Proc. NatL Acad. Sci. USA Vol. 79, pp. 1668-1672, March 1982 Neurobiology

Substance.P-irnmunoreactive peripheral branches of sensory innervate guinea pig sympathetic neurons (immunofluorescence/immunocytochemistry/prevertebral ganglia/capsaicin/ lesion) MARGARET R. MATTHEWS* AND A. CLAUDIO CUELLO*t Departments of *Human Anatomy and tPharmacology, University of Oxford, Oxford, England Communicated by Torsten N. Wiesel, November 2, 1981 ABSTRACT The presence of substance P-immunoreactive to preganglionic fibers (3) and demonstrated immunohis- *(SPI) varicose nerve networks and nerve fiber bundles in guinea tochemically in nerve terminal boutons (6). pig prevertebral sympathetic ganglia has been confirmed by flu- In the case of the prevertebral ganglia, there are a number orescence immunohistochemistry. No SPI, neurons have been ofpossible sources for the SPI nerve networks and nerve bun- found in sympathetic ganglia, including lumbar paravertebral dles. No SPI nerve cell bodies have been found in these ganglia ganglia. Peroxidase-antiperoxidase immunocytochemical methods or in other sympathetic ganglia (1). Possible extraganglionic have shown that SPI nerve terminal varicosities in the inferior sources of the SPI elements include the following. (a) SPI pri- mesenteric (IMG) form morphologically identifiable syn- mary sensory neurons (7, 8). The prevertebral ganglia are placed apses on dendritic shafts. Cutting the intermesenteric nerve pro- on a between the alimentary tract and pelvic organs and duces no obvious change in SP immunoreactivity in the IMG; cut- pathway ting the lumbar splanchnic produces nearly total depletion the central and are known to be traversed by which becomes virtually complete ifthe two lesions are combined; fibers (9, 10). (b) SPI neurons in the enteric nerve SP immunoreactivity accumulates in the central ends of the lum- plexuses (11-14); Nerve fibers of enteric origin also enter the bar-splanchnic nerves and in the cranial end ofthe intermesenteric ganglia (15-18). (c) SPI neurons inside the spinal cord. Small nerve. Cuttinghypogastric nerves or.colonic branches ofthe IMG numbers of SPI neurons have been observed after colchicine leads to accumulation of SP immunoreactivity in their ganglionic pretreatment in the intermediate region ofthe spinal cord, from stumps and to build-up (colonic nerve lesion) rather than depletion which the preganglionic sympathetic fibers originate (19). of SP immunoreactivity in the IMG. Capsaicin treatment leads to Bundles ofnonvaricose SPI nerve fibers in the ganglia might total loss of SP immunoreactivity from the prevertebral ganglia be fibers, ofpassage (for example, afferent fibers); but the pres- and dorsal root ganglia, severe depletion in laminae I and II and ence of varicose networks of SPI fibers raises questions: Do dorsolateral fasciculus ofthe spinal cord, and total loss from pen- these networks represent nerve terminals and, if so, are they vascular and paravascular networks of the ileum and mesentery, derived from enteric. or from spinal ganglia or intraspinal neu- with sparing of the SP immunoreactivity ofthe enteric.nerve plex. rons? Do the SPI varicosities establish synaptic contacts with uses. Capsaicin is.thought 'to deplete sensory neurons selectively. the sympathetic neurons? The present experiments were de- Removal of the spinal cord belowT7 without damage to the dorsal signed to explore these possibilities. The results support the root ganglia leaves the intraganglionic SPI nerve networks and scheme shown in Fig. 1. bundles intact. We conclude' that these are derived from periph- eral processes of sensory neurons and we propose that the SPI MATERIALS AND METHODS in the IMG arise from collateral branches of these sen- sory peripheral processes. This implies a novel role for these pro- Young albino guinea pigs were used in all experiments. cesses, in forming intraganglionically in the prevertebral ganglia Nerve Lesions. In male guinea pigs (250-700 g) the lumbar synapses which may take part in the reflex control of the viscera, splanchnic, intermesenteric, colonic, or hypogastric nerves independently of the centralnervous system. were