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The role of substance P-containing fibers in sympathetic ganglia: Effect of capsaicin
Peptides, Vol. 4, pp. 769-774, 1983. ©AnkhoInternationalInc. Printedin the U.S.A.
The Role of Substance P-Containing Fibers in Sympathetic Ganglia: Effect of Capsaicin L A W R E N C E D. W I L K I N , L E E O. F A G R E , J E A N Y. J E W A N D T E R E N C E H. W I L L I A M S
Department of Anatomy, University of Iowa College of Medicine Iowa City, IA 52242
WILKIN, L. D., L. O. FAGRE, J. Y. JEW AND T. H. WILLIAMS. The role of substance P-containingfibers in sympathetic ganglia: Effect ofcapsaicin. PEPTIDES 4(5)769-774, 1983.--Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8--10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and tong-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogenous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc. Substance P
Capsaicin
Sympathetic ganglia
Mesenteric ganglia
I M M U N O H I S T O C H E M I C A L studies have revealed a dense network of nerve fibers containing substance P-like immunoreactivity (SP-I) in the celiac/superior mesenteric ganglion (CMG) and inferior mesenteric ganglion (IMG) of several mammals [13]. The immunoreactive fibers show numerous varicosities and clusters of terminals that frequently surround principal ganglionic neurons (PGNs) in a basket-like fashion, suggesting that these fibers and terminals may have a role in ganglionic neurotransmission. The basket-like SP-I arrangements of terminals have been shown at the ultrastructural level to make axodendritic synapses on PGNs in the guinea pig celiac ganglion, while other varicose fibers engage in frequent axo-axonic contacts possessing abundant vesicles in both axon profiles but no distinct pre- or postsynaptic membrane specializations [17]. The cell bodies of at least some IMG fibers containing SP-I have been localized in the dorsal root ganglia [6]. It is uncertain whether the basket-like arrangements and the varicose fibers originate from the same or different sources, however, as the afferent/efferent role of the different SP fibers and terminals in the CMG and IMG has not been clarified. Capsaicin, the active ingredient in capsicum peppers, is a potential tool for discriminating between afferent and efferent SP fibers. Capsaicin has been shown to deplete substance P (SP) content in spinal cord, dorsal root ganglia, and celiac/superior mesenteric ganglia [10, 23, 26]. It has been proposed that within the spinal cord and central nervous system, capsaicin depletes SP levels only in fibers of primary sensory neurons and their terminals but not in other types of
Guinea pig
neurons or fibers [3, 9, 12, 22]. Recently, it has also been demonstrated in the peripheral nervous system that capsaicin selectively damages only sensory afferents while leaving unmyelinated efferent fibers and intrinsic SP-I neurons of the gut intact [2,14]. In the present study, therefore, capsaicin administration has been used to resolve the afferent/efferent nature of SP-I fibers in the CMG and IMG. Specifically, do these fibers represent collaterals of a homogenous population of sensory afferents or do the basket-like PGN contacts and the other, axo-axonic contacts [ 17] represent functionally divergent fiber populations? EXPERIMENTALPROCEDURES Twelve young male guinea pigs weighing approximately 350 g were used in the present study. The experimental animals received a total dose of 125 mg of capsaicin given in 20 subcutaneous injections over 2 days in gradually increasing doses from 0.1 to l0 mg. To ameliorate the severe respiratory impairment caused by capsaicin, theophylline (0.5 mg doses up to 4-5 mg) was given IP 5 min prior to the first few capsaicin injections and an isoproterenol inhalant spray (Medihaler-Iso) was used as necessary. Control animals received no treatment, as previous studies have shown no difference in ganglionic SP between vehicle/theophyllinetreated and untreated controls [10]. Following capsaicin treatment, experimental animals were allowed to survive for 2 days or 8-10 days. Under Nembutal/Innovar anesthesia, the guinea pigs were perfused through the descending aorta
769
C A P S A I C I N A N D S U B S T A N C E P IN S Y M P A T H E T I C G A N G L I A
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FIG. 2. "'Basket-like" clusters of SP-I terminals surround principal ganglionic neurons of the inferior mesenteric ganglion (arrows).
