Distribution and origin of peptide-containing nerve fibers in the celiac superior mesenteric ganglion of the guinea-pig

0306-4522/88$3.00 + 0.00

A’euroscience Vol. 26, No. 3, pp. 1037-1071, 1988

Pergamon Press plc 0 1988IBRO

Printed in Great Britain

DISTRIBUTION AND ORIGIN OF PEPTIDE-CONTAINING NERVE FIBERS IN THE CELIAC SUPERIOR MESENTERIC GANGLION OF THE GUINEA-PIG B. LINDH, T. H~KFELT and L.-G. ELFVIN Departments of Anatomy and Histology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden Abstract-The origin of the peptidergic nerve fibers and terminals in the celiac superior mesenteric ganglion of the guinea-pig was studied. The distribution of immunoreactivity to enkephalin, substance P, calcitonin gene-related peptide, cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin was analysed in intact animals and in animals subjected to various denervation and ligation procedures. The present results show that each of the connected nerve trunks carries peptidergic pathways and contributes to the peptidergic networks in the celiac superior mesenteric ganglion. Thus, the thoracic splanchnic nerves contain enkephalin-, substance P- and calcitonin gene-related peptide-immunoreactivity of which substance P and calcitonin gene-related peptide coexist in the same nerve fibers. In addition, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine- and dynorphin-immunoreactivity is present in some fibers. All of these immunoreactivities are present in sensory neurons except enkephalin which probably originates in the spinal cord. The mesenteric nerves carry enkephalin-, calcitonin gene-related peptide-, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine-, bombesin- and dynorphin-immunoreactive fibers from the intestine and are the main source for cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin fibers. Double-staining experiments indicate that many of these peptides are synthesized in the same enteric neurons. Also the intermesenteric nerve contains peptide-immunoreactive fibers to the celiac superior mesenteric ganglion from different sources, probably including the distal colon as well as dorsal root ganglia and spinal cord at lower thoracic and lumbar levels. The results are discussed in relation to earlier morphological and physiological studies supporting the view of a role of the celiac superior mesenteric ganglion in local reflex mechanisms involved in regulation of gastrointestinal functions.

sympathetic ganglia supply several target organs within the abdominal and pelvic cavity. In accordance with the pioneer studies of Langley, these ganglia were earlier considered as being largely relay stations controlled by sympathetic preganglionic neurons in the spinal cord. More recent physiological and morphological studies have revealed that dorsal root ganglion cells as well as neurons in visceral intramural ganglia and neighboring autonomic ganglia also project to the prevertebral sympathetic ganglia. Today we know that the prevertebra1 sympathetic ganglia possess a high degree of integrative ability and participate in coordinated peripheral reflex activities (for reviews see Refs 21, 92 and 94). Prevertebral

Abbreviations:

ACh, acetylcholine; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; CP, celiac pole; CSMG, celiac superior mesenteric ganglion; ENK, enkephalin; FITC, fluorescein isothiocyanate; GRP, gastrin releasing peptide; IMG, inferior mesenteric ganglion; LI, -like immunoreactivity; NA, noradrenaline; NPY, neuropeptide tyrosine(Y); PHI, peptide histidine isoleucine; SIF, small intensely fluorescent; SMP, superior mesenteric pole; SP, substance P; TRITC, tetramethylrhodamine isothiocyanate; VIP, vasoactive intestinal polypeptide.

Acetylcholine (ACh) and noradrenaline (NA) are the classical transmitters in the autonomic nervous system. Recent immunohistochemical investigations have made it clear that a large number of pathways in the sympathetic nervous system consist of neurons containing small, biologically active peptides. Thus, in the guinea-pig prevertebral sympathetic ganglia nerve fibers and terminals have been characterized with antisera raised to vasoactive intestinal polypeptide (VIP),% substance P (SP),5’,73.74enkephalin (ENK),88*89cholecystokinin (CCK),@ bombesin (gastrin releasing peptide, GRP),*6 dynorphin,‘6,98 and calcitonin gene-related peptide (CGRP)27*66using immunohistochemical techniques. In many cases these peptides seem to coexist with the classical transmitters. Furthermore, neurons with multiple peptide contents have been demonstrated, and physiological studies suggest that these peptides may have a neuromodulator and even a neurotransmitter function (for reviews see Refs 53 and 69). The origin of the peptidergic nerve fibers and terminals found in the inferior mesenteric ganglion (IMG) of the guinea-pig has been analysed in detai1,‘s’5.73 whereas the celiac superior mesenteric ganglion (CSMG) has so far mainly been studied with regard to the topography of its peptide-containing

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1038 neurons.67.7i

The majority

of the principal

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ganglion

cells contain neuropeptide Y-like immunoreactivity (NPY-LI) or somatostatin-11. These two peptides have a complementary ganglionic distribution with the NPY-cells located mainly in the celiac poles (CP) and the somatostatin cells in the superior mesenteric pole (SMP) of the ganglion. The vast majority of the NPY- and somatostatin-bells also contain tyrosine hydroxylase- and dopamine ~-hydroxylase-LI, confirming their catecholaminer~c phenotype. The very dense network of VIP/peptide histidine leucine (PHI)positive fibers observed in well-defined areas of the

CSMG50 has been revealed to be generally confined to areas where the somatostatin-containing cells predominate.67,70 The present study is an attempt to make a systematic characterization of the origin and distribution of the known peptidergic nerve fibers projecting to the CSMG of the guinea-pig. The indirect immunofluorescence technique of Coons and collaborators’ was used to analyse the dist~bution of nerve fibers containing ENK,& SP,6,u CGRP,‘~M CCK,?* VIP,79,85 PHI?’ bombesin2 and dynorphin4’>42 in the CSMG of the guinea-pig. In the present study an antiserum raised to the 14 amino-acid-peptide bombesin, originally isolated from frog skin by Anastasi ef ~l.,~ was used. Its mammalian counterpart may, in fact, be the 27 amino-acid-peptide GRP, isolated by McDonald et ~1.‘~~‘~ In the following we will, however, use the term bombesin for this immunoreactivity. The results

presented on the origin of the peptidergic nerve fibers and terminals are based on denervation and crush or ligation experiments.

EXPERIMENTAL

PROCEDURES

All experiments were performed on male adult guineapigs (body weight 20&3OOg) under anesthesia with diazepam (Stesolid, Dumex, Copenhagen, Denmark) and Hypnorm Vet (Leo, Helsingborg, Sweden), alternatively ketamine (Ketalar, Parke-Davis, Gwent, U.K.) and xylazine (Rompun Vet, Bayer, Leverkusen, F.R.G.). Two groups of five animals each were used to analyse the origin of peptidecontaining fibers in CSMG with respect to three peripheral nerves: the thoracic splanchnic, the mesenteric and the intermesenteric nerves. In each group three animals were partially denervated by cutting two out of the three abovementioned nerve trunks, leaving one intact. In one animal all three nerve trunks were transected, and one animal was unoperated. Figure IA is a schematic drawing showing the CSMG and the connected nerve trunks. The various denervation procedures are also illustrated (Fig. lB-E). In addition, nerve ligation and crush experiments were performed in two groups of three animals. The CSMG and one of the connected nerve trunks were identified in each experiment. The connected nerve trunk was either ligated (mesenteric nerves) or crushed with watchmaker’s forceps (thoracic splanchnic and intermesenteric nerves) close to the ganglion. In the case of the mesenteric nerve ligation, the thoracic splanchnic nerves were cut bilaterally 7 days prior to the ligation experiment. After a post-operative survival time of 5-7 days, or 24 h in the case of the nerve ligation and nerve crush experiments, the animals were anesthetized and perfused transcardiaily with lOOmI of Tyrode’s solution. After perfusion, the

Fig. 1. Schematic drawing of the guinea-pig celiac superior mesenteric ganglion (CSMG) with the connected nerve trunks (tsn, thoracic splanchnic nerves; mn, mesenteric nerves; imn, intermesenteric nerve). A shows the situation in the unoperated animal. B-E show the various denervation procedures performed. In B the ganglion is totally denervated; in C the thoracic splanchnic supply is intact; in D the mesenteric supply is intact; and in E the intermesenteric supply is intact.

CSMG or the appropriate nerve trunks were rapidly dissected out and immersed in 10% formalin with 0.4% picric acid in 0.16 M sodium phosphate bufferlo for 90 min. The specimens were transferred to a phosphate-buffered 15% sucrose solution and stored overnight at 4°C. They were cut on a cryostat (Dittes, Heidelberg, F.R.G.) at 14pm and mounted on slides. The sections were processed for indirect immunohist~hemistry according to Coons and collaborators.’ In brief, the sections were incubate in a humid atmosphere at 4°C for 24 h with polyclonal antisera raised in rabbits towards Met-El%@ (dilution I : 200), SP (dilution 1:200; Emson, unpublished observations), CGRP (dilution I :400; Peninsula, Belmont, U.S.A.), VIP or PH12’~24~25~~6 (dilution 1:400),bombesins6 (dilution I : 200). dynorphir? (dilution 1:400), and CCK(l-8) (will be referred to as CCK-8 antiserum**; dilution 1: 400). A second rabbit antiserum for analysis of CCK systems was also used. This antiserum was raised against porcine CCK(l-33) and reacts approximately with amino acids 17-23. This will be referred to as CCK-Mid antiserum.83 Polyctonal antiserum raised in sheep towards NPY4 (dilution I: 100) was also used. Monoclonal antibodies to somatostatin’ (dilution 1: IOO), SP” (dilution 1: 10) and CCK (dilution 1:800, antibody 28.2 directed against midportion of CCK(I-8), but not seeing the C-terminal amino acid of CCK(I-8)“) were also used.