exposed and were. cut a few millimeters from the inferior mesenteric ganglion, alone or in various combinations. The pro- In sympathetic ganglia of the guinea pig, rat, and cat, Hokfelt cedures were performed with aseptic precautions and the ani- et al. (1) have demonstrated bundles and varicose networks of mals were under anesthesia induced by intraperitoneal injec- nerve fibers immunoreactive for substance P (SP). These are tion of chloral hydrate. Care was, taken not to injure the blood particularly abundant in the prevertebral ganglia (celiac, su- vessels of the region. Four or 6 days later the animals were re- perior mesenteric, inferior mesenteric). There is strong elec- anesthetized and were perfused through the heart with oxy- trophysiological evidence that SP plays a transmitter role in the genated Krebs-Henseleit solution to wash out blood and then prevertebral ganglia (2). Interest has recently been focused on with 4% freshly depolymerized paraformaldehyde in 0.1 M so- the possibility ofpeptidergic transmission in autonomic ganglia dium phosphate buffer (pH 7.2-7.4), both at room temperature after the demonstration by Kuffler and associates (3) that lu- (20-22°C). Control guinea pigs ofthe same weight and age were teinizinghormone-releasing hormone has a transmitter function similarly perfused. Twenty-eight animals were used for these in a bullfrog sympathetic ganglion. Mammalian prevertebral experiments. ganglia have been shown to contain nerve networks immuno- Fixation and Tissue Processing for SP Immunofluorescence reactive for enkephalin and for vasoactive intestinal' polypep- Histochemistry. The inferior mesenteric ganglion and the ce- tide, in addition to the substance P-immunoreactive (SPI) net- liac-superior mesenteric ganglion complex' were dissected out works (4, 5). In the case of the bullfrog sympathetic ganglion, and kept in fresh fixative for a total fixation time of 21/2-3 hr. the luteinizing hormone-releasing hormone has been localized The ganglia were then washed -with three changes of 0.1 M phosphate buffer containing 5% sucrose, at 4°C, for at least 3 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: SP, substance P; SPI, SP-immunoreactive; Pj'NaCl, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. phosphate-buffered saline. 1668 Neurobiology: Matthews and Cuello Proc. Natl. Acad. Sci. USA 79 (1982) 1669 buffer, conventional dehydration, and flat embedding in Ar- aldite under plastic coverslips. Slices were assessed by light microscopy and then embedded for electron microscopy. Ul- S P trathin sections were examined unstained or lightly stained with lead citrate and uranyl acetate in a Philips EM 200 electron microscope. Capsaicin Injections. Guinea pigs (340-400 g) were injected subcutaneously with capsaicin under cover oftheophylline, ac- cording to the dosage and schedule described in ref. 23. Control animals were injected with theophylline and with vehicle on the same schedule. Six days after the last injection the animals were anesthetized and fixed by perfusion as described above. The inferior mesenteric and celiac-superior mesenteric ganglia, spinal ganglia, spinal cord, and pieces of ileum and the mes- entery were taken for determination ofspecific SP immunoflu- orescence. Three capsaicin-treated and three control animals were examined in this way. Spinal Cord Lesions. In guinea pigs (75-250 g) ofages rang- ing from a few days to a few weeks, anesthesia was induced by intraperitoneal administration of chloral hydrate and main- tained with ether. Laminectomy was performed from the mid- thoracic level to the sacrum, the spinal cord was divided be- FIG. 1. Schematic drawing showing the organization of sensory tween ligatures at the level of the T7 segment or between T7 SPI peripheral nerve fibers at lumbar spinal levels as suggested by and T8, and the caudal section of the spinal cord was excised. evidence derived from the present study and from refs. 1,2, and 14. The Care was taken not to injure the spinal ganglia. After careful example shown is that of a sensory of lumbar origin inner- hemostasis, the erector spinae muscles were approximated over vating the colon by way