with 50 ml of warm McIlwain's solution (pH 7.4, 37 °) followed by 500 ml of 4% paraformaldehyde in 0.167 M phosphate buffer, pH 7.4, for 15 min. The celiac/superior mesenteric and the inferior mesenteric ganglia were dissected out, immersed in the fixative for 2 hr, and incubated in 10% sucrose in phosphate-buffered saline (PBS) overnight. The tissues were then frozen and sectioned at 15/zm thickness in a cryostat. The tissue sections were mounted on chrome-alum coated slides and processed for indirect immunofluorescence according to Coons [4]. The slides were incubated with rabbit anti-SP antiserum (Immuno Nuclear Corp.) at a dilution of I:1000 in PBS/I% normal goat serum for 20 hr at 4°C. Immunostaining control slides were incubated in antiserum which had been preincubated with 1 /xg/ml of synthetic SP (Sigma). All slides were then washed in PBS and incubated with fluorescein isothyocyanate (FITC)-linked goat antirabbit gamma globulin (1:16 dilution), washed again and coverslipped with glycerin-PBS (3:1). The slides were viewed with a Leitz fluorescence microscope system equipped with BG 38/BG 12 excitation and K495 suppression filters and an Ortholux camera system. RESULTS
Control Animals Substance P-immunoreactivity was observed within discrete nerve fibers in the celiac/superior mesenteric and inferior mesenteric ganglia of non-capsaicin treated control animals. These fibers exhibited numerous varicosities and formed a dense network throughout the ganglia (Fig. 1) and were also contained within axon bundles entering and leaving the ganglia. In several areas within each ganglion, SP-I
fibers coursed in parallel rows. There were also f r e q u e n t examples of varicose, SP-I fibers surrounding principal ganglionic neurons in basket-like terminal arrangements (Fig. 2). These latter varicosities appeared to terminate on the principal ganglionic neurons. Tissues incubated in SP antiserum that was previously immunoadsorbed with SP showed no immunoreactive fibers. Capsaicin Treatment Capsaicin treatment resulted in an almost complete loss of SP-I nerve fibers in both the celiac/superior mesenteric and inferior mesenteric ganglia including the basket-like terminals as well as varicose fiber networks and axon bundles. Occasional traces of faint SP-I, suggestive of nerve fibers, could be found near the periphery of the ganglia in both 2 day and 8-10 day survival animals (Figs. 3--4). A single, varicose, brightly fluorescent fiber was seen in one 8 day animal (Fig. 5). DISCUSSION
The present results give evidence, based on combined pharmacological and morphological criteria, that SPcontaining fibers in the celiac/superior mesenteric and inferior mesenteric ganglia represent a homogenous population of sensory afferent fibers. These results, added to findings of recent fluorescent tracer and degeneration studies showing that the cell bodies of cat celiac and guinea pig mesenteric SP fibers are located in spinal dorsal root ganglia [6,11] and to biochemical results showing almost total depletion of assayable ganglionic SP by capsaicin [10], indicate that ganglionic SP fibers are sensory afferents. This hypothesis is further
CAPSAICIN AND SUBSTANCE P IN SYMPATHETIC G A N G L I A
773
FIG. 5. Superior mesenteric ganglion 10 days following capsaicin treatment. A single SP-I fiber is seen close to the edge of the ganglion.