Peptide fibers in the celiac superior mesenteric ganglion After rinsing, the sections were incubated at 37°C for 30 min with green fluorescent fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit antibodies (Dakopatts, Copenhagen, Denmark; dilution 1: 10) or, alternatively, FIT&conjugated goat anti-rabbit antibodies (Boeringer Mannheim Scandinavia, Stockholm, Sweden; dilution 1:40) for rabbit antisera. Red fluorescent tetramethylrhodamine isothiocyanate (TRITC)-conjugated swine anti-rabbit antibodies (Dakopatts; dilution 1: 10) and TRITCconjugated goat anti-rabbit antibodies (Boeringer, dilution 1:40) were also used for rabbit antisera. FITC-conjugated donkey anti-goat antibodies (Nordic, Tilburg, The Netherlands; dilution 1: 10) were used: for sheep antisera. In the case of monoclonal antibodies, the following secondary antisera were used: for somatostatin, FITCconjugated sheep anti-mouse antibodies (Amersham, Dagenham, U.K.; dilution I : 10); for SP, TRITC-conjugated goat anti-rat antibodies (American Qualex, Miranda, California, U.S.A.; dilution 1: IO); and for CCK, FITCconjugated goat anti-mouse antibodies (Boeringer; dilution 1:40). Experiments with simultaneous staining (see Refs 80 and 100) of two antigens with antisera raised in different species were also performed. The following combinations of antigens were analysed in the double-staining experiments: somatostatin/CCK, somatostatin/PHI, somatostatimbombesin, somatostatin/dynorphin, NPY/CCK, NPY/PHI, NPY/bombesin. NPYidvnorphin. CCK/SP. CCK/CGRP, CCK/PHI, CCK/bomb&in, CCK/dynorphin and SP/CGRP. Combinations of appropriate secondary antibodies described above labeled with different fluorophores were used to visualize the primary antibodies. All sera used in this study contained 0.3% Triton X-100.” All sections were examined in a Zeiss fluorescence microscope equipped with a dark-field oil condenser. To visualize the FITC-induced fluorescence, a Schott KP 500 excitation filter and an LP 520 stop filter were used. For the TRITCinduced fluorescence, a Schott BP 546 excitation filter and an LP 590 stop filter were used. For photography, Kodak Tri-X black-and-white film (Rochester, N.Y., U.S.A.) was used. For control purposes all antisera were preabsorbed with the respective peptide for 2 h at room temperature or for 24 h at 4°C. All peptides were purchased from Peninsula. In addition, cross-absorption experiments were carried out in some cases. For details, see Results. RESULTS Enkephalin-like

immunoreactivity

In the unoperated strongly fluorescent

animals (Fig. 1A) patches of ENK-immunoreactive nerve

fibers were found after incubation with the [Met]enkephalin antiserum 336 (Fig. 2A). These ENKpositive fibers did not seem to have any preferred localization, with the patches principally found in all parts of the ganglion often surrounding single cells. In addition, a dense network of medium strongly to weakly fluorescent fibers was seen. The latter plexus consisting of fine varicose fibers was almost exclusively confined to the superior mesenteric pole (SMP) of the ganglion (Fig. 2A). Analysis of adjacent sections revealed that the SMP mainly contained somatostatin-positive cells, as compared to the NPYdominated celiac poles (CP) (cf. Lindh et aL6’). No ENK-immunoreactive principal ganglion ceils were seen, whereas many of the small intensely fluorescent (SIF) cells were ENK-positive (Fig. 2A).

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After total denervation (Fig. 1B) virtually no ENK-positive nerve fibers were observed in the ganglion. When the mesenteric and intermesenteric nerves were cut, leaving the thoracic splanchnic nerves intact (Fig. lC), a strongly fluorescent ENK-immunoreactive nerve fiber network with a patchy distribution similar to the one seen in the unoperated animal was observed (Fig. SA), whereas most of the weakly fluorescent fibers in the SMP pole had disappeared (Fig. SB). When the thoracic splanchnic nerves were crushed, accumulation of ENK-immunoreactive material was seen mainly central (spinal cord side) to the compression (Fig. 6A). In cases where only the mesenteric nerves were left intact (Fig. ID), only the weakly fluorescent, dense nerve fiber network in the SMP pole was seen (Fig. 7A). In fact, the ENK immunoreactivity in these fibers seemed to increase after these transections. Occasional, strongly fluorescent ENK-positive nerve fibers could be observed (Fig. 7A). Ligation of the mesenteric nerves in animals where the thoracic splanchnic nerves had been cut 7 days previously, resulted in an accumulation of ENK-immunoreactive material distal (intestinal side) to the ligature (Fig. 9A). When the supply from the intermesenteric nerve was studied (Fig. lE), some ENK-immunoreactive nerve fibers were observed in the SMP (Fig. 10A) and occasional fibers in the CP. When the intermesenteric nerve was crushed, ENK-LI could be observed to accumulate on both sides of the compression (Fig. 11A). Substance

P-like

immunoreactivity

In the unoperated animals a moderately dense network of varicose SP-positive fibers was seen in the CSMG (Fig. 3A). Although the SP-containing fibers were found in all parts of the ganglion, they seemed to be somewhat more prevalent in the SMP (Fig. 3A). Furthermore, many non-varicose SP-positive nerve fibers were seen in bundles, particularly in the CP, close to the entrance sites of the theracic splanchnic nerves (Fig. 3A). Double-staining experiments revealed that many of the SP-positive fibers and terminals also contained CGRP-LI (cf. Fig. 12A and B). No SP-immunoreactive principal ganglion cells were seen, whereas several of the SIF cells were SP-positive (Fig. 3A). Some of the SP-positive SIFcells also contained CGRP-LI. After total denervation (Fig. 1B) only occasional SP-positive nerve fibers could be observed in the ganglion. Cutting the mesenteric and intermesenteric nerves, leaving the thoracic splanchnic nerves intact (Fig. lC), resulted in an accumulation of SP-LI in fiber bundles within the ganglion (Fig. 5D). Varicose SP-positive nerve fibers surrounding the principal ganglion cells were also observed with a distribution similar to that seen in the unoperated animals (cf. Figs 3A and 5D). Crushing the thoracic splanchnic nerves resulted in an accumulation of SP-immunoreactive material proximally (spinal cord side)

Fig. 2. Immunofluorescence micrographs (montages) of sections of the CSMG of an unoperated animal after incubation with antisera to ENK (A) and CCK (CCK-Mid-antiserum (B) and CCK-8-antiserum (C)). B and C show two consecutive sections. In this figure and in Figs 3-5, 7 and 8 the superior mesenteric pole (SMP) represents the lower half of the micrographs, whereas one of the celiac poles (CP) can be seen at the top. In A, patches of strongly ENK-positive fibers can be observed in all parts of the ganglion (arrows). In addition, a plexus of fine varicose fibers of a somewhat weaker fluorescence can-also be distinguished (arrowheads) in A. This plexus is mainly confined to the SMP of the ganglion. Double arrows point to ENK-immunoreactive &F-cells (A). The two dense CCK-positive nerve fiber networks have a similar distribution and are mainly confined to the SMP of the CSMG (compare B and C). In C, a sparse network of weakly fluorescent nerve fibers is seen also in the CP. Bars = 50 pm. 1040