of a lumbar splanchnic nerve and a colonic branch of the inferior mesenteric ganglion (IMG). DH, dorsal horn; the vertebral canal by a continuous suture and the skin wound VH, ventral horn; sg, substantia gelatinosa; DRG, dorsal rootganglion; was closed (method adapted from ref. 24). LSG, lumbar sympathetic ganglion of the paravertebral chain; LSN, In three animals, survivals of 21/2, 4, and 51/2 days were ob- lumbar splanchnic nerve; imn, intermesenteric nerve; CN, colonic tained, at the end of which tissues were fixed for immunoflu- nerve fascicle; HNN, hypogastric nerves. orescence either by in vivo perfusion under anesthesia or by immersion fixation shortly after death. Normal control animals hr. Cryostat (Dittes, Heidelberg) sections were cut at 8 Am and were perfused at the same time. In order to control for the rate were collected on gelatinized glass slides for localization of spe- ofdisappearance of SP immunoreactivity after nerve lesions, in cific SP immunofluorescence by the indirect technique with a two guinea pigs the lumbar splanchnic and intermesenteric monoclonal anti-SP antibody (NCI/34-HL) (20). The cryostat nerves were cut and the animals were perfused after 21/2 and sections were processed as described (21). Dilutions were 1:200 3 days, respectively; a third animal was perfused 46 hr after the for the monoclonal antibody and 1:10 for the fluorescein iso- intermesenteric, lumbar splanchnic, and hypogastric nerves thiocyanate-conjugated anti-rat IgG (Miles). The preparations had been cut. The prevertebral ganglia, spinal ganglia, and lum- were viewed in a Leitz fluorescence microscope and photo- bar paravertebral ganglia were processed for specific SP im- graphed on Kodak Tri-X film. A Leitz Variomat system was munofluorescence as described above, together with spread used, and the same exposure intervals were used for both ex- preparations of mesentery, omentum, and mesocolon. perimental and control material. Fixation and Tissue Processing for Electron Microscopy. RESULTS Young male guinea pigs were anesthetized and perfused with As briefly reported elsewhere (25), immunofluorescence con- 4% paraformaldehyde and 0.01-0.05% glutaraldehyde in 0.1 M firmed the presence of SPI varicose perineuronal networks and phosphate buffer. Within 30-60 min the prevertebral ganglia coarser nonvaricose fibers in normal celiac, superior mesen- were removed and kept in fresh fixative for 2 hr at 40C. After teric, and inferior mesenteric ganglia; groups of SIF cells (the preliminary washing in 0.1 M phosphate buffer containing 5% catecholamine-rich, small, intensely fluorescent cells) were sucrose, the ganglia were kept for 2-4 hr in the same buffer devoid of SPI networks. This confirms previous observations containing 30% sucrose, rapidly and briefly frozen in isopentane by Hokfelt et al. (1). No SPI neurons were observed in these cooled by liquid nitrogen, then chopped into 50-gm slices on ganglia or in ganglia ofthe lumbar sympathetic chain, which are a Smith-Farquhar tissue chopper. Immunoenzyme staining was traversed by the lumbar splanchnic nerves (Fig. 1). The SP im- performed with monoclonal anti-SP antibody (NCV/34-HL) munofluorescence of normal inferior mesenteric ganglia is il- (20), by the peroxidase-antiperoxidase technique (22). lustrated in Fig. 2 A and C. Slices were incubated overnight at 40C in the monoclonal Peroxidase-antiperoxidase preparations showed rows of var- antibody diluted 1:100 in phosphate-buffered saline (PJNaCl). icosities in intercellular and pericellular arrays (Fig. 2F). At the Control incubations were routinely performed as for the im- ultrastructural level, this method showed specific SP immu- munofluorescence. Subsequent processing was at room tem- noreactivity in vesicle-containing nerve varicosities or terminal perature. The slices were washed for 1 hr in PJNaCl, incubated profiles in the inferior mesenteric ganglion, some ofwhich were 1 hr in intermediate antibody [rabbit anti-rat serum (Miles) seen to make synaptic contact with in the ganglion diluted 1:50], washed 1 hr in PjNaCl, incubated 1 hr in rat (Fig. 2G). These synapses formed by SPI terminals showed all peroxidase-antiperoxidase (Cappel Laboratories, Cochranville, the features of typical "axo"-dendritic synapses, including pre- PA) diluted 1:50, and washed 1 hr in PjNaCl. At this stage they clustering and apposition ofcytoplasmic dense were examined as wet preparations by light microscopy, before material to the postsynaptic membrane. No such labeling of osmication for 1 hr in 1% osmium tetroxide/0. 1 M phosphate nerve terminal profiles was found when the incubation with 1670 Neurobiology: Matthews and Cuello Proc. Natl. Acad. Sci. USA 79 (1982)