supported by recent findings that neither visceral efferent activity [2] nor intrinsic SP neurons and efferents of the small intestine [14] are affected by capsaicin. Although afferent nerve fibers have previously been assumed to traverse the sympathetic ganglia without making functional connections [8,20], it has recently been shown that SP-I fibers traversing the abdominal sympathetic ganglia make axodendritic synaptic contacts with principle ganglionic neurons [13,17]. Additionally, a series of physiological studies has shown that SP can exert a "neuromodulatory" effect on membrane ionic conductance of principal ganglionic neurons. This effect resembles a non-cholinergic slow excitatory postsynaptic potential (EPSP) in these neurons, also resembles pre- or post-ganglionic nerve root stimulation, and is abolished by capsaicin pretreatment [7, 18, 19, 21, 26]. Thus, a mechanism exists for a subspinal reflex arc involving afferent SP fibers and PGNs. Evidence from several previous studies has suggested that somatic SP sensory fibers are involved in pain transmission [15, 16, 22, 24, 25]. I f S P fibers have a similar function in visceral pathways, it would suggest that ganglionic SP FIGURE AT RIGHT FIG. 6. Schematic drawing of SP-containing primary afferents passing through the inferior mesenteric ganglion (IMG) and their possible synaptology. Enkephalin (Enk)-containing preganglionic neurons from the intermediolateral column of the lumbar spinal cord are also included. A SP-containing afferent collateral forms synaptic contact with the principal ganglionic neuron, and both afferents have several varicosities. It is possible that the preganglionic endings establish contact with the axon collateral or with the main afferent fibers from the SP-containing primary sensory neurons.
FIG. 6.
774
W I L K 1 N El" A L .
fibers or their collaterals m a y exert a direct modulatory effect on gut activity in r e s p o n s e to visceral irritation. This n e u r o m o d u l a t o r y f e e d b a c k effect would o c c u r prior to afferent synaptic contact within the spinal cord. The f e e d b a c k o f SP afferent collaterals within the I M G may be modulated by enkephalinergic efferents. C o m b i n e d retrograde tracing and i m m u n o f l u o r e s c e n c e has r e v e a l e d that at least s o m e of the enkephalin-containing fibers in the I M G originate in the sympathetic preganglionic intermediolateral cell column of the spinal cord [6]. An i m m u n o e l e c t r o n - m i c r o s c o p i c a l analysis o f SP-I nerve fibers in the m e s e n t e r i c ganglion has indicated that axo-axonic contacts may o c c u r b e t w e e n enkephalin and SP terminals [17]. O n e physiological study by Konishi et al. [18] demonstrated that SP-mediated slow E P S P s in the inferior mesenteric ganglion are subject to enkephalinergic presynaptic inhibition. Figure 6 illustrates possible synaptic relationships of SP-containing afferents and afferent collaterals in the I M G . F u r t h e r electrophysiological, anatomical and ultrastructural investigations o f possible interactions b e t w e e n SP-ergic and
enkephalinergic systems in the sympathetic ganglia are necessary in order to completely understand the interaction bet w e e n visceral nociception and motility. Although the findings in the present study indicate that all of the SP-I nerve fibers in the mesenteric ganglia represent visceral afferents, it is yet possible that a divergence of function exists regarding synaptic contacts within the ganglia. E l e c t r o n m i c r o s c o p i c a l l y , K o n d o and Yui [17] distinguished clearly b e t w e e n SP terminals that contact principal ganglionic neurons and SP varicosities that engage in axoaxonic contacts with other presumed peptidergic or enkephalinergic varicosities. As illustrated in Fig. 6, it is unclear whether or not axo-axonic synaptic activity can affect axo-somatic activity of SP terminals. This information would be very useful in understanding the inhibitory action o f enkephalin on SP-induced slow EPSPs in the mesenteric ganglia. The relationships of the different types of SP terminals to each other, to the ganglion cells, and to enkephalinand other peptide-containing fibers represent areas for future research.