Peptide fibers in the celiac superior mesenteric ganglion Fig. 3. Immunofluorescence micrographs (montages) of consecutive sections of the CSMG of an unoperated animal after incubation with antiserum to SP (A), CGRP (B) and somatostatin (SOM) (C). Varicose SP- (A} and CGRP- (B) positive fibers are found both in the SMP and the CP of the CSMG. The SP- (A) and CGRP- (B) positive fibers seem to be somewhat more prevalent in the SMP. Many non-varicose SP- (A) and CGRP- (B) positive fibers can be seen in bundles (arrowheads), particularly in the CP, where the thoracic splanchnic nerves enter the ganglion. Double arrows point to SP-immunoreactive SIF-cells (A). The somatostati~-positive cell bodies are mainly found in the SMP of the CSMG (C). Bars = 50 pm. Fig. 4. Immunofluorescence micrographs (montages) of consecutive sections of the CSMG of an unoperated animal after incubation with antiserum to CCK (CCK-Mid antiserum) (A), PHI (B), bombesin (BOM) (C) and dynorphin (DYN) (D). Sections in A-D are semiconsecutive (every two or three sections) to the sections seen in Fig. 3A-C. Dense overlapping networks of CCK- (A), PHI/VIP- (B), bombesin(C) and dynorphin- (D) positive fibers can be seen, mainly confined to the SMP of the ganglion. Sparse ~pt~de-~mmunor~ctive nerve fiber networks can be distinguished in the CP (A-D). Non-varicose bombesin-positive fibers can be seen in bundles (C) particularly in the CP {arrowheads}. In D a double arrow points to dynorphin-positive SIF-cells. Bars = SOpm. Fig. 5. Immunofluorescence micrographs (D, E and F are montages) of sections of the CSMG of an operated animal (only thoracic splanchnic supply intact) after incubation with antisera to ENK (A and 3), PHI (C and F), SP (D) and CGRP (E). Micrographs D and E show two consecutive sections, whereas the section seen in F is semiconsecutive to D and E. Strongiy fluorescent ENK-positive fibers are seen (A) with a patchy distribution similar to the one seen in the unoperated animal (cf. A with Fig. 2A), Also some more weakly fluorescent fibers are observed (B). A sparse network of PHI/VIP-positive fibers can be observed in the SMP (C and F). Some PHI/VIP fibers can also be seen in the CP (F). In the sections incubated with SP- and CGRP-antiserum (D and E) accumulation of immunareactivity is seen in fiber bundles (arrowheads) in the inner parts of the ganglion. Varicose SP-positive fibers with a similar distribution to the one seen in the unoperated animal are also observed (cf. D with Fig. 3A). The varicose CGRP-positive nerve fiber network within the SMP appears to be less dense as compared to the unoperated animal (cf. E with Fig. 3B). Bars = 50pm. Fig. 6. Immunofluorescence micrographs of sections of a crushed thoracic splanchnic nerve (A-N) of the guinea-pig (arrowhead in A points towards the CSMG) after incubation with antisera to ENK (A), SP (B), CGRP (C), CCK (CCK-8 antiserum) (D), PHI (E), bombesin (BOM) (F), dynorphin (DYN) (G) and somatostatin (SON) (H). In the crushed nerve accumulations of ENK- (A), SF- (B), CGRP- (C), CCK(D), PHI/VIP- (E), bombesin- (F) and dynorphin- (G) LE are seen on the proximal side (spinal cord side) of the compression. On the distal side (CSMG side) of the crush, a few fibers containing immunor~ctivity to SP (B), CGRP fC) and CCK (D) can be seen (arrows). Whether these latter a~umulations are due to retrograde accumulation or to anterograde accumulation in fibers with cell bodies located distal to the crush cannot be established with the present techniques. The main accumulation of somatostatin-LI (H) occurs on the distal (CSMG) side (double arrowheads point to site of crush). Bars = $0 ,~m. Fig. 7. Immunofluorescence micrographs (montages) of sections of the CSMG of an operated animal (only mesenteric supply intact) after incubation with antisera to ENK (A), SP (B), CGRP (C) and PHI (D). Sections seen in A-D and in Fig. 8A and B are semiconsecutive. A network of weakly fluorescent ENK-positive nerve fibers is seen, mainly confined to the SMP (A). Occasional, strongly fluorescent ENK-positive fibers can he observed (arrows) in A. Many of the SIF-cells contain ENK-LI (double arrows) (A). Some SP-positive fibers (arrows) are found in the SMP of the CSMG (B). A network of CGRP-positive nerve fibers can be observed in the SMP (C). A dense network of PHI/VIP-positive fibers is seen in the SMP (D). These PHI-VIP-positive fibers have a similar distribution to that in the unoperated animal (cf. D with Fig. 4B). Bars = 50 pm. Fig. 8. Immunofl~~r~en~ micrographs (montages) of sections of the CSMG of two operated animals (only mesenteric supply intact in A and B and only intermesenteric supply intact in C and D) incubated with antisera to CCK (CCK-Mid antiserum in A and CCK-8 antiserum in B), CGRP (C) and PHI (D). Micrographs A and B show consecutive sections. These sections are semiconsecutive to the sections seen in Fig. 7A-D. A dense network of CCK-positive nerve fibers is observed in the SMP of the CSMG with the CCK-Mid antiserum (A). A more strongly fluorescent network with a similar distribution can be seen after incubation with the CCK-8 antiserum (B). When the intermesenteric supply is intact (C and D), a sparse CGRP-positive nerve fiber network mainly confined to the SMP can be seen (C). After incubation with PHI-antiserum a sparse network of PHI/VIP-~siti~e nerve fibers is present in the SMP (D). A few PHI~VIP-immuno~active fibers can also be seen in the CP (D). 3ars = 50pm. Fig. 9. Immunofluorescence micrographs of sections of ligated mesenteric nerves (A-H) (the thoracic splanchnic nerves were cut 7 days before the ligation experiment) of the guinea-pig (arrowhead in A points toward the CSMG) after incubation with antisera to ENK (A), SP (B), CGRP (C), CCK (CCK-Mid antiserum) 0, PHI {E), bon&sin (BOM) (F), dynorphin (DYN) (G) and somatostatin (SOM) (H). Accumulation of ENK-LI can be seen on the distal (intestinal) side (A). Accumulation of SP- (B) and of CGRP- (C) I.J is observed on the proximal {CSMG) side with some SP- (8) and many CGRP- (C) ~mrnuno~a~tive fibers on the distal (intestinal) side. A heavy accumulation of CCK- (D)+ PHI/VIP- (E), bombesin- (F) and dynorphin- (G) LI is present on the distal (intestinal) side. A few CCK- (D) and some PHI/VIP- (E) and bombesin- (F) immunoreactive fibers can be seen on the proximal (CSMG) side of the ligation. A heavy accumulation of dynorphin-LI can be observed on the proximal (CSMG) side (G). Somatostatin-LI can be Seen to accumulate on both sides of the ligation with most of the immunoreactive material on the proximal (CSMG) side (H). Bars = 5Opm.

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Fig. 3 1042

Fig. 4. 1043

Fig. 5

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Fig. 6. NSC26,3--K

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Fig. 7 1046

Fig. 8. 1047

Fig. 9 1048

Peptide fibers in the celiac superior mesenteric ganglion

Fig. 10. Immunofluorescence micrographs of sections of the CSMG (SMP pole) of an operated animal (only intermesenteric supply intact)8Rer incubation with antisera to.ENKfA); SP (B), ?ZGRP (C and HI. CCK (CCK-Mid antiserum in D and monoclonal CCK-antiserum in G. I and KI. PHI (El. dvnorohin (DYN) (F-and L) and bombesin (BOM) (J). Micrographs G-L am taken from doubl~~staini~~‘~x~~~ents and G and H, I and J, and K and L, respectively, show the same section. In G arrows point to CCK-positive varicosities also containing CGRP-LI (H). In I CCK-~mmunoreactive terminals (arrows) also containing bombesin-Ll (J) are indicated. In K, arrows point to C~K~ontaining nerves which are also immunoreactive to dyno~hin-LI (L). Bars = 50 pm. Fig. I I. Immunofluorescence micrographs of sections of a crushed intermesenteric nerve (A-H) of the guinea-pig (arrowhead in A points towards the CSMG) after incubation with antisera to ENK (A), SP (B), CGRP (C), CCK (CCK-Mid antiserum) (D), PHI (E), bombesin (BOM) (F), dynorphin (DYN) (G) and somatostatin (SON) (H). With all antisera accumulation of immunoreactive materials can be observed on both sides of the compression. In the case of ENK- (A), CCK- (D) and bombesin-11 (F) the heaviest accumulation is seen on the distal (IMG) side of the compression, whereas in the case of SP- (B), CGRP(C) and somatostatin- (H) LI most of the immunoreactive materials accumulate on the proximal (CSMG) side. Bars = 50 pm.

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f.ig IO. 1050

II. 1051

B. hiDH

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(Fig. 6B). When the ganglion supply from the mesenteric nerves was studied by cutting the thoracic splanchnic and intermesenteric nerves (Fig. lD), some varicose SP-containing nerve fibers were observed in the SMP pole of the ganglion (Fig. 7B). Some of these SP-positive nerves were found to surround the principal ganglion cells. Ligation of the mesenteric nerves resulted in accumulation of SP-LI proximal (ganglion side) to the ligation (Fig. 9B). Some SP-immunoreactive material was also accumulated on the distal side (intestinal side). Cutting the thoracic splanchnic and mesenteric nerves, leaving the intermesenteric nerve intact (Fig. lE), resulted in an almost total depletion of SP-immunoreactivity in the CSMG with a few SP-positive fibers in the SMP (Fig. 10B) and only occasional fibers in the CP. When the intermesenteric nerve was crushed SP-LI was found on both sides of the compression with most of the SP-immunoreactive material proximally, i.e. on the CSMG side (Fig. 11B). Calcitonin gene-related

peptide-like

immunoreactivity

In the unoperated animals a moderately dense network of varicose CGRP-positive nerve fibers was observed (Fig. 3B). The CGRP-positive nerve fibers and terminals were found in all parts of the ganglion but they seemed to be more prevalent in the SMP. Many non-varicose CGRP-containing fibers were seen in bundles, particularly in the CP (Fig. 3B). Many CGRP-immunoreactive nerve fibers and terminals also contained SP-LI, as revealed with double staining (cf. above). Double staining revealed that the CGRP- and CCK-immunoreactivity in the SMP of the ganglion in some cases appeared to coexist (cf. Fig. 12C and D). No CGRP-immunoreactive neuronal cell bodies were seen, but a few SIF-cells were CGRP-positive. After total denervation (Fig. 1B) only occasional CGRP-positive nerve fibers were observed in the ganglion. Cutting the mesenteric and intermesenteric nerves, leaving the thoracic splanchnic nerves intact (Fig. lC), resulted only in minor changes, with accumulation of CGRP-immunoreactive material in non-varicose fiber bundles (Fig. 5E) and a less dense fiber network within the SMP as compared to the unoperated animals (cf. Figs 3B and 5E). Crushing the thoracic splanchnic nerves resulted in accumulation of CGRP-LI proximally (spinal cord side) (Fig. 6C). When the supply from the mesenteric nerves was studied by cutting the thoracic splanchnic and intermesenteric nerves (Fig. lD), a network of varicose CGRP-positive fibers was still observed in the SMP (Fig. 7C). Double-staining experiments revealed that these CGRP-positive nerve fibers contained CCK-LI. Ligation of the mesenteric nerves resulted in an accumulation of CGRP-LI both proximal and distal to the ligation (Fig. 9C). In cases where only the intermesenteric nerve was left intact (Fig. 1E) a sparse network of varicose CGRP-positive nerve fibers was seen in the SMP of the ganglion

et

al.