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FIG. 2. (A) Pattern of varicose and nonvaricose fibers as revealed by a monoclonal antibody (20) in the inferior mesenteric ganglion of a normal guinea pig, perfused and processed together with the tissue in B. (B) Inferior mesenteric ganglion of a capsaicin-treated guinea pig, showing total loss of specific SP immunofluorescence, apart from a small residual trace (asterisk). (C) SP immunofluorescence in the inferior mesenteric ganglion of a normal guinea pig, perfused and processedtogether with the tissue inD. (D) Almost total disappearance of SP immunofluorescence in the guinea pig inferior mesenteric ganglion at 4 days after lumbar nerves were severed. (E) Persistence of varicose and nonvaricose SPI fibers in the inferior mesenteric ganglion of a guinea pig in which the spinal cord had been removed (below T7) 4 days previously. (F) Trails of varicosities immuno- reactively labeled by the SP/peroxidase-antiperoxidase method in a 50-pm slice of celiac-superior mesenteric ganglion photographed before os- Neurobiology: Matthews and Cuello Proc. Natl. Acad. Sci. USA 79 (1982) 1671 specific antibody was omitted or the specific antibody was normal in the various mesenteries below the level ofthe lesion preabsorbed with SP. These synaptic endings correspond in size as well as above it. To test whether the survival intervals were and in general situation with varicosities of the perineuronal adequate to display changes in SP immunoreactivity after loss networks seen in the ganglion with the light microscope (Fig. ofneurons oforigin, the lumbar splanchnic and intermesenteric 2F). nerves were cut in three control animals. In these animals, it Nerve Lesion Experiments. These experiments have already was confirmed that alteration and depletion of SP immuno- been briefly reported (25). In normal ganglia, the attached lum- reactivity in the inferior mesenteric ganglion and mesentery was bar splanchnic nerves contained many SPI nerve fibers. At 4 beginning to be evident after 46 hr and, apart from a single days after division of lumbar splanchnic nerves, marked accu- minute surviving fascicle in the inferior mesenteric ganglion, mulation of SPI material was seen in their central stumps, and depletion was complete by 3 days postoperatively. their distal stumps showed a total loss of SP immunofluores- cence (results not illustrated); the inferior mesenteric ganglion DISCUSSION was totally or almost totally depleted of nonvaricose SPI fibers and of varicose SPI networks (Fig. 2D). The variable extent of These experiments show that the SPI nerve fibers and networks the depletion seemed to be inversely correlated with the pres- in the inferior mesenteric ganglion of the guinea pig are abol- ence of varying numbers of SPI fibers in the intermesenteric ished subtotally by section ofthe lumbar splanchnic nerves and nerve. that the loss is virtually complete when the intermesenteric Sectioning of hypogastric nerves or of the intermesenteric nerve is also severed. Sensory nerve fibers are known to reach nerve caused no gross change in the appearance of the intra- this ganglion from as high as the lowest thoracic levels (10). ganglionic networks after 4 days, but there was dramatic ac- Radioimmunoassay has indicated a partial loss of SP immuno- cumulation of SPI material in the ganglionic stumps of the hy- reactivity from the guinea pig inferior mesenteric ganglion after pogastric nerves, and accumulation was seen in some fibers of nerve lesions that should interrupt sensory and spinal connec- the cranial stump ofthe divided intermesenteric nerve. Cutting