REFERENCES 1. Baker, S. C,, A. C. Cuello and M. R. Matthews. Substance P-containing synapses in a sympathetic ganglion, and their possible origin as collaterals from sensory nerve fibers. J Physiol (Lond) 308: 76--77P, 1980. 2. Cervero, F. and H. A. McRitchie. Neonatal capsaicin does not affect unmyelinated efferent fibers of the autonomic nervous system: functional evidence. Brain Res 239: 283-288, 1982. 3. Cuello, A. C., R. Gamse, R. Holzer and F. Lembeck. Substance P immunoreactive neurons following neonatal administration of capsaicin. Naunyn Schmiedebergs Arch Pharmaeol 215: 185194, 1981. 4. Coons, A. H. Fluorescent antibody methods. In: General Cytochemical Methods, edited by J. F. Danielli. New York: Academic Press, 1958, pp. 399-422. 5. Dalsgaard, C. J., T. HOkfelt, L.-G. Elfvin, L. Skirboll and P. Emson. Substance P containing primary sensory neurons projecting to the inferior mesenteric ganglion: evidence from combined retrograde tracing and immunohistochemistry. Neuroseience 7: 647-654, 1979. 6. Dalsgaard, C. J., T. HOkfelt, L.-G. Elfvin and L. Terenius. Enkephalin-containing sympathetic preganglionic neurons projecting to the inferior mesenteric ganglion: Evidence from combined retrograde tracing and immunohistochemistry. Neuroscience 7: 2039-2050, 1982. 7. Dun, N. J. and A. G. Karczmar. Actions of substance P on sympathetic neurons. Neuropharrnaeology 18:215-218, 1979. 8. Gabella, G. Structure o f the Autonomic Nerw~us System. London: Chapman and Hall, 1976. 9. Gamse, R., P. Holzer and F. Lembeck. Decrease of substance P in primary afferent neurones and impairment of neurogenic plasma extravasation by capsaicin. Br J Pharmaeol 68: 207-213, 1980. 10. Gamse, R., A. Wax, R. E. Zigmond and S. E. Leeman. lmmunoreactive substance P in sympathetic ganglia: distribution and sensitivity towards capsaicin. Neuroscience 6: 437-441, 1981. 11. Hayashi, H,, K. Oshumi, N. Veda, M. Fujiwara and N. Mizuno. Effect of spinal ganglionectomy on substance P-like immunoreactivity in the gastroduodenal tract of cats. Brain Res 232: 227-230, 1982. 12. Helke, C. J., J. A. DiMicco, D. M. Jacobowitz and I. J. Kopin. Effect of capsaicin administration to neonatal rats on the substance P content of discrete CNS regions. Brain Res 222: 428431, 1981.
13. HOkfelt, T., F. G. Elfvin. M. Schultzberg, M. Goldstein and G. Nilson. On the occurrence of substance-P containing fibers in sympathetic ganglia: immunohistochemical evidence. Brain Res 132: 29-41, 1977. 14. Holzer, P., R. Gamse and F. Lembeck. Distribution of substance P in the rat gastrointestinal tract--lack of effect of capsaicin. Eur J Pharmaeol 61: 303-307, 1980. 15. Jessell, T. M., L. L. Iversen and A. C. Cuello. Capsaicininduced depletion of substance P from primary sensory neurones. Brain Res 152: 183-188, 1978. 16. Juan, H., F. Lembeck, S. Seewann and U. Hack. Nociceptor stimulation and PGE release by capsaicin. Naunyn Schmiedebergs Arch Pharmacol 312: 139-143, 1980. 17. Kondo, H. and R. Yui. An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs. Brain Res 222: 134--t37, 1981. 18. Konishi, S.. A. Tsunoo and M. Otsuka. Substance P and noncholinergic excitatory synpatic transmission in guinea pig sympathetic ganglia. Proc" Jpn A('ad 55: Set B, 525-530, 1979. 19. Konishi, S., A. Tsunoo, N. Yanaihara and M. Otsuka. Peptidergic excitatory and inhibitory synapses in mammalian sympathetic ganglia: roles of substance P and enkephalin. Biomed Res 1: 528-536, 1980. 20. Kuntz, A. The Autonomic Nervous System. London: Tidall and Cox, 1946. 21. Minota, S., N. 1. Dun and A. G. Karczmar. Substance P-induced depolarization in sympathetic neurons: not simple K-inactivation. Brain Res 216: 224--228, 1981. 22. Nagy, J. I., S. R. Vincent, W. A. Staines, H. C. Fibiger, T. D. Reisine and H. I. Yamamura. Neurotoxic action of capsaicin on spinal substance P neurons. Brain Res 186: 435-444, 1980. 23. Nagy, J. I., S. P. Hunt, L. L. lversen and P. C. Emson. Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin. Neuroscience 6: 1923-1934, 1981. 24. Piercy, M. F., L, A. Schroeder, K. Folkers, J.-C. Xu and K. Horig. Sensory and motor functions of spinal cord substance P. Science 214: 1361-1363, 1977. 25. Randic, M. and V. Miletic. Effect of substance P in cat dorsal horn neurones activated by noxious stimuli. Brain Res 128: 164-169, 1977. 26. Tsunoo, A., S. Konishi and M. Otsuka. Substance P as an excitatory transmitter of primary afferent neurons in guinea-pig sympathetic ganglia. Neuroscienee 7: 2025-2037, 1982. 27. Virus, R. M. and G. F. Gebbart. Pharmacologic actions of capsaicin: apparent involvement of substance P and serotonin. Lift, Sei 25: 1273-1284. 1979.