(Figs 8C, 1OC and H). A few CGRP-immunoreactive fibers were also seen in the CP (Fig. 8C). When the intermesenteric nerve was crushed, CGRPimmunoreactive material was accumulated on both sides of the compression (Fig. 11C). Cholecystokinin-like

immunoreactivity

In the unoperated animals a very dense network of varicose CCK-positive fibers was seen (Figs 2B, C and 4A) confined to the SMP of the ganglion. Networks with a similar distribution were observed with both the antisera used, which were directed, respectively, to the C-terminal (CCK-8 antiserum) and mid-portion (CCK-Mid antiserum) of CCK( l-33). In addition, a sparse, weakly fluorescent fiber network was seen in the CP, which was less distinct with the mid-portion directed antiserum. Double-staining experiments revealed that in the SMP the CCK-positive nerve fibers were mainly located around the somatostatin-positive cell bodies (cf. Fig. 13A and B), although some CCK-positive nerves also surrounded somatostatin-negative ganglion cells. Double-staining experiments in the SMP showed most of the CCK-positive fibers to be present around the NPY-negative cell bodies. Only occasional NPY-positive cells were surrounded by CCKcontaining nerves (cf. Fig. 14A and B). Double staining with antisera to CCK combined with CGRP, VIP/PHI, bombesin or dynorphin, respectively, showed that at least some of the CCK-positive fibers appeared to also contain immunoreactivity to each of these peptides (cf. Fig. 12D, E, G and I with 12C, F, H and J, respectively). The density of these fiber networks, however, made a close analysis difficult. No evidence for coexistence of CCK- and SP-LI was obtained (cf. Fig. 12K and L). No CCK-LI was seen in the principal ganglion cells or in the SIF-cells. After total denervation (Fig. 1B) almost no CCKpositive fibers were seen. When only the thoracic splanchnic nerves were left intact (Fig. lC), the ganglion contained a sparse network with most fibers in the SMP, and this network was best seen with CCK-8 antiserum. A marked accumulation of CCKimmunoreactive material could be observed on the central side of the crushed thoracic splanchnic nerves with both the CCK-8 antiserum (Fig. 6D) and the CCK-Mid antiserum. Cutting the thoracic splanchnic and intermesenteric nerves, leaving the mesenteric nerves intact (Fig. lD), resulted in a similar distribution of the dense CCK network in the SMP to that seen in the unoperated animals with both antisera (cf. Figs 8A and B with 2B and C respectively). Ligation of the mesenteric nerves resulted in a heavy accumulation of CCK-8- and CCK-Mid-L1 distally (intestinal side) (Fig. 9D). In cases where only the intermesenteric nerve was left intact (Fig. IE), a sparse network of CCK-positive nerve fibers was seen in the SMP (Fig. lOD, G, I and K) with a few fibers in the CP. Double-staining experiments revealed that in these animals the CCK-positive fibers in some cases

Peptide fibers in the celiac superior mesenteric ganglion

Fig. 12. Immunofluorescence micrographs of sections of the CSMG of an unoperated animal after in&bation with antisera to SP (mo&&nal antiserum in A and polyclonal antiserum in L), CGRP (B and C). CCK (monoclonal antiserum) (D. E. G. I and K). PHI IF). bombesin (BOM) (H) and dvnorohin (DYN) (J). Ali micrographs (A-L) are‘taken from double-staining experiments, and A and B, C and D, E and F, G and H, I and J, and K and L, respectively, show the same section. In A, arrows point to SP-positive fibers also containing CGRP-LI (B). Arrowheads indicate a dense CGRP-positive nerve fiber network (B) which lacks SP-LI (A). In C, arrows indicate CGRP-positive varicosities also containing CCK-LI (D). Arrows in E point to CCK-positive terminals which also contain PHI/VIP-L1 (F). In G, arrows indicate CCK-containing varicosities which are also bombesin-immunoreactive (H). In I, CCK-positive terminals (arrows) also containing dynorphin-11 (J) are indicated. Coexistence of CCK- (K) and SP- (L) LI in nerve fibers cannot be established. Bars = 50pm. Fig. 13. Immunofluorescence micrographs of sections of the CSMG of an unoperated animal after incubation with antisera to somatostatin (SOM) (A, C, E and G), CCK (CCK-Mid antiserum) (B), PHI (D), bombesin (BOM) (F) and dynorphin (DYN) (H). Micrographs A-H are taken from double-staining experiments, and A and B, C and D, E and F, and G and H, respectively, show the same section. In A, arrows point to somatostatin-positive cells which are surrounded by CCK-positive fibers (B). Some somatostatin-negative cells (arrowheads) (A) are also surrounded by CCK-containing fibers (B). In C, somatostatin-positive (arrows) and somatostatin-negative (arrowhead) cells are surrounded by PHI/VIPimmunoreactive fibers (D). Somatostatin-positive (arrows) and somatostatin-negative (arrowheads) cells (E) can be observed with bombesin-containing fibers distributed around them (F). In G are indicated somatostatin-positive (arrows) and somatostatin-negative (arrowheads) cells surrounded by dynorphincontaining fibers (H). Bars = 50 pm. Fig. 14. Immunofluorescence micrographs of sections of the CSMG of an unoperated animal after incubation with antisera to NPY (A, C, E and G), CCK (CCK-Mid antiserum) (B), PHI (D), bombesin (BOM) (F) and dynorphin (DYN) (H). Micrographs A-H are taken from double-staining experiments, and A and B, C and D, E and F, and G and H, respectively, show the same section. This plate shows rare examples, where intestinal afferents can be seen around NPY-positive cells in the SMP. In A, an NPY-positive cell (arrow) is partly surrounded by CCK-positive fibers (B). The CCK-positive fibers (B) are mainly found around NPY-negative cells (arrowheads) (A). In C, arrows point to two NPY-positive cells which are near some PHI/VIP-immunoreactive fibers (D). Most of the PHI/VIP-immunoreactive fibers (D) are found around NPY-negative cells (arrowheads) (C). In E, two NPY-containing cells (arrows) are surrounded by bombesin-immunoreactive fibers (F). The bombesin-containing fibers (F) are generally found around NPY-negative cells (arrowheads) (E). In G. the arrow ooints to an NPY-oositive cell surrounded by dynorphin-positive fibers (H). Most’of the dynorphin-containing fibers (H) can be seen around NPY-negative cells (arrowheads) (G). Bars = 50 pm. Fig. 15. Immunofluorescence micrographs of sections of thoracic dorsal root ganglia of the guinea-pig after incubation with antisera to CGRP (A-C) and CCK (D-1) (CCK-8 antiserum in D and E and CCK-Mid antiserum in F-I). CGRP antiserum (B), CCK-8 antiserum (E) and CCK-Mid antiserum (I) have all been pretreated with an excess of CGRP peptide. CGRP antiserum (C) and CCK-Mid antiserum (H) have both been preabsorbed with CCK(l-8). CCK-Mid antiserum (G) has been pretreated with CCK (l-33). In the dorsal root ganglia CGRP-LI is observed in cell bodies and fibers (A). The CGRP staining is completely abolished by CGRP peptide (IO-‘M) (B) but not with CCK(l-8) (10m4M) (C). After incubation with CCK-8 antiserum CCK-immunoreactive cell bodies and fibers can be seen (D). The CCK-8 staining diminishes after preabsorption with CGRP peptide (10m4M) (E). The staining observed after incubation with CCK-Mid antiserum (F) is abolished by preabsorption with CCK(l-33) (lo-’ M) (G) but not with CCK(l-8) (10d4 M) (H) or CGRP peptide (10e4 M) (I). Bars = 50 pm. Fig. 16. Immunofluorescence micrographs of sections of the CSMG of the guinea-pig after incubation with antisera to CGRP (A and B) and CCK (C-I) (CCK-8 antiserum in C-E and CCK-Mid antiserum in F-I). CGRP antiserum(B), CCK-8 antiserum (E) and CCK-Mid antiserum (I) were all pretreated with an excess of CGRP. CCK-8 antiserum (D) and CCK-Mid antiserum (H) were both preabsorbed with CCK( l-8). CCK-Mid antiserum (G) was pretreated with CCK(l-33). Many CGRP-positive nerve fibers are seen in the CSMG (A). The CGRP-staining is completely abolished after pretreatment with CGRP peptide (IO-‘M) (B). The staining with CCK-8 antiserum (C) is abolished by preabsorption with CCK(I-8) (10e6 M) (D) and is diminished after pretreatment with CGRP peptide (10e4 M) (E). The CCK-Mid positive fiber staining (F) is abolished by preabsorption with CCK(l-33) (IO-‘M) (G) but not with CCK(l-8) (10e4 M) (H) or CGRP peptide (lO-4 M) (I). Bars = 50pm.

NSC 26,3--K’

1053

Fig 12. 1054

Fig. 13 1055

Fig

14.

1056

Fig. 15. 1057

Fig. 16 1058

Peptide fibers in the celiac superior mesenteric ganglion

Fig. 17. Immuno~uor~n~ micrographs of sections of the dorsat horn of the thoracic spinal cord of the guinea-pig after incubation with antisera to CGRP (A) and CCK (3 and C) (CCK-8 antiserum in B and CCK-Mid antiserum in C). A dense plexus of CGRP-positive fibers can be seen mainly confined to the superticial laminae but also in deeper layers of the dorsal horn (A). In sections incubated with CCK-8 antiserum (B) a plexus with a similar distribution can be observed, but in addition a network of fine distinct fluorescent fibers can be seen in all parts of the dorsal horn but mainly in layers III-V. A similar CCK-positive plexus can be seen after incubation with CCK-Mid antiserum (C). Bars = 50 pm.

