NK 2 receptors in guinea-pig bladder also has been demonstrated by Shinkai et al. Ž1993.. We found that 1 nM substance P, the NK 1 receptor agonist, Sar 9 MetŽO 2 .11 -SP, or the NK 1 receptor antagonist, SR140333, all completely inhibited BHSP binding in this tissue. Although the NK 2 receptor agonist, Nle10 NKAŽ4–10., had no significant effect on BHSP binding at any concentration, the non-peptide NK 2 antagonist, SR48968, partially inhibited BHSP binding Ž53% inhibition at 10y7 M; FŽ1,2. s 32.1, P - 0.05.. The NK 3 receptor agonist, senktide, and the non-peptide antagonist, SR142801, had no significant effect on BHSP binding. These observations are consistent with previous reports ŽBurcher and Buck, 1986; Shinkai et al., 1993. and support the reliability of our observations on the sympathetic ganglia.
4. Discussion 4.1. Distribution of substance P binding sites Substance P binding sites, localized with 125 I Bolton Hunter-Substance P ŽBHSP., were distributed in a pattern that varied considerably in each of the sympathetic ganglia analysed. The greatest density of BHSP binding sites was found in the prevertebral ganglia. The inferior mesenteric ganglion had the highest overall density of BHSP binding sites with almost the entire area of the ganglion covered in autoradiographic grains. The coeliac ganglion also had a very high density of autoradiographic grains over large regions of the ganglion. A moderate density of autoradiographic grains was seen in the superior cervical ganglion and in the lumbar chain ganglia, and the lowest average density of BHSP binding sites was found in the stellate ganglion. There was also considerable variation in the distribution of BHSP grains within individual ganglia. In most ganglia,
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BHSP binding sites were patchy, or clumped, suggesting that only subsets of postganglionic neurons express high densities of these binding sites. In the bi-lobed coeliac ganglion, large clumps of BHSP binding sites were found in the medial regions of each of the two lobes, and little binding was observed in the lateral poles. This distribution of BHSP binding sites corresponds to the distribution of neurons projecting to the enteric plexuses. These neurons are located preferentially in the medial poles of the coeliac ganglion, and many of them contain SOM-IR ŽCosta and Furness, 1984; Keast et al., 1993; Lindh et al., 1986; Mantyh et al., 1992.. A similar close association between BHSP binding sites and SOM-IR neurons also was observed in the inferior mesenteric ganglion, although there is no obvious polarity in that ganglion. Thus, substance P binding sites in the coeliac ganglion must be located predominantly on neurons regulating gastrointestinal motility and secretory activity, and are likely to have a similar selective localization in the inferior mesenteric ganglion. BHSP binding sites in the lumbar chain ganglia were notable in that they tended to be more clumped than in the other ganglia, and that the clumps were distributed irregularly throughout the ganglia. Labelling for VIP-IR neurons on alternate sections indicated that there was only a loose association between the clumps of BHSP grains and the distribution of VIP-IR neurons. In principle, BHSP binding sites also could be located on different populations of nerve fibres within the sympathetic ganglia. For example, the medial region of the coeliac ganglion, which contains neurons projecting to the enteric plexuses, is also the region where most of the intestinofugal fibres terminate ŽLindh et al., 1986; Macrae et al., 1986.. Consequently, the high density of BHSP binding sites in the medial region of the ganglion could be associated with these fibres themselves, rather than with the neurons they surround. However, there is no functional evidence to indicate that substance P receptors are present on any population of nerve fibres within sympathetic ganglia. 4.2. Characterization of substance P binding sites Tachykinin receptors are characterized as either neurokinin NK 1 , NK 2 or NK 3 receptors ŽBurcher et al., 1991; Regoli et al., 1988, 1994.. We have used selective tachykinin receptor agonists and antagonists to characterize the binding sites that are present in guinea-pig sympathetic ganglia. In all ganglia, at concentrations as low as 1 nM, substance P completely inhibited BHSP binding. Low concentrations of the specific NK 1 receptor peptide agonist Sar 9 MetŽO 2 . 11 -SP and the non-peptide antagonist SR140333 also completely inhibited BHSP binding, in each of the five ganglia. These results indicate that BHSP is predominantly binding to NK 1 receptors. R e s u lts w ith p e p tid e a n d n o n -p e p tid e agonistsrantagonists to the NK 2 and NK 3 receptors indi-
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cated that there were few NK 2 or NK 3 binding sites in guinea-pig sympathetic ganglia. The low levels of inhibition of BHSP binding seen in the presence of high concentrations of these agents most likely were due to non-specific binding to NK 1 receptors ŽPetitet et al., 1993.. This conclusion is consistent with electrophysiological studies showing that most effects of tachykinins on the membrane properties of coeliac ganglion neurons are mediated by NK 1 receptors. However, these studies have also shown some effects mediated by NK 3 receptors ŽZhao et al., 1995, 1996.. Our observations would suggest either that relatively few neurons express NK 3 receptors, or that the level of NK 3 receptor expression is low overall compared with that of NK 1 receptors.
a loose spatial correlation between VIP-IR neurons and substance P binding sites. Although clusters of BHSP binding sites tended to be include regions of the ganglion containing VIP-IR neurons, they often included neurons without VIP-IR. Conversely, many groups of VIP-IR neurons apparently lacked binding sites. Taken together, these observations suggest that although the distribution of SP binding sites corresponds well to the overall density of SP-IR fibres at a ganglionic scale, there is not necessarily such a good correlation at the level of individual neurons within ganglia.
4.3. Substance P binding sites and the distribution of SP-IR fibres
We have found a heterogeneous distribution of BHSP binding sites in guinea-pig sympathetic ganglia. The majority of those binding sites most probably are NK 1 receptors. Although there is some association of binding sites with well-defined functional populations of neurons, it is not the case universally. Furthermore, the distribution of BHSP binding sites only loosely follows that of SP-IR fibres within the ganglia. In the spinal cord, substance P can diffuse for large distances from its sites of release, especially in the presence of CGRP ŽDuggan and Furmidge, 1994; Schaible et al., 1992.. It seems equally likely that substance P Žand other tachykinins. released from intraganglionic terminals of either preganglionic or unmyelinated sensory neurons also could diffuse for relatively large distances within the ganglia. This may mean that the sites of action of tachykinins in sympathetic ganglia are determined primarily by the distribution of their receptors rather than their release sites. In other words, any specificity in the actions of neurally released substance P within sympathetic ganglia is more likely to be due to the patterns of receptor expression, rather than to particulars of the circuitry of nerve fibres with SP-IR. One consequence of this conclusion is that neurally released tachykinins are likely to act heterosynaptically to increase the excitability of sympathetic neurons expressing NK 1 receptors.