The Role of Substance P-Containing Fibers in Sympathetic Ganglia: Effect of Capsaicin L A W R E N C E D. W I L K I N , L E E O. F A G R E , J E A N Y. J E W A N D T E R E N C E H. W I L L I A M S
Department of Anatomy, University of Iowa College of Medicine Iowa City, IA 52242
WILKIN, L. D., L. O. FAGRE, J. Y. JEW AND T. H. WILLIAMS. The role of substance P-containingfibers in sympathetic ganglia: Effect ofcapsaicin. PEPTIDES 4(5)769-774, 1983.--Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8--10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and tong-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogenous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc. Substance P
Capsaicin
Sympathetic ganglia
Mesenteric ganglia
I M M U N O H I S T O C H E M I C A L studies have revealed a dense network of nerve fibers containing substance P-like immunoreactivity (SP-I) in the celiac/superior mesenteric ganglion (CMG) and inferior mesenteric ganglion (IMG) of several mammals [13]. The immunoreactive fibers show numerous varicosities and clusters of terminals that frequently surround principal ganglionic neurons (PGNs) in a basket-like fashion, suggesting that these fibers and terminals may have a role in ganglionic neurotransmission. The basket-like SP-I arrangements of terminals have been shown at the ultrastructural level to make axodendritic synapses on PGNs in the guinea pig celiac ganglion, while other varicose fibers engage in frequent axo-axonic contacts possessing abundant vesicles in both axon profiles but no distinct pre- or postsynaptic membrane specializations [17]. The cell bodies of at least some IMG fibers containing SP-I have been localized in the dorsal root ganglia [6]. It is uncertain whether the basket-like arrangements and the varicose fibers originate from the same or different sources, however, as the afferent/efferent role of the different SP fibers and terminals in the CMG and IMG has not been clarified. Capsaicin, the active ingredient in capsicum peppers, is a potential tool for discriminating between afferent and efferent SP fibers. Capsaicin has been shown to deplete substance P (SP) content in spinal cord, dorsal root ganglia, and celiac/superior mesenteric ganglia [10, 23, 26]. It has been proposed that within the spinal cord and central nervous system, capsaicin depletes SP levels only in fibers of primary sensory neurons and their terminals but not in other types of
Guinea pig
neurons or fibers [3, 9, 12, 22]. Recently, it has also been demonstrated in the peripheral nervous system that capsaicin selectively damages only sensory afferents while leaving unmyelinated efferent fibers and intrinsic SP-I neurons of the gut intact [2,14]. In the present study, therefore, capsaicin administration has been used to resolve the afferent/efferent nature of SP-I fibers in the CMG and IMG. Specifically, do these fibers represent collaterals of a homogenous population of sensory afferents or do the basket-like PGN contacts and the other, axo-axonic contacts [ 17] represent functionally divergent fiber populations? EXPERIMENTALPROCEDURES Twelve young male guinea pigs weighing approximately 350 g were used in the present study. The experimental animals received a total dose of 125 mg of capsaicin given in 20 subcutaneous injections over 2 days in gradually increasing doses from 0.1 to l0 mg. To ameliorate the severe respiratory impairment caused by capsaicin, theophylline (0.5 mg doses up to 4-5 mg) was given IP 5 min prior to the first few capsaicin injections and an isoproterenol inhalant spray (Medihaler-Iso) was used as necessary. Control animals received no treatment, as previous studies have shown no difference in ganglionic SP between vehicle/theophyllinetreated and untreated controls [10]. Following capsaicin treatment, experimental animals were allowed to survive for 2 days or 8-10 days. Under Nembutal/Innovar anesthesia, the guinea pigs were perfused through the descending aorta
769
C A P S A I C I N A N D S U B S T A N C E P IN S Y M P A T H E T I C G A N G L I A
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FIG. 2. "'Basket-like" clusters of SP-I terminals surround principal ganglionic neurons of the inferior mesenteric ganglion (arrows).