1059

1060

8. LINDH et al.

also contained CGRP- (cf. Fig. 10G and H), VIP/ PHI-, bombesin- (cf. Fig. 101 and J) and dyno~hinLI (cf. Fig. 1OK and L). When the intermesenteric nerve was crushed, CCK-8- and CCK-Mid-immunoreactive material was accumulated on both sides of the compression, with most of the CCK-LI found on the distal side (IMG side) (Fig. 1ID). Vasoactive intestinal polypeptide-/peptide isoleucine-like immunoreactivity

histidine

In the unoperated animals a very dense network of VIP/PHI-positive fibers was observed in the SMP of the ganglion (Fig. 4B). In additon, a sparse VIP/PHIimmunoreactive nerve fiber network was seen in the CP. Double-staining experiments with antisera to VIP/PHI and somatostatin showed that in the SMP VIP/PHI-containing nerve fibers surrounded not only the somatostatin-positive cell bodies (cf. Fig. 13C and Df but also a number of somatostatin-negative ganglion cells. Furthermore the VIP/PHI-immunoreactive fibers mainly surrounded NPY-negative and only occasionally NPY-positive cells (cf. Fig. 14C and D). In addition to the VIP/PHI-positive fibers, a small number of VIP/PHI-immunoreactive cell bodies was observed bilaterally in the CP of the ganglion. Double-staining experiments revealed that many of these VIP/PHI-containing cells also contained NPY-LI and somatostatin-11. The SIF-cells appeared to be VIP/PHI-negative. After total denervation (Fig. 1B) an almost complete depletion of VIP/PHI-L1 in fibers was seen, but the VIP/PHI-positive cells in the celiac poles appeared somewhat more strongly fluorescent than in controls. When only the thoracic splanchnic nerves were left intact (Fig. lC), a sparse network of VIPfPHI positive fibers could be observed in the SMP and to a lesser extent in the CP (Fig. SC and F). Crushing the thoracic splanchnic nerves resulted in an accumulation of VIP/PHI-immunoreactive material proximally (spinal cord side) (Fig. 6E). When only the mesenteric nerves were left intact (Fig. lD), a similarly dense network of VIP/PHI-immunoreactive fibers to that in the non-dene~ated animals was observed in the SMP (Fig. 7D). Ligation of the mesenteric nerves resulted in a ‘,heavy accumulation of VIP/PHI-immunoreactive material distal to the ligature (intestinal side) (Fig. 9E). Some VIP/PHI-L1 was also a~umulated proximally (ganglion side) (Fig. 9Ef. In cases where only the intermesenteric nerve was left intact (Fig. lE), a sparse network of VIP/PHI-positive nerve fibers was seen in the SMP (Figs 8D and 1OE) of the ganglion with some fibers also in the CP (Fig. 8D). When the intermesenteric nerve was crushed, VIP/PHI-immunoreactive material was accumulated on both sides of the compression (Fig. I 1E). Bombesin -like immunoreactivity

In the unoperated animals a very dense network of varicose bombesin-containing nerve fibers was

seen, in principal in the SMP of the ganglion with low numbers of fibers in the CP. The latter part in addition contained strongly immunoreactive fiber bundles (Fig. 4C). Double-staining experiments showed that the bombesin-containing nerve fibers in the SMP were mainly dist~buted around the somatostatin-positive cell bodies (cf. Fig. 13E and F) but also around some somatostatin-negative ganglion cells, and that most of the bombesin-positive nerves were present around NPY-negative cells and only occasionally did they lie around NPY-positive cells {cf. Fig. 14E and F). No bombesin-positive principal ganglion cells could be observed, and the SIF-cells appeared to be ~rn~sin-negative. After total denervation (Fig. lB), almost no bombesin-immunoreactive nerve fibers could be observed in the ganglion. In cases where only the thoracic splanchnic nerves were left intact (Fig. IC), a sparse network of bom~sin-positive fibers could be observed in the CP and somewhat higher numbers in SMP. In these cases immunoreactive material could also be observed in fiber bundles. Crushing the thoracic splanchni~ nerves resulted in an accumulation of bombesin-11 proximally (spinal cord side) (Fig. 6F). When only the mesenteric nerves were left intact (Fig. ID), a dense network with bombesin-LI was seen in the SMP, similar to that seen in intact animals. Ligation of the mesenteric nerves resulted in a heavy accumulation of bombesin-immunoreactive material distally (intestinal side) (Fig. 9F) and some proximally (Fig. 9F). In cases where only the intermesenteric nerve was left intact (Fig. lE), a sparse network of bombesin-containing fibers was observed in the SMP (Fig. 1OJ) and a few fibers in the CP. When the intermesenteric nerve was crushed, bombesin-immunorea~tive material was accumulated on both sides of the compression with most of the bombesin-11 found on the distal side (IMG side) (Fig. 11F). Dynorphin -like ~munoreactivity

In the unoperated animals a very dense network of dynorphin-positive fibers was seen in the SMP of the ganglion with low numbers of fibers in the CP (Fig. 4D). In double-staining experiments it was revealed that in the SMP the dynorphin-containing nerve fibers surrounded mainly the somatostatinpositive cell bodies (cf. Fig. 13G and H) but also somatostatin-negative ganglion cells, and that the dynorphin-positive fibers surrounded mainly the NPY-negative and only occasionally NPY-positive cells (cf. Fig. 14G and H). Occasional dynorphinpositive principal ganglion cells were observed in the CP of the ganglion. In addition, many dynorphincontaining SIF-cells were found (Fig. 4D). After total denervation (Fig. IBf, almost no dynorphin-containing fibers were seen in the CSMG. In cases where only the thoracic splanchnic nerves were left intact (Fig. lC), dynorphin-positive fibers were seen only in low numbers in CP and somewhat more

1061

Peptide fibers in the celiac superior mesenteric ganglion frequently in SMP. Crushing the thoracic splanchnic nerves resulted in an accumulation of dynorphinimmunoreactive material proximally (spinal cord side) (Fig. 6G). When the thoracic splanchnic and intermesenteric nerves were cut, leaving the mesenteric nerves intact (Fig. lD), a dense dynorphin-

positive nerve fiber network could be observed in the SMP similar to the one seen in the unoperated animals. Ligation of the mesenteric nerves resulted in a heavy accumulation of dynorphin-immunoreactive material both proximal and distal to the ligature (Fig. 9G). In cases where only the intermesenteric nerve was left intact (Fig. lE), a sparse network of dynorphin-positive nerve fibers was observed in the SMP (Fig. 10F and L) of the ganglion and a few fibers in the CP. When the intermesenteric nerve was crushed, dynorphin-immunoreactive material was accumulated on both sides of the compression with most of the immunoreactive material distally (IMG side) (Fig. 11G).

Somatostatin-like

immunoreactivity

For reference, adjacent sections were always incubated with somatostatin antiserum. As described previously (Lindh et aL6’), somatostatin-positive cells mainly occupied the SMP of the ganglion (Fig. 3C). Somatostatin-LI accumulated mainly on the CSMG side after crushing the thoracic splanchnic nerves (Fig. 6H). After ligation of the mesenteric nerves most somatostatin-11 was observed on the CSMG side, but several fibers also showed accumulation on the intestinal side (Fig. 9H). After crushing the intermesenteric nerve, somatostatin-11 accumulated on both sides with the heaviest immunoreactivity on the CSMG side (Fig. 11H).

mostly at lo-’ M (see also Table 1). An exception was CCK-8 antiserum, where some fibers remained after preabsorption with lo-’ M CCK(l-8) (Table 1). The results from the analysis of the spinal cord, spinal ganglia and CSMG for establishing the identity of the CCK peptides are summarized in Table 1. Briefly, CGRP staining (Figs 15A, 16A and 17A) was abolished by CGRP peptide at lo-’ M (Figs 15B and 16B), but in addition very high concentrations (10m4M) of CCK(l-8) peptide reduced the staining somewhat (Fig. 15C) (Table 1). CCK-8 staining (Figs 15D, 16C and 17B) was abolished by CCK( 1-8) (Fig. 16D) and CCK(l-33) peptides, but was also diminished by CGRP peptide at 10m4M (Figs 15E and 16E) and also to a lesser extent by 10e5 M. This effect was seen in both CSMG and spinal cord and was most pronounced in the dorsal root ganglia (Table 1). The staining seen after incubation with the CCK-Mid antiserum (Figs 15F, 16F and 17C) was abolished by preabsorption with CCK(l-33) (Figs 15G and 16G) but not with CCK(l-8) (Figs 15H and 16H) or CGRP peptide (Figs 151 and 161). Finally, the bombesin staining was completely abolished by bombesin peptide at lo-‘M, but was also affected by SP peptide at 10m4M and 10m5M, both with regard to the dense fibers in the SMP, the sparse network in CP and the accumulation of bombesin-11 in the thoracic splanchnic nerve after crushing. DISCUSSION

The present results confirm and extend earlier findings on the chemical anatomy of the cell bodies and fibers in the CSMG of the guinea-pig. Thus, it is now established that the noradrenergic principal ganglion cells can be divided into subpopulations on the basis of a coexisting peptide and that these populations occupy specific domains within the

Absorption experiments The staining patterns observed above in the CSMG were completely abolished for all antisera after incubation with the appropriate peptide at 10m6M and

Table 1. The effects of preabsorption of calcitonin gene-related peptide- and cholecystokinin-antisera gene-related peptide and cholecystokin peptides Dorsal horn 4+ Antiserum Preabsorption CGRP IO-’ 1o-6 lO-5 1O-4 CCK (1-8) IO-’ lO-6 1o-5 lO-4 CCK (l-33) IO-’ 1O-6 10-5 1o-4

CSMG 4+

Dorsal root ganglia 4+

CGRP

CCK-8

CCK-Mid

CGRP

CCK-8

CCK-Mid

0 0 0 0

4 4 3 2

4 4 4 4

0

0 0 0

4 2 *;2

4 4 4 4

4 4 4 3

1 0 0 0

4 4 4 4

4 4 4 3

l/2 0 0 0

with calcitonin

CGRP

CCK-8

CCK-Mid

4 4 3 2

4 4 4 4

1 0 0 0

4 4 4 4

(M)

-

112 0 0 0

The intensity of the immunoreaction

-

0 0 0

0

-

was subjectively indicated on a scale from 0 to 4+ (controls).