tions of the ganglion (2). the colonic nerves led to marked accumulation of SPI material The present experiments have also shown that the SPI in- in their ganglionic stumps and these accumulations extended traganglionic nerve fibers and networks are totally depleted by back into the distal (colonic) pole of the ganglion where large capsaicin, which appears to act selectively upon sensory neu- immunofluorescent knobs and enlarged nonvaricose SPI fibers rons (30-34). Capsaicin treatment has been found to produce were seen (results not illustrated). severe depletion of radioimmunoassayable SPI material in the Capsaicin Experiments. Capsaicin treatment resulted in guinea pig celiac-superior mesenteric ganglion (35). The pres- complete loss of the normal pattern of brilliant specific SP im- ent capsaicin experiments have confirmed the sensory origin munofluorescence from the prevertebral sympathetic ganglia of the SPI nerve fibers on the submucosal blood vessels of the (Fig. 2B) and from the spinal ganglia (not illustrated). Minute small intestine (14) and have demonstrated a similar origin for residual traces of faintly SPI material were occasionally seen the SPI nerve elements of the mesenteric blood vessels and (Fig. 2B). In the spinal cord, SP immunofluorescence was se- mesenteries, an anatomical/pharmacological observation that verely depleted in laminae I and II and in the adjacent dorso- supports the idea of a vasoactive role for this peptide, as first lateral fasciculus. SP immunoreactivity survived in the inter- indicated by the work of von Euler and Gaddum (36). mediolateral zone and around the central canal and also in the The experiments involving removal of the spinal cord from ventral horn. In the small intestine, perivascular and paravas- a level (T7) above those segments (T13 to L4) that are known cular SPI networks and nerve bundles were lost from the serosal to project to the inferior mesenteric ganglion (37) have excluded and mesenteric blood vessels and from the blood vessels in the intraspinal neurons as the principal origin of the SPI networks submucosa (not illustrated). No major change was noticed in the in the guinea pig inferior mesenteric ganglion. In control ex- intramuscular networks, in the myenteric or submucous gan- periments in which lumbar or lumbar and intermesenteric glionated plexuses, or in the subepithelial villous network or nerves were cut, changes in the intraganglionic SPI elements the pericryptal network ofthe mucosa, except for a loss of were just beginning to be evident after 46 hr, but loss was es- large sentially complete after 3 days. The longer two of the survival SPI varicosities in the submucous plexus. intervals (2'/2, 4, and 51/2 days) obtained after spinal cord lesions Excision ofSpinal Cord. These experiments were performed were thus certainly long enough, even allowing for the extra to test whether the SP immunoreactivity of the inferior mes- distance ofa few millimeters from the spinal cord to the lumbar enteric ganglion was attributable to intraspinal neurons. Dorsal splanchnic nerves, to have permitted changes to develop in SP root ganglia from levels both above and below the lesion showed immunoreactivity in the inferior mesenteric ganglion after loss SP immunoreactivity in neurons scattered throughout the gan- of the neurons of origin. No SPI neurons were observed in the glion and in the processes of the neurons. Ganglia below the lumbar sympathetic ganglia, and they are very unlikely to be level of the lesion showed accumulations of SPI material in the the source of SP immunoreactivity in the inferior mesenteric severed ends ofthe dorsal roots. In the inferior mesenteric gan- ganglion, but the remote possibility remains and must be tested of these animals with ablation of the spinal cord caudal to more strictly. The experimental evidence presented here, how- T7, no obvious difference from the normal pattern and intensity ever, strongly suggests that the nerve fibers and nerve networks was observed in the SPI nerve fibers and networks at any sur- showing SP immunoreactivity in the inferior mesenteric gan- vival interval (Fig. 2E). Patterns of SP