The overall density of BHSP binding sites approximately matches the distribution of SP-IR fibres in the guinea-pig sympathetic ganglia. BHSP binding sites and SP-IR fibres both are much more dense in the prevertebebral ganglia than they are in the paravertebral ganglia. Nevertheless, within individual ganglia, there is generally a poor spatial correlation between the density of BHSP binding sites and the distribution of nerve fibres with SP-IR. For example, in the coeliac ganglion, BHSP binding sites are clumped preferentially over the medial region of the ganglion, whereas SP-IR fibres are distributed densely and uniformly throughout the ganglion. This means that a large proportion of SP-IR fibres in the coeliac ganglion, particularly in the lateral poles, are likely to innervate neurons which lack significant expression of neurokinin binding sites. On the other hand, there are abundant neurokinin binding sites associated with the medial region of the ganglion, which is enriched in SOM-IR neurons. Most of these neurons project to the enteric plexuses where they inhibit motility and secretomotor activity ŽCosta and Furness, 1984.. The excitability of these neurons could be enhanced by tachykinins released from the collaterals of unmyelinated sensory fibres, following excessive distension of the intestine, for example ŽKreulen and Peters, 1986; Matthews and Cuello, 1984.. These fibres also must be associated with the vasomotor neurons that lie predominantly in the lateral poles of the ganglion ŽCosta and Furness, 1984; Macrae et al., 1986.. However, in the absence of any significant receptor expression on these neurons, it is unlikely that they would be under the direct influence of tachykinins released from sensory collaterals. In the lumbar sympathetic chain ganglia, this apparent mismatch is more subtle. Within these ganglia, preganglionic SP-IR fibres are selectively associated with clusters of VIP-IR neurons and are rare throughout most of the rest of the ganglion ŽGibbins, 1992.. Nevertheless, there is only
5. Conclusions
Acknowledgements This project was supported by grants from the National Health and Medical Research Council of Australia, and the Flinders Medical Centre Foundation. Antibodies to somatostatin were provided by J.C. Brown, Medical Research Council of Canada Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver, B.C., Canada. The compounds SR140333, SR48968 and SR142801 were a generous gift from Sanofi, Sydney, Australia. Thanks are extended to Dr Judy Morris for the opportunity to do the autoradiography in her labora-
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tory and to Pat Vilimas for help in performing the experiments. Both provided invaluable expertise. JPM is a National Health and Medical Research Council Australian Postdoctoral Fellow.
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vasoconstrictor and pilomotor neurons and their relations with neuropeptide-containing preganglionic fibres in lumbar sympathetic ganglia of guinea-pigs. Neuroscience 70, 999–1012. Gibbins, I.L., Furness, J.B., Costa, M., 1987. Pathway-specific patterns of coexistence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig. Cell Tissue Res. 248, 417–437. Heym, C., Webber, R., Horn, M., Kummer, W., 1990. Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry. Histochemistry 93, 547–557. Heym, C., Liu, N., Gleich, A., Oberst, P., Kummer, W., 1993. Immunohistochemical evidence for the different pathways immunoreactive to substance P and calcitonin gene-related peptide ŽCGRP. in the guinea-pig stellate ganglion. Cell Tissue Res. 272, 563–574. Hokfelt, T., Kellerth, J.-O., Nilsson, G., Pernow, B., 1975. Substance P: ¨ localization in the central nervous system and in some primary sensory neurons. Science 190, 889–890. Hokfelt, T., Elfvin, L.G., Schultzberg, M., Goldstein, M., Nilsson, G., ¨ 1977. On the occurrence of substance P-containing fibres in sympathetic ganglia: immunohistochemical evidence. Brain Res. 132, 29–41. Hudson, A.L., 1993. Autoradiographic techniques. In: Wharton, J., Polak, J.M. ŽEds.., Receptor Autoradiography. Oxford Univ. Press, Oxford, 1993, pp. 57–79. James, S., Burnstock, G., 1988. Autoradiographic localization of binding sites for 125 Iysubstance P on neurones from cultured rat superior cervical ganglion. Brain Res. 458, 205–211. Ju, G., Hokfelt, T., Brodin, E., Fahrenkrug, J., Fisher, J.A., Frey, P., ¨ Elde, R.P., Brown, J.C., 1987. Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells. Cell Tissue Res. 247, 417–431. Keast, J.R., McLachlan, E.M., Meckler, R.L., 1993. Relation between electrophysiological class and neuropeptide content of guinea-pig sympathetic prevertebral neurons. J. Neurophysiol. 69, 384–394. Konishi, S., Otsuka, M., 1985. Blockade of slow excitatory post-synaptic potential by substance P antagonists in guinea-pig sympathetic ganglia. J. Physiol. London 361, 115–130. Konishi, S., Otsuka, M., Folkers, K., Rosell, S., 1983. A substance P antagonist blocks non-cholinergic slow excitatory postsynaptic potential in guinea-pig sympathetic ganglia. Acta Physiol. Scand. 117, 157–160. Konishi, S., Song, S.-Y., Ogawa, T., Kanazawa, I., 1992. Fast and slow depolarizations produced by substance P and other tachykinins in sympathetic neurons of rat prevertebral ganglia. Neurosci. Res. 14, 81–95. Konishi, S., Song, S.-Y., Yamakuni, T., Saito, K., 1993. Multiple actions of substance P and other tachykinins on sympathetic neurons in rat prevertebral ganglia. Regul. Pept. 46, 257–259. Kreulen, D.L., Peters, S., 1986. Non-cholinergic transmission in a sympathetic ganglion of the guinea-pig elicited by colonic distension. J. Physiol. London 374, 315–334. Krier, J., Szurszewski, J.H., 1982. Effect of substance P on colonic mechanoreceptors, motility, and sympathetic neurons. Am. J. Physiol. 243 Ž6., G259–G267, Gastrointest. Liver Physiol. Lindh, B., Hokfelt, T., Elfvin, L.-G., Terenius, L., Fahrenkrug, J., Elde, ¨ R., Goldstein, M., 1986. Topography of NPY-, somatostatin-, and VIP-immunoreactive neuronal subpopulations in the guinea-pig celiac-superior mesenteric ganglion and their projections to the pylorus. J. Neurosci. 6, 2371–2383. Lindh, B., Lundberg, J.M., Hokfelt, T., 1989. NPY-, galanin-, VIPrPHI-, ¨ CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections. Cell Tissue Res. 256, 259–273. Macrae, I.M., Furness, J.B., Costa, M., 1986. Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig. Cell Tissue Res. 244, 173–180.