1062

B. LINDH et al

ganglion complex.49~“~67~7”~7’ Also, the peptidergic nerve fibers and terminals exhibit a heterogeneous distribution with, for example, the VIP/PHI-immunoreactive nerve fiber network” surrounding mainly noradrenergic cell bodies containing somatostatin-11 and lacking NPY-LI.66,67.70.7’A distribution similar to that of the VIP/PHI-immunoreactive fibers has been suggested for other peptides such as CCK, bombesin and dynorphin.‘0.66,67.71 In contrast, fibers containing SP5’.” 74and CGRP-L166 are more evenly distributed within the ganglion, surrounding both somatostatinand NPY-positive cells. The ENK-LI exhibits a somewhat more complex pattern with a sparse plexus of strongly fluorescent fibers all over the ganglion and a denser, medium strong to weak fluorescent network with a regional distribution.“’ The results of the denervation and ligation experiments carried out in the present study indicate that each of the connected nerve trunks carries peptidergic pathways and contributes to the peptidergic network in the CSMG. Two approaches have been employed to try to define the origin of the various nerve fibers in the ganglion complex: (I) transection of nerves with recording of disappearance of immunoreactive material due to nerve fiber degeneration, and (2) compression of nerves and analysis of accumulation of peptides around the lesion. In a similar way, the IMG of the guinea-pig has been analysed previously.” The survival time after transection, 7 days, appears to be a sufficient period to assure complete degeneration as seen in earlier studies.” The analysis of accumulation is more complex. It is known from early studies on accumulation of catecholamine-containing granules in the sciatic nerve that accumulation occurs both central and peripheral to a ligation, representing anterograde and retrograde transport, respectively (see Ref. 12). It is therefore not possible to decide definitely whether accumulation on both sides in fact indicates that cell bodies are located on both sides, or whether one of the accumulations represents retrograde transport. To circumvent this problem we have, in one case, transected the thoracic splanchnic nerves 7 days before ligating the mesenteric nerve. In this way the anterograde and retrograde accumulation of peptides in fibers originating in spinal cord and spinal ganglia at thoracic levels and travelling through the CSMG is prevented. A summary of the results obtained is presented in Fig. 18. Thus, the thoracic splanchnic nerves contain ENK-, SP-, CGRP-, CCK-, VIP/PHI-, and dynorphin-immunoreactive fibers from sensory ganglia and/or of central origin. All of these peptides are present in sensory neurons except ENK, which probably originates in the spinal cord. The mesenteric nerves carry ENK-, CGRP-. CCK-, VIP/PHI-, bombesinand dynorphin-immunoreactive fibers from the intestine. Many of these peptides appear to be synthesized in the same intestinal neurons.‘0.30 The intermesenteric nerve carries ENK-, SP-, CGRP-,

CCK-, VIP/PHI-, bombesinand dynorphin-immunoreactive fibers from different sources probably including the distal colon, and dorsal root ganglia and spinal cord at lower thoracic and lumbar levels. Although no attempts were made to analyse the projection of the somatostatin-containing ganglion cells in this paper, our results suggest that somatostatin neurons may project into the thoracic splanchnit nerve, in addition to their major projection to the intestine.’ The above conclusions are based on integration of the two experimental approaches (transections, ligations/crushes) plus knowledge of localization of cell bodies. The conclusions must, however, still be regarded as tentative in many cases. Enkephalin

-immunoreactive

fibers

The present results confirm earlier observations” of two populations of ENK-immunoreactive nerve fibers in the CSMG of the guinea-pig, one strongly fluorescent network with a patchy distribution and another more weakly fluorescent, forming a denser network. The present results show that the latter fibers are mainly localized in the SMP of the ganglion. Furthermore the two types of ENK-positive fibers seem to reach the CSMG via different nerve routes. The experiment of cutting the mesenteric and intermesenteric nerves leaving the thoracic splanchnic nerves intact suggests that the strongly fluorescent network reaches the ganglion via the thoracic splanchnic nerves. These fibers are probably preganglionic sympathetic fibers originating in the spinal cord, since ENK-LI has not been described in cell bodies in guinea-pig paravertebral sympathetic or dorsal root ganglia. Such cells, however, have been described previously in the sympathetic lateral column of the guinea-pigI and the rat,” as well as in the cat spinal cord zona intermedia. In the rat some of these neurons may be cholinergic.60 Comparison of the distribution of preganglionic chohnergic and ENK fibers in the superior cervical and stellate ganglia of the guinea-pig suggests that these two neuroactive compounds in these ganglia and in this species at least in part are present in separate systems6’ In contrast, the weakly fluorescent ENKimmunoreactive nerve fiber network in the SMP seems to originate in the intestine. Thus, ligation of the mesenteric nerves resulted in an accumulation of ENK-immunoreactive material distal to the ligature, i.e. on the intestinal side. In earlier studies ENKcontaining nerve cell bodies have been demonstrated in the myenteric plexus of the guinea-pig (for review see Ref. 30) some of which have been suggested to project to the CSMG.“,” Interestingly, partial denervation of the ganglion seems to increase the level of ENK-LI in this system. It has been suggested that ENK-LI in myenteric neurons coexists with dynorphin-11.” This could imply that these neurons produce opioid peptides from different precursors, the preproenkephahn and preprodynorphin. This

Peptide fibers in the celiac superior mesenteric ganglion

1063

Fig. 18. A schematic drawing of the celiac superior mesenteric ganglion (CSMG) with the connected peptidergic pathways reaching the ganglion through three different nerve trunks; thoracic splanchnic nerves (tsn) containing sympathetic prega~glionic neurons from the thoracic spinal cord (TSC) and primary afferent neurons originating in the dorsal root ganglia (DRG); mesenteric nerves (mn) containing axons of enteric neurons with their cell bodies located in the enteric plexus of the proximal gastrointestinal tract (PCIT); intermesenteric nerve (imn) containing sympathetic preganglionic neurons from the lumbar spinal cord (LSC) and primary af!erent neurons originating in lumbar DRG (via the lumbar splanchnic nerves (Isn)) and the inferior mesenteric ganglion (IMG) and enteric neurons from the distal colon (DC) (via colonic nerves and IMG). For simplifi~tion, the peripheral courses of nerves traversing the CSMG have been omitted from the schematic drawing and only the terminal ramifications within the ganglion of these nerves have been indicated in the drawing. Also, the intermesenteric nerve may contribute to the celiac pole innervation with sparse fibers, as indicated in the text, but these fibers are not indicated here. The thick lines (B-H) represent the main pathway to the CSMG for each peptide. The interrupted lines (C and G) represent hypothet!ca! pathways. ENR, enkephalin-; SP, substance P-; CGRP, &citonin gene-related peptide-; CCK, cholecystokinin-; VIP, vasoactive intestinal poly~ptide/~ptide histidine isoleucine-; BOM, bombesin-; and DYN, dynorphin-iike immunorea~tivity.

has been shown in medullary neurons of the rat.43 However, it should be noted that the identity of the ENK-LI in intestinal neurons projecting to the CSMG has not definitely been established. Studies on striatonigral dynorphin systems may give some clues to this question. Thus, evidence from biochemical studies combined with lesion experiments suggests that some striatonigral neurons may partly process their dynorphin precursor peptide to an enkephalin pentapeptide (leucine-ENK) as suggested by Zamir et ~21.‘~~ and others.7.46 It may therefore be speculated that the ENK-LI seen in the SMP after cutting the splan~hnic and inte~esente~~ nerves, and thus orig inating in the intestine, is present in dynorphin nerves. In agreement the dynorphin neurons in the intestinal wall and the dynorphin fibers in the SMP have a distribution which overlaps markedly with that of the ENK-positive ones (see also below). Although our ENK antiserum was raised against methionine-ENK, it is known to cross-react with

leueine-ENK.” Thus, leucine-ENK may be a Product not only in some central dynorphin neurons, but also in certain intestinal dynorphin neurons projecting to the CSMG. The observations that occasional ENK-positive fibers remained in the CSMG, when only the intermesenteric nerve was left intact, and that ENKimmunoreactive material accumulated on both sides of the compression of the intermesenteric nerve, suggest that ENK-containing fibers project to the CSMG also via the inte~esente~c nerve. At least two interpretations are possible. Lumbar ENK-positive pre~nglionic neurons may have axons running through the IMG in the intermesenteric nerve to the CSMG, and/or ENK-positive myenteric neurons in the distal colon may project to the CSMG via the colonic and intermesenteric nerves. In sympathetic ganglia ENK has been shown to inhibit cholinergic transmission by a preganglionic mechanism.20.6i This effect on the cholinergic

1064

B. LINDH et al.

excitatory input may be exerted by ENK-containing preganglionic neurons through axoaxonic contacts with other preganglionic neurons.‘4,59 In the rat, coexistence of immunoreactivity to the ACh synthesizing enzyme choline a~tyitransferase and ENK has been demonstrated in neurons in the sympathetic lateral column, indicating that a population of the preganglionic sympathetic neurons contains both ACh and enkephalin.@’ In the guinea-pig, as mentioned above, it is uncertain whether or not ACh and ENK coexist in preganglionic neurons.68 Substance P-immunoreactiue

fibers

The results of the present study indicate that most of the SP-positive fibers observed in the CSMG of the guinea-pig reach the ganglion via the thoracic splanchnic nerves. Both varicose and non-varicose SP-positive fibers could be observed in the ganglion. Early suggestions” and subsequent analyses by Dalsgaard et af.“~” and Matthews and Cuello”~“’ have provided strong evidence that both varicose and non-varicose SP-positive nerve fibers of prevertebral ganglia originate from dorsal root ganglion cells. These fibers presumably traverse the CSMG to continue into the mesenteric nerves and to a lesser extent into the intermesenteric nerve. The varicose fibers have been assumed to represent axon collaterals forming terminal ramifications in contact with ganglion cells5’.58and this has, in fact, been demonstrated in the elegant analysis of Matthews and collaborators.72m74This is also supported by the present study, where compression or ligation of the thoracic splanchnic, mesenteric and intermesenteric nerves resulted in an accumulation of SP-immunoreactive material on the proximat side of these nerves. Moreover, when the mesenteric and intermesenteric nerves were cut, leaving the thoracic splanchnic nerves intact, there was an accumulation of SP-LI in nerve fibers in the CSMG. SP has been demonstrated in paravertebral and prevertebrat sympathetic ganglia with radioimmunoassay.62*82The biochemical studies of Gamse and collaborators strongly suggest that SP in CSMG is since a marked decrease present in sensory nerves, 35,36 in SP-content could be demonstrated after capsaicin treatment. Also in immunohistochemical studies a depletion of substance-P immunoreactivity has been observed in prevertebral sympathetic gangtia after capsaicin treatment.“.7~,74 A few varicose SP-immunoreactive nerve fibers in the SMP may have a different origin, as suggested by the denervation and ligation or crush experiments. One possible source is the gastrointestinal tract which contains numerous SP-immunoreactive neurons (see Ref. 30), some of which might contribute to an SP-positive nerve fiber network via the mesenteric nerves. Previous morphological studies have clearly shown that a population of the enteric neurons in the However, it remains to cat projects to the CSMG. 63.97 be shown that this is the case also for SP-containing