immunoreactivity were glion of the guinea pig arise from sensory neurons of the spinal mication and embedding for electron microscopy. (G) Electron micrograph showing dense deposits revealing SPI material over a nerve terminal (star) making a synaptic contact with a (D) of a sympathetic postganglionic neuron. The nerve terminal, of a type commonly seen in the guinea pig inferior mesenteric ganglion, contains a large cluster of small clear synaptic vesicles which are massed in the presynaptic zone and fewer large dense-cored vesicles which lie chiefly at the periphery of the cluster of small vesicles, away from the presynaptic membrane, together with a group of mitochondria. SP immunoreactivity within the nerve terminal is localized over the cores of the large dense-cored vesicles and is associated also with the cytoplasmic (external) surfaces of the small vesicles; both these features have been reported for SPI nerve terminals in the substantia gelatinosa of the spinal cord (26-29). N, nucleus of satellite cell; s, satellite cell cytoplasm; ct, space. (Scale bars represent 100 ,um for A-F and 0.5 gm for G.) 1672 Neurobiology: Matthews and Cuello Proc. Natl. Acad. Sci. USA 79 (1982) ganglion. This conclusion supports hypotheses advanced earlier 6. Jan, L. Y., Jan, Y. N. & Brownfield, M. S. (1980) Nature (Lon- by other investigators (1, 2, 35). don) 288, 380-382. The SPI nerve varicosities and synaptic terminals seen with 7. Hbkfelt, T., Kellerth, J.-O., Nilsson, G. & Pernow, B. (1975) Brain Res. 100, 235-252. the electron microscope in the prevertebral ganglia correspond 8. Cuello, A. C., Del Fiacco, M. & Paxinos, G. (1978) Brain Res. with rows of peroxidase-antiperoxidase-immunoreactive knobs 152, 499-509. seen by light microscopy and with the varicose nerve networks 9. Gabella, G. (1976) Structure of the Autonomic Nervous System seen by the immunofluorescence technique. It is therefore (Chapman and Hall, London). highly probable that they are derived from the peripheral pro- 10. Elfvin, L.-G. & Dalsgaard, C.-J. (1977) Brain Res. 126, 149-153. cesses of sensory neurons. We suggest that they arise from col- 11. Franco, R., Costa, M. & Furness, J. B. (1979) Naunyn-Schmiede- lateral branches of the sensory nerve fibers traversing the gan- berg's Arch. Pharmacol. 306, 195-201. 12. Jessen, K. R., Polak, J. M., van Noorden, S., Bloom, S. R. & glia (Fig. 1). Extrinsic SPI nerve fibers have recently been Burnstock, G. (1980) Nature (London) 283, 391-393. reported in the submucosa of the guinea pig ileum (14), and the 13. Schultzberg, M., Hokfelt, T., Nilsson, G., Terenius, L., Reh- present results show that capsaicin abolishes their SP immu- feld, J. F., Brown, M., Elde, R., Goldstein, M. & Said, S. (1980) noreactivity. SP induces a slow depolarization ofneurons in the 5, 689-744. guinea pig inferior mesenteric ganglion (2, 38, 39); a similar 14. Costa, M., Cuello, A. C., Furness, J. B. & Franco, R. (1980) noncholinergic slow depolarization is induced Neuroscience 5, 323-332. by repetitive 15. Kuntz, A. (1938)J. Comp. Neurol. 69, 1-12. stimulation of either the lumbar splanchnic or the hypogastric 16. Kuntz, A. (1940)J. Comp. Neurol. 72, 371-382. nerves (2, 40, 41), and potassium depolarization induces a cal- 17. Kuntz, A. & Saccomanno, G. (1944)J. Neurophysiol 7, 163-170. cium-dependent release of SP from the ganglion (2). The pres- 18. Ungvdry, G. Y. & Leranth, C. S. (1970) Z. Zellforsch. Mikrosk. ent study demonstrates that SPI nerve endings form synapses Anat. 110, 185-191. upon dendrites in the ganglion and provides an immunocyto- 19. Ljungdahl, A., Hokfelt, T. & Nilsson, G. (1978) Neuroscience 3, these 861-944. chemical, ultrastructural substrate for physiological 20. Cuello, A. C., Galfre, G. & Milstein, C. (1979) Proc. Natl. Acad. observations. Sci. USA 76, 3532-3536. Complex reflex interactions are demonstrable between gut 21. Cuello, A. C., Milstein, C. & Priestley, J. V. (1980) Brain Res. and ganglion (42-44), involving ganglionic excitation attribut- Bull. 5, 575-587. able to cholinergic neurons in the intestinal nerve plexuses. 22. Sternberger, L. A., Hardy, P. H., Cuculis, J. J. & Meyer, H. G. However, most of the preganglionic inputs to the inferior mes- (1970) J. Histochem. Cytochem. 