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Mantyh, P.W., Catton, M.D., Allen, C.J., Labenski, M.E., Maggio, J.E., Vigna, S.R., 1992. Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia. Neuroscience 46, 739–754. Matthews, M.R., Cuello, A.C., 1982. Substance P-immunoreactive peripheral branches of sensory neurons innervate guinea-pig sympathetic neurons. Proc. Natl. Acad. Sci. USA 79, 1668–1672. Matthews, M.R., Cuello, A.C., 1984. The origin and possible significance of substance P immunoreactive networks in the prevertebral ganglia and related structures in the guinea-pig. Philos. Trans. R. Soc., London Ser. B 306, 313–324. Morris, J.L., Gibbins, I.L., 1987. Neuronal colocalization of peptides, catecholamines, and catecholamine-synthesizing enzymes in guineapig paracervical ganglia. J. Neurosci. 7, 3117–3130. Morris, J.L., Gibbins, I.L., 1992. Co-transmission and neuromodulation. In: Burnstock, G. ŽEd.., Autonomic Neuroeffector Mechanisms. Harwood Academic Publishers, Chur, pp. 33–119. Morris, J.L., Murphy, R., Furness, J.B., Costa, M., 1986. Partial depletion of neuropeptide Y from noradrenergic perivascular and cardiac axons by 6-hydroxydopamine and reserpine. Regul. Pept. 13, 147–162. Niwa, M., Shigematsu, K., Plunkett, L., Saavedra, J.M., 1985. High-affinity substance P binding sites in rat sympathetic ganglia. Am. J. Physiol. 249 Ž18., H694–H697, Heart Circ. Physiol. Petitet, F., Beaujoun, J.-C., Saffroy, M., Torrens, Y., Glowinski, J., 1993. The nonpeptide NK-2 antagonist SR48968 is also a NK-3 antagonist in the guinea-pig but not in the rat. Biochem. Biophys. Res. Commun. 191, 180–187. Regoli, D., Drapeau, G., Dion, S., Couture, R., 1988. New selective
agonists for neurokinin receptors: pharmacological tools for receptor characterization. Trends Pharmacol. Sci. 9, 290–295. Regoli, D., Rouissi, N., D’Orleans-Juste, P., 1994. Pharmacological ´ characterization of receptor types. In: Buck, S.H. ŽEd.., The Tachykinin Receptors. Humana Press, NJ, USA, pp. 367–395. Schaible, H.G., Hope, P.J., Lang, C.W., Duggan, A.W., 1992. Calcitonin gene-related peptide causes intraspinal spreading of substance P released by peripheral stimulation. J. Neurosci. 4, 750–757. Shinkai, M., Takayanagi, I., Kato, T., 1993. Tachykinin receptors of the NK 2 type involved in the acetylcholine release by nicotine in guineapig bladder. Br. J. Pharmacol. 108, 759–762. Tsunoo, A., Konishi, S., Otsuka, M., 1982. Substance P as an excitatory transmitter of primary afferent neurons in guinea-pig sympathetic ganglia. Neuroscience 7, 2025–2037. Wharton, J., Walsh, D.A., Rutherford, R.A.D., Knock, G.A., Polak, J.M., 1993. In vitro autoradiographic localization and characterisation of binding sites. In: Wharton, J., Polak, J.M. ŽEds.., Receptor Autoradiography. Oxford Univ. Press, Oxford, pp. 79–105. Zhao, F.-Z., Saito, K., Yoshioka, K., Guo, J.-Z., Murakoshi, T., Konishi, S., Otsuka, M., 1995. Subtypes of tachykinin receptors on tonic and phasic neurons in the coeliac ganglion of the guinea-pig. Br. J. Pharmacol. 115, 25–30. Zhao, F.-Z., Saito, K., Yoshioka, K., Guo, J.Z., Murakoshi, T., Konishi, S., Otsuka, M., 1996. Tachykininergic synaptic transmission in the coeliac ganglion of the guinea-pig. Br. J. Pharmacol. 118, 2059–2066. Zoli, M., Agnati, L.F., 1996. Wiring and volume transmission in the central nervous system: the concept of closed and open synapses. Prog. Neurobiol. 49, 363–380.
Differential distribution of substance P binding sites in guinea-pig sympathetic ganglia J.P. Messenger ) , I.L. Gibbins Department of Anatomy and Histology, and Centre for Neuroscience, Flinders UniÕersity of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia Received 3 October 1997; revised 25 November 1997; accepted 28 November 1997
Abstract We have used a combination of autoradiographic and immunohistochemical techniques to investigate the distribution of binding sites for substance P in relation to the distribution of substance P-immunoreactive nerve fibres and specific functional populations of neurons in the sympathetic ganglia of guinea-pigs. There was considerable heterogeneity in the density of binding sites for Bolton Hunter labelled 125 I y substance P ŽBHSP.. Binding sites were more dense in the prevertebral ganglia, such as the coeliac and inferior mesenteric ganglia, than in the paravertebral ganglia, such as the superior cervical or lumbar chain ganglia. The binding sites tended to be clumped within the ganglia. Within the prevertebral ganglia, they were associated predominantly with neurons projecting to the enteric plexuses. Many of these neurons contained somatostatin immunoreactivity. In the lumbar sympathetic chain ganglia, there was a weak association of binding sites with neurons containing immunoreactivity to vasoactive intestinal peptide. Overall, the density of binding sites matched the density of nerve fibres containing immunoreactivity to substance P in different ganglia. However, within particular ganglia, there was little, if any, correlation between the distribution of binding sites and nerve fibres containing substance P. Most of the binding sites in the ganglia had the pharmacological characteristics of NK 1 receptors. Our results show that there is considerable heterogeneity in the expression of NK 1 receptors in the sympathetic ganglia of guinea-pigs. However, given the relatively poor spatial correlation between the distribution of binding sites and potential sites of substance P release from intraganglionic nerve fibres, we suggest that substance P may diffuse for relatively large distances through the ganglia, with actions only on those neurons selectively expressing NK 1 receptors. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Substance P; Neurokinin receptor; Sympathetic ganglia; Sensory; Preganglionic; Autoradiography
1. Introduction Immunoreactivity to substance P ŽSP-IR. is widespread in peripheral neurons of common laboratory mammals. Nerve cell bodies with SP-IR occur in dorsal root ganglia, cranial and pelvic parasympathetic ganglia, sympathetic and enteric ganglia ŽCosta et al., 1981; Gibbins, 1990, 1992; Gibbins et al., 1987; Hokfelt et al., 1975; Ju et al., ¨ 1987; Lindh et al., 1989.. Usually, SP-IR is expressed together with other neuropeptides in specific subpopulations of neurons projecting to well-defined peripheral target tissues ŽGibbins, 1990, 1992; Gibbins et al., 1987; Lindh et al., 1989.. Many autonomic ganglia also contain ) Corresponding author. Department of Anatomy and Histology, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5042, Australia. Tel.: q61 8 82044261; fax: q61 8 82770085; e-mail: [email protected]
nerve fibres with SP-IR ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins, 1992; Gibbins et al., 1987; Hokfelt et al., 1977; Matthews and Cuello, 1982, 1984; ¨ Morris and Gibbins, 1987.. Within the sympathetic ganglia of guinea-pigs, SP-IR fibres can arise from two different sources. One population of fibres contains immunoreactivity to calcitonin gene-related peptide ŽCGRP-IR. in addition to SP-IR and almost certainly represents collaterals of capsaicin-sensitive, unmyelinated sensory neurons with cell bodies in the dorsal root ganglia ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins et al., 1987; Matthews and Cuello, 1982, 1984.. The other population of SP-IR fibres lacks CGRP-IR and is most likely to represent the endings of preganglionic neurons that presumably also contain acetylcholine ŽGibbins, 1992; Heym et al., 1990, 1993.. In the prevertebral ganglia, sensory fibres containing both SP-IR and CGRP-IR predominate, while in the lumbar sympathetic chain ganglia, preganglionic SP-IR
0165-1838r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 0 7 - 1