enteric neurons. Ligation of the mesenteric nerves in the present study gave no conclusive evidence for a projection of SP-positive nerve fibers from the gastrointestinal tract to the CSMG. Although the thoracic splanchnic nerves were cut 7 days prior to the ligation experiment, the main accumulation of SP-immunoreactive material occurred on the ganglion side. Another explanation for the remaining SP-positive fibers in the CSMG is that at least some of them may be processes of the intrinsic SP-containing SIF-ceils. SIF-cell processes may sometimes resemble neurites.” A third possible explanation is that the denervation experiments were not completely successful and that a small branch of the thoracic splanchnic nerves was left intact. In this context the findings of Matthews and Cuello73,74and Dalsgaard et aLI are interesting. They found that a small proportion of SP-immunoreactive fibers in guinea-pig prevertebral ganglia were unaffected by capsaicin treatment. In the biochemical study of Gamse and collaborators36 it was also shown that the depletion of SP in dorsal root ganglia after capsaicin treatment was not total, but about 90%. The remaining SP-immunoreactivity in prevertebrat sympathetic ganglia after capsaicin treatment may thus either originate in a population of SP-containing neurons which is not located in dorsal root ganglia or derive from a population of capsaicin-resistant primary sensory neurons. Moreover, it has been shown that the SP-containing enteric neurons are not sensitive to capsaicin.33.47,74 A second route for SP-fibers to the CSMG is the intermesenteric nerve. These SP-containing fibers are probably also of sensory nature, originating in lumbar dorsal root ganglia projecting to the gastrointestinal tract via the lumbar splanchnic and intermesenteric nerves traversing the IMG. The varicose SP-positive fibers seen in the CSMG, when only the intermesenteric nerve was left intact, were probably collaterals of these nerves. It has recently been shown with retrograde axonal transport techniques (Lindh et al., unpublished observations) as well as with anterograde axonal transport methodology (Aldskogius and Elfvin, unpubtished observations) that lumbar dorsal root ganglion cells project to the CSMG. The functional significance of the SP-containing neurons innervating the prevertebral sympathetic gangtia is not clear, but it is known from the studies of Konishi and collaboratorsbi that the SP release from the guinea-pig IMG is calcium dependent. Moreover, SP causes an atropine-resistant, slow membrane depolarization of the principal ganglion cells in the IMG.‘*,‘9.62 Ca~c~to~~ngene -related peptic

-imm~noreact~~~efibers

The results of the present study indicate that the CGRP-positive nerve fibers in the CSMG of the guinea-pig represent at least two nerve populations, originating respectively in dorsal root ganglia and in

Peptide fibers in the celiac superior mesenteric ganglion the intramural ganglia of the gastrointestinal tract. Both varicose and non-varicose CGRP-positive fibers were observed in the CSMG. The non-varicose CGRP-positive fibers were often seen in bundles particularly in the CP of the ganglion. These nonvaricose CGRP-containing fibers probably represent axons from thoracic dorsal root ganglion cells traversing the CSMG into the mesenteric nerves or into the intermesenteric nerve as has been proposed for the non-varicose SP-containing fibers in the CSMG.5’.72-74These assumptions are supported by the present finding that compression or ligation of the thoracic splanchnic, mesenteric and intermesenteric nerves all resulted in an accumulation of CGRP-LI on the proximal (i.e. dorsal root ganglion) side of these nerves. Furthermore, in cases where the mesenteric and intermesenteric nerves were transected, leaving the thoracic splanchnic nerves intact, there was an accumulation of CGRP-LI in fiber bundles in the CSMG. Only a slight reduction of the density of the varicose CGRP-positive nerve fiber network was observed in the SMP of the ganglion in these cases as compared to the unoperated animals. Taken together, these findings suggest that many of the varicose CGRP-positive fibers in the CSMG are collaterals of primary afferents originating in thoracic dorsal root ganglia. In fact, the similarity in response between CGRP-LI and SP-LI to various perturbations as well as the findings in our double-staining experiments support the results of many earlier studies showing that SP-containing primary sensory neurons also contain CGRP-L13*.39s’o’(see also Refs 37 and 56). A high degree of coexistence of SP and CGRP in the fibers of the CSMG is especially supported by the studies of Gibbins et a1.38 on guinea-pig sensory ganglia, showing that more than 80% of the SP cells contain CGRP-LI, while more than 95% of the CGRP neurons were immunoreactive for SP. In contrast, for example both cat3’ and rats6 contain considerably more CGRP- than SP-positive neurons. Terminals with CGRP-LI forming synaptic contacts with principal ganglion cells in the rat celiac ganglion have recently been demonstrated at the electron microscope level by Lee et aI.65 who suggested that they originate from dorsal root ganglia. The second population of CGRP fibers probably originates in the intestine, as indicated by animals with only the mesenteric nerves intact. They had a network of varicose CGRP-positive fibers in the SMP of the ganglion. Ligation of the mesenteric nerves resulted in accumulation of CGRP-immunoreactive material on both sides of the ligature. The CGRP accumulation on the intestinal side was much more pronounced than the one seen for SP. Since the ligation experiment was preceded by a transection of the thoracic splanchnic nerves, the accumulation of CGRP-LI distal to the ligature in all probability was caused by an anterograde accumulation in nerves

1065

running from the gut wall to the CSMG. Doublestaining experiments indicate that these fibers, at least in some cases, also contained CCK-LI. Earlier immunohistochemical studies have shown that CGRP-positive cell bodies occur in both the myenteric and the submucous plexus of the guinea-pig.32 It has been proposed that one of the histochemically identified populations of the myenteric neurons in the guinea-pig containing immunoreactivity to ENK, CCK, VIP/PHI, GRP (bombesin) and dynorphin projects to the CSMG (see Refs 10 and 30). The results of the present study suggest that CGRP-containing neurons in the intramural ganglia of the guinea-pig project via the mesenteric nerves to the CSMG. Our results indicate that these neurons also contain at least CCK-LI, and previous work of Costa, Furness and collaborators’0,30 suggests that they may in addition contain ENK, VIP/PHI, GRP (bombesin) and dynorphin. As discussed above for SP, denervation and crush experiments indicate that some CGRP-positive fibers reach the CSMG via the intermesenteric nerve and that these fibers are of sensory nature and originate in lumbar dorsal root ganglia. Another possible explanation is that they originate in the distal colon. Thus, CGRP-immunoreactive enteric neurons in the distal colon would project to the CSMG via the colonic and intermesenteric nerves traversing the IMG. For discussion of CGRP-CCK cross-reactivity, see below. Fibers immunoreactive to cholecystokinin, vasoactive intestinal polypeptidelpeptide histidine isoleucine, bombesin and dynorphin

Since the fibers with immunoreactivity to these four peptides show a high degree of overlap and in many instances seem to originate from the same neurons as judged from analysis of the myenteric plexus,‘0*30they are being discussed together. However, when studying the CSMG with double staining it is difficult to establish the exact degree of coexistence in view of the very high density of these fiber networks, as experienced in the present study. The varicose nerve fiber networks containing these peptides are mainly confined to the SMP of the CSMG surrounding the principal ganglion cells. The results of the double-staining experiments show that the fibers surround two of the three main populations of noradrenergic neurons in the ganglion, i.e. noradrenergic neurons containing somatostatin-11 (NA/ somatostatin neurons) and noradrenergic neurons so far apparently lacking peptide immunoreactivity (NA/neurons). The fibers are only occasionally found around NPY-LI neurons containing (NA/NPY neurons). The overwhelming majority of fibers containing immunoreactivity to CCK, VIP/PHI, bombesin and dynorphin appear to originate in the intramural plexuses of the gastrointestinal tract and reach the ganglion via the mesenteric nerves, since cutting