18, 315-333. 23. Gamse, R., Wax, A., Zigmond, R. E. & Leeman, S. E. (1981) enteric ganglion, whether peripheral or central, operate by way Neuroscience 6, 437-441. of the integration of subthreshold excitatory inputs to the post- 24. Zelend, J. & Soukup, T. (1974) Cell Tissue Res. 153, 115-136. ganglionic neuron-hence, the potential modulatory impor- 25. Baker, S. C., Cuello, A. C. & Matthews, M. R. (1980)J. Physiol. tance of slow depolarization which can raise the excitability of (London) 308, 76P-77P. a neuron to above the firing threshold for these inputs (41). The 26. Pickel, V. M., Reis, D. J. & Leeman, S. E. (1977) Brain Res. 122, present and earlier (1, 2) results indicate the possibility of SP- 534-540. 27. Cuello, A. C., Jessell, T. M., Kanazawa, I. & Iversen, L. L. mediated intraganglionic modulation, operating through a (1977)J. Neurochem. 29, 747-751. short-loop system ofcollaterals from peripheral branches ofthe 28. H6kfelt, T., Johansson, O., Kellerth, J.-O., Ljungdahl, A., Nils- primary sensory neurons of the spinal ganglia, with sensory, son, G., NygArds, A. & Pernow, B. (1977) in Substance P, eds. probably nociceptive, terminals in the gut. Such an arrange- von Euler, U. S. & Pernow, B. (Raven, New York), pp. 117-145. ment need not be confined to the SPI pathway, and would sup- 29. Barber, R. P., Vaughn, J. E., Slemmon, J. R., Salvaterra, P. M., plement, rather than replace, centrally generated reflexes. Roberts, E. & Leeman, S. (1979)J. Comp. Neurol 184, 331-352. 30. Jancs6, G., Jancs6-Gabor, A. & Szolcsdnyi, J. (1967) Br. J. Phar- In the case of SP, the evidence presented here indicates that macol. 31, 138-151. such a loop may operate through a morphologically specialized 31. Jessell, T. M., Iversen, L. L. & Cuello, A. C. (1978) Brain Res. synapse and that classical synapses may be formed by peripheral 152, 183-188. branches of mammalian primary sensory neurons. 32. Lembeck, F., Gamse, R., Holzer, P. & Molnar, A. (1980) in Neu- ropeptides and Neural Transmission, eds. Ajmone Marsan, C. & Traczyk, W. Z. (Raven, New York), pp. 51-72. We thank Mr. S. C. Baker for collaboration in the initial part of the 33. Cuello, A. C., Gamse, R., Holzer, P. & Lembeck, F. (1981) Nau- work, during a Medical Research Council Dr. Zelend for nyn-Schmiedeberg's Arch. Pharmacol 315, 185-194. scholarship, J. 34. Lawson, S. N. & Nickels, S. M. (1980) J. Physiot (London) 303, demonstration ofsurgical technique, Messrs. P. J. Belkand S. Bramwell 12P. for technical assistance, Messrs. B. Archer and T. Barclay and Miss J. 35. Gamse, R., Wax, A., Zigmond, R. E. & Leeman, S. E. (1981) Lloyd for photographic work, and Miss J. Ballinger and Mrs. E. Iles Neuroscience 6, 437-442. for secretarial assistance. We gratefully acknowledge grants from the 36. von Euler, U. S. & Gaddum, J. H. (1931)J. Physiol. (London) 72, Medical Research Council, the Wellcome Trust, the Royal Society, and 74-81. the E. P. Abraham Cephalosporin Fund. 37. Dalsgaard, C.-J. & Elfvin, L.-G. (1979) Brain Res. 172, 139-143. 38. Dun, N. J. & Karczmar, A. G. (1979) Neuropharmacology 18, 215-218. 1. Hokfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. & 39. Krier, J. & Szurszewski, J. H. (1979) Fed. Proc. Fed. Am. Soc. Nilsson, G. (1977) Brain Res. 132, 29-41. Exp. Biol. 38, 958. 2. Konishi, S., Tsunoo, A. & Otsuka, M. (1979) Proc. Jpn. Acad. 55, 40. Neild, T. 0. (1978) Brain Res. 140, 231-239. 525-530. 41. Weems, W. A. & Szurszewski, J. H. (1978) J. Neurophysiol 41, 3. Jan, Y. N., Jan, L. Y. & Kuffler, S. W. (1979) Proc. Natl. Acad. 305-321. Sci. USA 76, 1501-1505. 42. Crowcroft, P. J., Holman, M. E. & Szurzewski, J. H. (1971) 1. 4. Schultzberg, M., Hokfelt, T., Terenius, L., Elfvin, L.-G., Lund- Physiol (London) 219, 443-461. berg, J. M., Brandt, J., Elde, R. P. & Goldstein, M. (1979) Neu- 43. Szurszewski, J. H. & Weems, W. A. (1976)J. Physiol (London) roscience 4, 249-270. 256, 541-556. 5. Hokfelt, T., Elfvin, L.-G., Schultzberg, M., Said, S. I., Mutt, V. 44. Kreulen, D. L. & Szurszewski, J. H. (1979)J. Physiol (London) & Goldstein, M. (1977) Neuroscience 2, 885-896. 295, 21-32.