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fibres predominate ŽDalsgaard et al., 1982, 1983; Gamse et al., 1981; Gibbins, 1992; Gibbins et al., 1987; Heym et al., 1990; Matthews and Cuello, 1982, 1984.. As has been seen for other peptide-containing inputs to sympathetic ganglia ŽGibbins, 1992; Lindh et al., 1986; Macrae et al., 1986; Morris and Gibbins, 1992., SP-IR fibres tend to be associated with specific populations of ganglionic neurons ŽGibbins, 1992; Heym et al., 1990.. For example, in the lumbar sympathetic chain ganglia of guinea-pigs, preganglionic fibres containing SP-IR are associated selectively with non-noradrenergic vasodilator neurons that contain immunoreactivity for vasoactive intestinal peptide ŽVIP-IR. and neuropeptide Y ŽNPY-IR. ŽGibbins, 1992.. Exogenously applied substance P generally has excitatory effects on sympathetic neurons, and there is evidence that it can mediate slow synaptic potentials in the prevertebral sympathetic ganglia ŽDun and Karczmar, 1979; Dun and Minota, 1981; Dun and Jiang, 1982; Konishi and Otsuka, 1985; Konishi et al., 1983, 1992; Kreulen and Peters, 1986; Krier and Szurszewski, 1982; Tsunoo et al., 1982; Zhao et al., 1995, 1996.. In the coeliac ganglia of guinea-pigs, these effects are mediated by NK 1 and NK 3 receptors, which appear to be present on both phasic and tonic neurons ŽZhao et al., 1995, 1996.. Hybridization experiments with synthetic DNA probes specific to each of the tachykinin receptor types indicate that NK 1 , NK 2 and NK 3 tachykinin receptors all are expressed in rat prevertebral ganglia ŽKonishi et al., 1993.. SP binding sites also have been localised autoradiographically in sympathetic ganglia ŽJames and Burnstock, 1988; Mantyh et al., 1992; Niwa et al., 1985.. Nevertheless, although the density of substance P binding sites varies within ganglia, it is not known how this variation relates to the distribution of intraganglionic SP-IR fibres or to particular functional populations of ganglionic neurons. It might be expected that variation in the density of substance P binding sites in sympathetic ganglia would be related to the differential distribution of SP-IR fibres in the ganglia. However, in many parts of the nervous system, there may be, at best, only a loose correlation between the distribution of peptide-containing nerve fibres and the receptors for that peptide ŽZoli and Agnati, 1996.. Therefore, this study aims to identify and map the distribution of substance P binding sites in the sympathetic ganglia of guinea-pigs, using autoradiographic methods, and to correlate the distribution of binding sites with immunohistochemically characterised populations of neurons and SP-IR fibres in these ganglia.
2. Materials and methods 2.1. Tissue preparation Male and female guinea-pigs weighing 200–400 g were used. The superior cervical ganglia, stellate ganglia, lum-
bar sympathetic chain ganglia, coeliac ganglion and inferior mesenteric ganglion were dissected from guinea-pigs that had been stunned by a blow to the head and bled via the carotid arteries. Ganglia were cleared of fat and connective tissue, placed in cryomoulds containing OCT ŽTissue-Tek, Miles, Elkhart, IN, USA., and rapidly frozen in isopentane cooled in liquid nitrogen. The tissue was sectioned at 10–12 m m on a cryostat at y208C. Sections were thaw mounted to slides coated with polyethylenimine ŽSigma, Castle Hill, NSW, Australia. and allowed to air dry. 2.2. Autoradiography Ganglia to be analysed both autoradiographically and immunohistochemically were processed in the following way ŽBurcher and Buck, 1986; Hudson, 1993; Wharton et al., 1993.. Alternate sections were dried onto different slides. Alternate slides were processed for autoradiographic localization of substance P binding sites or for immunohistochemical analysis Žsee Section 2.3.. Slides for autoradiography were pre-incubated in Tris–HCl-buffer Ž0.05 M, pH 7.4. containing 0.02% bovine serum albumin ŽBSA. for 2 = 15 min. This solution was removed and replaced with incubation solution ŽTris-buffer containing 0.02% BSA, 2 m grml chymostatin, 4 m grml leupeptin, 40 m grml bacitracin and 3 mM MgCl 2 . also containing Bolton Hunter-labelled 125 I y substance P ŽBHSP. for 2 h. Slides were washed for 6 = 90 s in ice cold Tris-buffer0.02% BSA followed by one 90-s wash in distilled water. Sections were air dried and fixed in paraformaldehyde vapour, at 608C for 90 min. Slides were then taken up through graded alcohols to xylene, and down through graded alcohols to distilled water. The slides were dried again and dipped in radiation sensitive emulsion ŽLM-1, Amersham Australia, Sydney, NSW., and left for 7–14 days in the dark at 48C, before developing with Kodak D19 developing solution ŽKodak ŽAustralasia., Coberg, Victoria.. The developed slides were coverslipped with DPX mountant ŽBDH, Poole, UK.. In all experiments, a control slide was incubated in solution that did not contain BHSP. Non-specific binding was determined on sections incubated in parallel by adding excess unlabelled substance P Ž10y6 M. to the incubation solution. The specific binding of BHSP was determined in a saturation binding assay, in each of the ganglia Ž n s 2, in duplicate.. Specific binding is defined as the total binding minus the non-specific binding. Total binding is that binding achieved in the presence of radioligand alone, while non-specific binding is the binding seen in the presence of radioligand plus excess unlabelled ligand. The optimum binding concentration was that dose of the radioligand that gave the highest percentage of specific binding. Several time points were tested for each concentration, giving the optimal incubation period. For all ganglia, the optimal
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binding concentration was 0.1 nM, the optimal incubation period was 120 min, and these parameters were used routinely. The aim of the first series of experiments was to determine the density of BHSP binding sites in each of the ganglia. To do this, the specific binding of BHSP was determined as described above. Sections that had been incubated with radioligand were wiped from the slides with moistened filter paper after the final wash in distilled water. The wiped sections and filter paper were placed in glass tubes and gamma counts per minute were recorded by a Riastar gamma counter ŽCanberra-Packard, Mt. Wa-
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verly, Australia.. Alternate sections, not incubated in radioligand, were fixed in 4% formaldehyde, and stained with cresyl violet. The stained sections were drawn with a camera lucida attachment on an Olympus BH2 microscope and digitised so that their area could be measured with NIH Image running on a Macintosh 8500 computer. This allowed us to derive an estimate of specific binding as gamma counts per squared millimeter for each of the ganglia Ž n s 3, done in duplicate.. The aim of a further series of experiments was to determine the type of tachykinin receptor to which BHSP was binding. Sections of ganglia were incubated in radiola-
Fig. 1. Distribution of substance P-IR fibres in guinea-pig sympathetic ganglia. Ža. Sparse SP-IR fibres in the superior cervical ganglion ŽSCG.. Žb. SP-IR fibres in the lumbar sympathetic chain ganglia ŽLCG. mostly form dense pericellular baskets around unlabelled neurons. Žc,d. A high density of SP-IR fibres is found in the coeliac ganglion ŽCG, c., and, in the inferior mesenteric ganglion ŽIMG, d.. Že,f. Coexistence of SP-IR and CGRP-IR in most fibres in the CG. Žg,h. In the LCG, however, SP-IR fibres forming baskets around neurons do not contain CGRP-IR. Scale bar: a–d, 50 m m; e–h, 20 m m.