1066

B. LINDH et

the thoracic splanchnic and intermesenteric nerves leaving the mesenteric nerves intact had little effect on their distribution in the SMP. Moreover, ligation of the mesenteric nerves resulted in a heavy accumulation of ~ptide-immunoreactive material distal to the ligature, suggesting an anterograde a~umulation of peptide-LI in nerves running from the gut. These ENK-, CCK-, VIP/PHI-, GRP (bombesin)and dynorphin-immunoreactivities in myenteric neurons10.30 may represent the afferent limb of the intestino-intestinal inhibitory reflex arc, of which the noradrenergic neurons in the ganglion constitute the efferent limb*’ (see also Ref. 94). The three peptides CCK, VIP/PHI and dynorphin may, however, to a small extent originate in sensory ganglia at the thoracic level, since after cutting the mesenteric and inte~esenteric nerves and leaving the thoracic splanchnic nerves intact, sparse networks of CCK-, VIP/PHI- and dynorphin-positive fibers were seen, mainly confined to the SMP. Furthermore, compression of the thoracic splanchnic nerves resulted in an accumulation of immunoreactivity to these three peptides central to the site of compression. This would be in agreement with the previous demonstration of CCK,?’ VIP/PHI” and dynorphi# in the guinea-pig sensory ganglia. The latter peptide has also been observed in cat primary sensory neurons.’ The presence of a bombesin-like peptide in the SMP of the CSMG has previously been described with immunohistochemical techniquesa (see also Ref. 45), and it was shown to originate in the intestinal wall.*’ Bombesin and substance P have the same amino acids in C-terminal position and antisera raised against these peptides may therefore give rise to cross-reactivity. In fact, absorption of bombesin antiserum with substance P at high concentration ( lO--5M) markedly reduced or completely abolished (10m4M) bombesin staining. It is therefore clear that our bombesin antiserum cross-reacts with substance P (see Refs 21a and 21b). However, it is unlikely that the bombesin-11 in the SMP represents crossreactivity with substance P, since the substance P fibers in the SMP form only a comparatively sparse plexus. The question of specificity may, however, be raised with regard to the few bombesin-immunoreactive fibers seen in the CSMG after transection of the mesenteric and intermesenteric nerves with the thoracic splanchnic nerves intact. These fibers may thus have a central origin as also indicated by the fact that compression of the thoracic splanchnic nerves resulted in accumulation of bombesin-LI central to the site of compression. Moreover, in the CP their distribution shows a similar pattern to that of the substance P fibers. This bom~sin-LI may therefore be present in primary sensory neurons as previously shown.34,8’ Whereas Fuxe et a1.34 observed co-localization of bombesin- and substance P-L1 in sensory ganglia, Panula et al.*’ found the two immunoreactivities in different cell populations.

al.

Moreover, Panula et al.” analysed both spinal cord and spinal ganglia with biochemical techniques and the results demonstrated the presence of genuine bombesin (GRP)-LI. It may be, therefore, that a population of sensory neurons projecting to the CSMG contains a bombesin~GRP-like peptide. Our findings indicate that fibers containing immunoreactivity to CCK, VIP/PHI, bombesin and dynorphin also project to the CSMG via the intermesenteric nerve and at least to some extent contribute to the peptide networks seen in the CSMG of unoperated animals. Thus, with only the intermesenteric nerve left intact, sparse networks of varicose nerve fibers of all four peptides were observed, mainly confined to the SMP of the ganglion. Furthermore, when the intermesenteric nerve was crushed, immunoreactive material was accumulated on both sides of the compression, indicating that at least some of the immunoreactive material was anterogradely accumulated in nerves running toward the CSMG. These fibers may originate in the distal colon and project to the CSMG via the colonic and intermesenteric nerves traversing the IMG. They may form part of the morphological substratum for the cola-colonic inhibitory reflex arc (see Refs 93 and 94). In addition, CCK, VIP/PHI and dynorphin sensory neurons in lumbar dorsal root ganglia may project to the CSMG. CCK- and VIP/PHI-L1 of central origin have been shown in earlier studies to accumulate in a few fibers of the lumbar splanchnic nerves of the guineapig after ligationn suggesting a sensory origin for these latter immunoreactivities. The identity of CCK-LI in the guinea-pig has been specifically analysed in the present study, since there is evidence that this immunoreactivity in dorsal root ganglion ceils may represent cross-reactivity with CGRP or a CGRP-like peptide.S4.56.57This is less likely in the guinea-pig, since the immunoreactive CCK fibers can be demonstrated with antisera directed to different sequences of the CCK(l-33) molecule, i.e. either to the C-terminal part or the midportion. This suggests that these neurons contain a CCK-like peptide. The fact that absorption of C-terminally directed CCK antiserum with CGRP in high concentrations reduced, but not abolished, the staining does not argue against this view, but indicates that one of our CCK-antisera, as shown earlier, has a population of antibodies directed against the C-terminal phenylalanine, which CCK and CGRP share. There is then presumably another population of antibodies which can detect the deeper parts of the guinea-pig CCK-but probably not of the rat CCK, which would account for the remaining staining. With respect to the VIP/PHI-L1 it is interesting to note that in the rat these immunoreactivities in primary sensory neurons may represent a special case. Thus, in a normal rat only a few primary sensory neurons contain VIP-L& at least in sufficient amount to be detected with immunohistochemical techniques, but these show a remarkable increase in

Peptide fibers in the celiac superior mesenteric ganglion VIP- and PHI-L1 after peripheral transection.52.77.90.9’ Whether or not crushes, ligations and transections as performed in the guinea-pig in this study also change levels of the VIP/PHI-L1 is at present unknown. Finally, it should be noted that the intrinsic VIP/PHI- and DYN-positive neurons in the CSMG which are mainly located in the celiac poles of the ganglion,66~67~7’ may have contributed to the immunoreactive nerve fiber networks observed. However, the VIP/PHI- and DYN-positive neurons comprise less than 1% of the principal ganglion cells. CONCLUSIONS

The present results show that all three main origins for afferents to the CSMG, spinal cord, dorsal root ganglia and intestine, contain one or more peptides. Some of these peptides are co-localized in certain systems. Only one peptide has convincingly been identified to be localized in central preganglionic neurons, namely ENK. However, there are two different types of ENK-positive nerve fiber networks, in agreement with earlier studies by Schultzberg et ~1.~~ The denervation experiments show that the strongly fluorescent fibers with a patchy distribution which are found both in the CP and in the SMP in all likelihood only originate in the spinal cord and thus represent preganglionic nerves, reaching the ganglion via the thoracic splanchnic nerves. With regard to primary sensory neurons, as shown with denervation experiments they contain SP- and CGRP-LI and reach the CSMG via the thoracic splanchnic nerves. The SP- and CGRP-LI in all probability coexist in most nerve fibers. To a lesser extent primary sensory neurons may also contain CCK-, VIP/PHI-, and dynorphin-11 and perhaps bombesin (GRP)-LI. Also lumbar dorsal root ganglia may via the intermesenteric nerve supply the CSMG with peptide-containing fibers. The most extensive peptidergic input to the CSMG is, however, provided by intestinal neurons. On the basis of double-staining experiments on cell bodies in the myenteric ganglia in the intestinal wall, it has been suggested that the CCK-, VIP/PHI-, GRP(bombesin)and dynorphin-immunoreactive fibers in the mesenteric nerves projecting to the guinea-pig CSMG in all probability originate in the same population of enteric neurons (for review see Refs 10 and 30). This is supported by the present double-staining experiments which show that at least some CCK-immunoreactive fibers in the CSMG also contain VIP/PHI-, bombesin-, and dynorphinLI, respectively. In fact, the present results indicate that some of these neurons also contain CGRPLI. Also the weakly fluorescent, dense ENKpositive network in the SMP originates in the intestine. The enteric neurons projecting to the CSMG may represent the afferent limb of the peripheral intestino-intestinal reflex arc (see above). These neurons may be cholinergic, since fast synaptic

1067

cholinergic inputs from the intestine have been recorded in prevertebral sympathetic ganglia (see Refs 93 and 94). Enteric neurons containing CCK-, VIP/PHI-, bombesin- and dynorphin-11 projecting to the CSMG terminate mainly around NA/neurons which project to myenteric ganglia, and around NA/somatostatin neurons which project to submucous ganglia. The peptide-containing myenteric neurons projecting to the CSMG only occasionally terminate around NA/NPY neurons which project to intestinal blood vessels (see also Refs 9, 10 and 31). This suggests that this peripheral reflex arc would be mainly of importance in controlling motility and mucosal functions but may not be involved in the regulation of the intestinal blood flo~.~~‘~ Although the dominating peptidergic input to the CSMG is from the proximal intestinal tract, enteric neurons in the distal colon appear to project to the CSMG via the colonic and intermesenteric nerves. These neurons seem to have a similar content of peptides to that of the enteric neurons projecting to the CSMG via the mesenteric nerves. The finding of a projection of enteric neurons from the distal colon to the CSMG supports earlier physiological experiments by Szurszewski and collaborators (see Refs 93 and 94) where neurons in the CSMG were shown to receive an input from the distal colon. Thus, there is also histochemical evidence for interaction between regions in the proximal and distal parts of the gastrointestinal tract. Future studies using, for example, retrograde axonal tracing in combination with immunohistochemistry, will be one way by which we can morphologically clarify the organization of such a communication pathway. Acknowledgemen&--The skilful technical assistance of MS A.-S. Hoijer, MS B. V&are, MS B. Dahlgren and MS W. Hiort and the excellent secreterial help of MS M. Jepsen and MS M. Gottfridsson are gratefully acknowledged. The present study was supported by grants from the Karolinska Institutet, the Swedish Medical Research Council (12x-5189, 04x-2887), the Swedish Society for Medical Sciences, Ruth and Richard Julins Stiftelse and Alice and Knut Wallenbergs Stiftelse. For generous supply of antisera we thank Drs W. W. Blessing, Department of Medicine (Neurology), Flinders Medical Centre, Bedford Park, South Australia (NPY); J. C. Brown, Medical Research Council of Canada, Regulatory Peptide Group, University of British Columbia, Vancouver, B.C., Canada (somatostatin); A. C. Cuello, Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada (SP); G. Dockray, Department of Physiology, University of Liverpool, Liverpool, U.K. (bombesin); P. Emson, Department of Neuroendocrinology, MRC Group, Institute of Animal Physiology, Babraham, Cambridge, U.K. (SP); J. Fahrenkrug, Bispebjerg Hospital, Copenhagen, Denmark (VIP and PHI); P. Frey, Wander Research Institute (Sandoz), Bern, Switzerland (CCK); J. F. Rehfeld, Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark (CCK); L. Terenius Department of Pharmacology, Uppsala University, Uppsala, Sweden (ENK and dynorphin); and J. H. Walsh, Center for Ulcer Research and Education (CURE), Los Angeles, California, U.S.A. (CCK).

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et al.

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