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belled substance P, with a tachykinin receptor agonist or antagonist. In these experiments, the BHSP binding also was measured by gamma counting. The sections were incubated in radioligand as described above, with or without the agonist or antagonist, and the sections were wiped from the slides, after washing, for gamma counting. The peptide agonists ŽRegoli et al., 1988. and non-peptide antagonists ŽEdmonds-Alt et al., 1992, 1993, 1994. used were, respectively: NK 1 receptor, Sar 9 MetŽO 2 .11 -SP and SR140333; NK 2 receptor, Nle 10 -NKA Ž4–10 . and SR48968; NK 3 receptor, senktide and SR142801. Each compound was used in concentrations ranging from 10y6 M to 10y9 M, on sections from each of the ganglia Ž n s 3, done in duplicate.. Results were analysed statistically with multivariate repeated measures analysis of variance, using the GLM procedure of SAS on a personal computer ŽRelease 6.04; SAS Institute, Cary, NC, USA.. The small size of the sympathetic ganglia was a limiting factor in the design of these experiments. It was not possible to obtain reliably more than 36 sections 10-m m thick from a single ganglion. Therefore, only a limited number of slides could be used for receptor characterization experiments. For each ganglion, four doses of each ligand were selected, plus a slide for total binding and a slide for non-specific binding. 2.3. Immunohistochemistry The aim of one series of experiments was to determine the chemical coding of the postganglionic neurons over which autoradiographic grains were localized. In the prevertebral ganglia, neurons can be differentiated by their immunoreactivity for somatostatin ŽSOM-IR. or NPY: neurons with NPY-IR tend to be located more laterally in the ganglia and project to blood vessels; neurons with SOM-IR or which lack both NPY-IR and SOM-IR tend to be located more medially and project to the enteric plexuses ŽCosta and Furness, 1984; Lindh et al., 1986; Macrae et al., 1986.. In the paravertebral ganglia, VIP-IR was used to localize neurons that are surrounded preferentially by SP-IR fibres ŽGibbins, 1992.. It was not possible to determine the relationship between clumps of BHSP binding sites and the distribution of SP-IR fibres directly, since the SP antiserum did not produce specific labelling on the freshly cut and post-fixed tissue used for autoradiography. However, double labelling immunohistochemistry for SP and CGRP was performed on freshly fixed tissue that was not processed for autoradiography. Sections to be immunohistochemically analysed were fixed in phosphate buffered 0.2% picric acid and 4% formaldehyde for 1 h then cleared with dimethylsulphoxide ŽDMSO. for 15 min and washed in phosphate buffered saline ŽPBS. for 15 min. Whole ganglia were fixed overnight and then cleared in ethanol followed by DMSO, and again in ethanol. Ganglia were embedded in polyethylene glycol, and sections were cut with a rotary
microtome. All sections were incubated in 10% normal donkey serum for 30 min, and then incubated in primary antiserum containing 10% normal donkey serum for 48 h. Primary antisera used were raised against VIP Žraised in rabbit; Incstar; code 9032039., SOM Žraised in mouse; source J.C. Brown; code SOMA 08., NPY Žraised in rabbit; source C. Maccarone and B. Jarrott; code JrM 264 ŽMorris et al., 1986.. substance P Žraised in rat; Sera Lab; code B8E035. and CGRP Žraised in goat; Arnel Laboratories, code F1124459.. Secondary antisera used were Cy3 or DTAF conjugated anti-rabbit, anti-rat, anti-sheep or anti-mouse IgG, all raised in donkey ŽJackson ImmunoResearch Laboratories, West Grove, PA, USA.. These antibodies have been used extensively in our laboratory and show no inappropriate cross-reactivity ŽGibbins, 1992; Gibbins and Matthew, 1996.. 2.4. Microscopy and digital imaging Autoradiographic grains were visualized using an Olympus BX-50 or BH-2 microscope, with either bright field or dark field optics. Immunofluorescent labelling on the sections adjacent to the autoradiographically labelled sections was analysed on the BX-50 microscope. Images were captured with a Sony CCD camera ŽModel SSCM370CE. and a Scion LG-3 frame grabber in a Macintosh Quadra 850AV computer, using NIH Image software. Digital enhancement and image analysis was performed on a Macintosh 8500 computer using NIH Image and Adobe Photoshop software ŽAdobe Systems, Mountain View, CA, USA.. Grey scale images were enhanced in Photoshop by altering brightness and contrast to appropriate levels for publication. Images of radiolabelled whole ganglia were thresholded and converting to binary format in NIH Image before assembling into montages using Powerpoint ŽMicrosoft..
Fig. 2. Density of BHSP binding sites in guinea-pig sympathetic ganglia. There is a significant overall difference in binding density. One-way ANOVA and post-hoc comparisons show Ž). that the stellate ganglion has less binding than all other ganglia and that the IMG has a greater binding density than all other ganglia Ž FŽ4,15. s 4.8, P s 0.01.. SCG, superior cervical ganglion; LCG, lumbar chain ganglia; CG, coeliac ganglion; IMG, inferior mesenteric ganglion.
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2.5. Chemicals The peptides Sar 9 MetŽO 2 .11 -SP, Nle10-NKAŽ4–10. and senktide were made by Auspep ŽParkville, Victoria, Australia.. Bolton Hunter 125 I y substance P was made by Amersham ŽNorth Ryde, NSW, Australia.. The peptidase inhibitors chymostatin, leupeptin and bacitracin were obtained from Sigma ŽAustralia.. The tachykinin receptor antagonists were a generous gift of Sanofi ŽSydney, Australia.. They were SR140333, Ž1-2-w3-Ž3,4-dichlorophenyl . -1- Ž 3-isopropoxyphenylacetyl .piperidin-3-yl xethyl4 -4-phenyl-1-azoniabicyclow2.2.2xoctone.; SR48968,
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Ž S .-N-methyl-N w4-acetylamino-4-phenylpiperidino.-2-Ž3,4dichloro phenyl.butylx benzamide and SR142801, ŽŽ S-Ž N .-Ž1-Ž3-Ž1-benzoyl-3-Ž3,4-dichlorophenyl.piperidin3-yl.-4-phenylpiperidin-4-yl.-N-methylacetamide. 3. Results 3.1. Distribution of SP-IR and CGRP-IR fibres in sympathetic ganglia Varicose nerve fibres with SP-IR were found in all sympathetic ganglia analysed, as has been reported previ-
Fig. 3. Distribution of BHSP binding sites Ždarkfield microscopy., NPY-IR neurons and SOM-IR neurons Žimmunofluorescence. in the inferior mesenteric ganglion ŽIMG. and coeliac ganglion ŽCG.. Binding sites are most dense where SOM-IR neurons are present and least dense where NPY-IR neurons occur. Ža,b. Low magnification montages of one lobe of an IMG showing Ža. clustered BHSP binding sites Žarrows. and Žb. corresponding clusters of SOM-IR neurons in the adjacent section Žarrows.. Žc,d. Micrographs showing the negative correlation between Žc. BHSP binding sites and Žd. NPY-IR neurons in the IMG. Že,f. micrographs showing a good positive correlation between Že. clustered BHSP binding sites Žarrow. and Žf. a corresponding group of SOM-IR neurons Žarrow. in the CG. Scale bar: a–b, 200 m m; c–f, 100 m m.
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ously ŽDalsgaard et al., 1983; Gamse et al., 1981; Heym et al., 1990, 1993; Hokfelt et al., 1977; Matthews and Cuello, ¨ 1982, 1984.. The density of the SP-IR fibres in each of the ganglia, and characteristics of their distribution within a ganglion, varied between ganglia ŽFig. 1.. The superior cervical ganglion had only sparse SP-IR fibres which did not appear to target selectively any structures within the ganglion ŽFig. 1a.. These fibres mostly contained CGRP-IR in addition to SP-IR. The stellate ganglion had a low density of SP-IR fibres, some of which formed pericellular baskets around the cell bodies of unlabelled neurons. SP-IR fibres in the lumbar chain ganglia were numerous. They mostly lacked CGRP-IR, and formed frequent baskets of boutons around unlabelled neurons ŽFig. 1b,g,h.. Such baskets of SP-IR boutons are associated selectively with VIP-IR neurons in the lumbar chain ganglia ŽGibbins, 1992.. SP-IR fibres in the coeliac and inferior mesenteric ganglia were densely distributed throughout the ganglia
ŽFig. 1c,d.. They formed obvious baskets less often than in the lumbar chain ganglia, and there was no obvious bias in the distribution of SP-IR fibres in the medial or lateral lobes of the coeliac ganglion. In both prevertebral ganglia, most SP-IR fibres also contained CGRP-IR ŽFig. 1e,f.. 3.2. Substance P binding sites Bolton Hunter 125 I y substance P ŽBHSP. binding was observed in all the sympathetic ganglia we examined. However, the density and distribution pattern of binding sites varied consistently from ganglion to ganglion ŽFig. 2.. 3.2.1. Inferior mesenteric ganglion This ganglion had a highest density of BHSP binding sites of all ganglia analysed ŽFig. 2.. Mean binding density over the entire ganglion was 36.2 " 9.9 cpmrmm2 Žmean
Fig. 4. Distribution of BHSP binding sites and VIP-IR neurons in two examples of lumbar sympathetic chain ganglia. Ža,b. Clusters of BHSP binding sites Žarrows, a. only loosely match with the groups of VIP-IR neurons in the adjacent section Žarrows, b.. Žc,d. A cluster of BHSP binding sites Žc. is associated with a VIP-IR neuron on the adjacent section Žd., but also extend into regions of the ganglion devoid of VIP-IR neurons. Scale bar: a–b, 200 m m; c–d, 100 m m.
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" SEM, n s 3 animals, experiments were done in duplicate.. The binding sites were distributed in large clumps throughout the ganglion ŽFig. 3a.. Subsequent immunohistochemistry for SOM-IR on adjacent sections revealed that the BHSP binding was closely correlated with the distribution of SOM-IR cells ŽFig. 3b.. The areas of sparse BHSP binding matched the distribution of NPY-IR neurons, seen in adjacent sections ŽFig. 3c,d.. Neurons without either SOM-IR or NPY-IR occurred both in areas with high and low density BHSP binding. 3.2.2. Coeliac ganglion The coeliac ganglion consists of two lobes. NPY-IR neurons tend to be located laterally in each lobe, while SOM-IR neurons and neurons lacking NPY-IR and SOMIR tend to be located more medially ŽCosta and Furness, 1984; Keast et al., 1993; Lindh et al., 1986; Mantyh et al., 1992.. In the coeliac ganglion as a whole, there was a high density of BHSP binding sites Ž29.0 " 3.4 cpmrmm2 ; Fig. 2.. The BHSP binding sites were distributed in a patchy manner over large regions of the ganglion. However, there was a distinct polarity across each lobe so that the greatest density of BHSP binding sites was found over the medial region of each lobe. This region corresponds to the part of the ganglion containing predominantly SOM-IR neurons ŽFig. 3e,f.. 3.2.3. Lumbar sympathetic chain ganglia The average density of BHSP binding sites for lumbar chain ganglia was 13.8 " 3.9 cpmrmm2 ŽFig. 2.. BHSP binding sites generally were concentrated into small
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clumps, scattered irregularly throughout the ganglia ŽFig. 4a,b.. Most ganglia had several clumps of grains, but some ganglia had no clumps of BHSP binding sites and other ganglia had a high density of binding sites uniformly over the ganglion. Alternate sections were labelled for VIP-IR and there was an inconsistent association between clumps of BHSP binding sites and VIP-IR cells. Although BHSP binding sites often were localized over VIP-IR cells ŽFig. 4c,d., clumps of BHSP binding sites also were associated with VIP immunonegative cells. Many VIP-IR cells were not associated with BHSP binding sites ŽFig. 4a,b.. 3.2.4. Superior cerÕical ganglion and stellate ganglion The average density of BHSP binding sites was 19.6 " 3.1 cpmrmm2 for the superior cervical ganglion and 7.0 " 2.4 cpmrmm2 for the stellate ganglion ŽFig. 2.. In the superior cervical ganglion, BHSP binding sites were distributed evenly throughout the ganglion without obvious clustering ŽFig. 5a.. In the stellate ganglion, occasional clumps of BHSP binding sites were seen ŽFig. 5b.. Labelling for VIP-IR on sequential sections of the stellate ganglion showed that there was no correlation between the distribution of VIP-IR cells and BHSP binding sites. 3.3. Characterization of BHSP binding sites In a further series of autoradiographic experiments, the effects of the proposed endogenous ligand for the NK 2 receptor, neurokinin A ŽNKA., and the proposed endogenous ligand for the NK 3 receptor, neurokinin B ŽNKB., on BHSP binding were tested. As seen in Fig. 6, an excess
Fig. 5. Distribution of BHSP binding sites in darkfield montages of the superior cervical ganglion ŽSCG, a. and the stellate ganglion Žb.. BHSP binding sites are patchy and relatively sparse. Scale bar: 500 m m.
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Fig. 6. BHSP binding in the coeliac ganglion ŽCG., inferior mesenteric ganglion ŽIMG. and the lumbar chain ganglia ŽLCG., and the effect on BHSP binding of adding excess unlabelled SP, NKA or NKB to the incubation solution. In all ganglia, 1 m M SP completely inhibited BHSP binding Žsecond column of micrographs.. However, an excess of NKA or NKB did not affect BHSP binding in any ganglia Žthird column of micrographs.. Scale bar: 500 m m.
Ž10y7 M. of unlabelled NKA or NKB in the incubation medium did not inhibit BHSP binding in any ganglia, suggesting that BHSP is binding predominantly to NK 1 receptors. In order to test this conclusion further, we examined the ability of selective agonists and antagonists for NK 1 , NK 2 and NK 3 receptors to inhibit BHSP binding, as determined by gamma counting on sections wiped from slides. In each of the ganglia analysed, unlabelled substance P Ž1 nM. in
the incubation medium completely inhibited BHSP binding. The selective NK 1 peptide agonist, Sar 9 MetŽO 2 .11 -SP Ž1 nM., also abolished BHSP binding in all ganglia, as did the NK 1 selective non-peptide antagonist SR140333 Ž1 nM.. These results confirm that most of the BHSP binding occurred at NK 1 receptors. Consistent with the preceding results, selective NK 2 and NK 3 agonists and antagonists only inhibited BHSP binding significantly at the highest concentrations tested
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Ž10y6 M. if at all. For example, in the inferior mesenteric ganglion, BHSP binding was partially inhibited by the NK 3 receptor agonist, senktide Ž45% inhibition at 10y7 M; FŽ1,2. s 47.7, P - 0.05., and the NK 3 receptor antagonist, SR142801 Ž39% inhibition at 10y7 M; FŽ1,2. s 25.4, P - 0.05.. In the stellate ganglion, the non-peptide NK 2 antagonist partially inhibited BHSP binding Ž61% inhibition at 10y7 M; FŽ1,2. s 278.4, P - 0.05., although the NK 2 agonist, Nle10 -NKAŽ4–10. had little or no effect on binding. Similarly, the non-peptide NK 3 antagonist, but not the peptide agonist, partially inhibited BHSP binding in the superior cervical ganglion Ž68% inhibition at 10y7 M; FŽ1,2. s 28.4, P - 0.05.. As a well-characterised control tissue, we examined BHSP binding in the urinary bladder, where the tachykinin binding sites have been described previously as representing predominantly NK 1 receptors ŽBurcher and Buck, 1986.. The presence of NK 2 receptors in guinea-pig bladder also has been demonstrated by Shinkai et al. Ž1993.. We found that 1 nM substance P, the NK 1 receptor agonist, Sar 9 MetŽO 2 .11 -SP, or the NK 1 receptor antagonist, SR140333, all completely inhibited BHSP binding in this tissue. Although the NK 2 receptor agonist, Nle10 NKAŽ4–10., had no significant effect on BHSP binding at any concentration, the non-peptide NK 2 antagonist, SR48968, partially inhibited BHSP binding Ž53% inhibition at 10y7 M; FŽ1,2. s 32.1, P - 0.05.. The NK 3 receptor agonist, senktide, and the non-peptide antagonist, SR142801, had no significant effect on BHSP binding. These observations are consistent with previous reports ŽBurcher and Buck, 1986; Shinkai et al., 1993. and support the reliability of our observations on the sympathetic ganglia.
4. Discussion 4.1. Distribution of substance P binding sites Substance P binding sites, localized with 125 I Bolton Hunter-Substance P ŽBHSP., were distributed in a pattern that varied considerably in each of the sympathetic ganglia analysed. The greatest density of BHSP binding sites was found in the prevertebral ganglia. The inferior mesenteric ganglion had the highest overall density of BHSP binding sites with almost the entire area of the ganglion covered in autoradiographic grains. The coeliac ganglion also had a very high density of autoradiographic grains over large regions of the ganglion. A moderate density of autoradiographic grains was seen in the superior cervical ganglion and in the lumbar chain ganglia, and the lowest average density of BHSP binding sites was found in the stellate ganglion. There was also considerable variation in the distribution of BHSP grains within individual ganglia. In most ganglia,
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BHSP binding sites were patchy, or clumped, suggesting that only subsets of postganglionic neurons express high densities of these binding sites. In the bi-lobed coeliac ganglion, large clumps of BHSP binding sites were found in the medial regions of each of the two lobes, and little binding was observed in the lateral poles. This distribution of BHSP binding sites corresponds to the distribution of neurons projecting to the enteric plexuses. These neurons are located preferentially in the medial poles of the coeliac ganglion, and many of them contain SOM-IR ŽCosta and Furness, 1984; Keast et al., 1993; Lindh et al., 1986; Mantyh et al., 1992.. A similar close association between BHSP binding sites and SOM-IR neurons also was observed in the inferior mesenteric ganglion, although there is no obvious polarity in that ganglion. Thus, substance P binding sites in the coeliac ganglion must be located predominantly on neurons regulating gastrointestinal motility and secretory activity, and are likely to have a similar selective localization in the inferior mesenteric ganglion. BHSP binding sites in the lumbar chain ganglia were notable in that they tended to be more clumped than in the other ganglia, and that the clumps were distributed irregularly throughout the ganglia. Labelling for VIP-IR neurons on alternate sections indicated that there was only a loose association between the clumps of BHSP grains and the distribution of VIP-IR neurons. In principle, BHSP binding sites also could be located on different populations of nerve fibres within the sympathetic ganglia. For example, the medial region of the coeliac ganglion, which contains neurons projecting to the enteric plexuses, is also the region where most of the intestinofugal fibres terminate ŽLindh et al., 1986; Macrae et al., 1986.. Consequently, the high density of BHSP binding sites in the medial region of the ganglion could be associated with these fibres themselves, rather than with the neurons they surround. However, there is no functional evidence to indicate that substance P receptors are present on any population of nerve fibres within sympathetic ganglia. 4.2. Characterization of substance P binding sites Tachykinin receptors are characterized as either neurokinin NK 1 , NK 2 or NK 3 receptors ŽBurcher et al., 1991; Regoli et al., 1988, 1994.. We have used selective tachykinin receptor agonists and antagonists to characterize the binding sites that are present in guinea-pig sympathetic ganglia. In all ganglia, at concentrations as low as 1 nM, substance P completely inhibited BHSP binding. Low concentrations of the specific NK 1 receptor peptide agonist Sar 9 MetŽO 2 . 11 -SP and the non-peptide antagonist SR140333 also completely inhibited BHSP binding, in each of the five ganglia. These results indicate that BHSP is predominantly binding to NK 1 receptors. R e s u lts w ith p e p tid e a n d n o n -p e p tid e agonistsrantagonists to the NK 2 and NK 3 receptors indi-
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cated that there were few NK 2 or NK 3 binding sites in guinea-pig sympathetic ganglia. The low levels of inhibition of BHSP binding seen in the presence of high concentrations of these agents most likely were due to non-specific binding to NK 1 receptors ŽPetitet et al., 1993.. This conclusion is consistent with electrophysiological studies showing that most effects of tachykinins on the membrane properties of coeliac ganglion neurons are mediated by NK 1 receptors. However, these studies have also shown some effects mediated by NK 3 receptors ŽZhao et al., 1995, 1996.. Our observations would suggest either that relatively few neurons express NK 3 receptors, or that the level of NK 3 receptor expression is low overall compared with that of NK 1 receptors.
a loose spatial correlation between VIP-IR neurons and substance P binding sites. Although clusters of BHSP binding sites tended to be include regions of the ganglion containing VIP-IR neurons, they often included neurons without VIP-IR. Conversely, many groups of VIP-IR neurons apparently lacked binding sites. Taken together, these observations suggest that although the distribution of SP binding sites corresponds well to the overall density of SP-IR fibres at a ganglionic scale, there is not necessarily such a good correlation at the level of individual neurons within ganglia.
4.3. Substance P binding sites and the distribution of SP-IR fibres
We have found a heterogeneous distribution of BHSP binding sites in guinea-pig sympathetic ganglia. The majority of those binding sites most probably are NK 1 receptors. Although there is some association of binding sites with well-defined functional populations of neurons, it is not the case universally. Furthermore, the distribution of BHSP binding sites only loosely follows that of SP-IR fibres within the ganglia. In the spinal cord, substance P can diffuse for large distances from its sites of release, especially in the presence of CGRP ŽDuggan and Furmidge, 1994; Schaible et al., 1992.. It seems equally likely that substance P Žand other tachykinins. released from intraganglionic terminals of either preganglionic or unmyelinated sensory neurons also could diffuse for relatively large distances within the ganglia. This may mean that the sites of action of tachykinins in sympathetic ganglia are determined primarily by the distribution of their receptors rather than their release sites. In other words, any specificity in the actions of neurally released substance P within sympathetic ganglia is more likely to be due to the patterns of receptor expression, rather than to particulars of the circuitry of nerve fibres with SP-IR. One consequence of this conclusion is that neurally released tachykinins are likely to act heterosynaptically to increase the excitability of sympathetic neurons expressing NK 1 receptors.
The overall density of BHSP binding sites approximately matches the distribution of SP-IR fibres in the guinea-pig sympathetic ganglia. BHSP binding sites and SP-IR fibres both are much more dense in the prevertebebral ganglia than they are in the paravertebral ganglia. Nevertheless, within individual ganglia, there is generally a poor spatial correlation between the density of BHSP binding sites and the distribution of nerve fibres with SP-IR. For example, in the coeliac ganglion, BHSP binding sites are clumped preferentially over the medial region of the ganglion, whereas SP-IR fibres are distributed densely and uniformly throughout the ganglion. This means that a large proportion of SP-IR fibres in the coeliac ganglion, particularly in the lateral poles, are likely to innervate neurons which lack significant expression of neurokinin binding sites. On the other hand, there are abundant neurokinin binding sites associated with the medial region of the ganglion, which is enriched in SOM-IR neurons. Most of these neurons project to the enteric plexuses where they inhibit motility and secretomotor activity ŽCosta and Furness, 1984.. The excitability of these neurons could be enhanced by tachykinins released from the collaterals of unmyelinated sensory fibres, following excessive distension of the intestine, for example ŽKreulen and Peters, 1986; Matthews and Cuello, 1984.. These fibres also must be associated with the vasomotor neurons that lie predominantly in the lateral poles of the ganglion ŽCosta and Furness, 1984; Macrae et al., 1986.. However, in the absence of any significant receptor expression on these neurons, it is unlikely that they would be under the direct influence of tachykinins released from sensory collaterals. In the lumbar sympathetic chain ganglia, this apparent mismatch is more subtle. Within these ganglia, preganglionic SP-IR fibres are selectively associated with clusters of VIP-IR neurons and are rare throughout most of the rest of the ganglion ŽGibbins, 1992.. Nevertheless, there is only
5. Conclusions
Acknowledgements This project was supported by grants from the National Health and Medical Research Council of Australia, and the Flinders Medical Centre Foundation. Antibodies to somatostatin were provided by J.C. Brown, Medical Research Council of Canada Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver, B.C., Canada. The compounds SR140333, SR48968 and SR142801 were a generous gift from Sanofi, Sydney, Australia. Thanks are extended to Dr Judy Morris for the opportunity to do the autoradiography in her labora-
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tory and to Pat Vilimas for help in performing the experiments. Both provided invaluable expertise. JPM is a National Health and Medical Research Council Australian Postdoctoral Fellow.
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