with 50 ml of warm McIlwain's solution (pH 7.4, 37 °) followed by 500 ml of 4% paraformaldehyde in 0.167 M phosphate buffer, pH 7.4, for 15 min. The celiac/superior mesenteric and the inferior mesenteric ganglia were dissected out, immersed in the fixative for 2 hr, and incubated in 10% sucrose in phosphate-buffered saline (PBS) overnight. The tissues were then frozen and sectioned at 15/zm thickness in a cryostat. The tissue sections were mounted on chrome-alum coated slides and processed for indirect immunofluorescence according to Coons [4]. The slides were incubated with rabbit anti-SP antiserum (Immuno Nuclear Corp.) at a dilution of I:1000 in PBS/I% normal goat serum for 20 hr at 4°C. Immunostaining control slides were incubated in antiserum which had been preincubated with 1 /xg/ml of synthetic SP (Sigma). All slides were then washed in PBS and incubated with fluorescein isothyocyanate (FITC)-linked goat antirabbit gamma globulin (1:16 dilution), washed again and coverslipped with glycerin-PBS (3:1). The slides were viewed with a Leitz fluorescence microscope system equipped with BG 38/BG 12 excitation and K495 suppression filters and an Ortholux camera system. RESULTS
Control Animals Substance P-immunoreactivity was observed within discrete nerve fibers in the celiac/superior mesenteric and inferior mesenteric ganglia of non-capsaicin treated control animals. These fibers exhibited numerous varicosities and formed a dense network throughout the ganglia (Fig. 1) and were also contained within axon bundles entering and leaving the ganglia. In several areas within each ganglion, SP-I
fibers coursed in parallel rows. There were also f r e q u e n t examples of varicose, SP-I fibers surrounding principal ganglionic neurons in basket-like terminal arrangements (Fig. 2). These latter varicosities appeared to terminate on the principal ganglionic neurons. Tissues incubated in SP antiserum that was previously immunoadsorbed with SP showed no immunoreactive fibers. Capsaicin Treatment Capsaicin treatment resulted in an almost complete loss of SP-I nerve fibers in both the celiac/superior mesenteric and inferior mesenteric ganglia including the basket-like terminals as well as varicose fiber networks and axon bundles. Occasional traces of faint SP-I, suggestive of nerve fibers, could be found near the periphery of the ganglia in both 2 day and 8-10 day survival animals (Figs. 3--4). A single, varicose, brightly fluorescent fiber was seen in one 8 day animal (Fig. 5). DISCUSSION
The present results give evidence, based on combined pharmacological and morphological criteria, that SPcontaining fibers in the celiac/superior mesenteric and inferior mesenteric ganglia represent a homogenous population of sensory afferent fibers. These results, added to findings of recent fluorescent tracer and degeneration studies showing that the cell bodies of cat celiac and guinea pig mesenteric SP fibers are located in spinal dorsal root ganglia [6,11] and to biochemical results showing almost total depletion of assayable ganglionic SP by capsaicin [10], indicate that ganglionic SP fibers are sensory afferents. This hypothesis is further
CAPSAICIN AND SUBSTANCE P IN SYMPATHETIC G A N G L I A
773
FIG. 5. Superior mesenteric ganglion 10 days following capsaicin treatment. A single SP-I fiber is seen close to the edge of the ganglion.
supported by recent findings that neither visceral efferent activity [2] nor intrinsic SP neurons and efferents of the small intestine [14] are affected by capsaicin. Although afferent nerve fibers have previously been assumed to traverse the sympathetic ganglia without making functional connections [8,20], it has recently been shown that SP-I fibers traversing the abdominal sympathetic ganglia make axodendritic synaptic contacts with principle ganglionic neurons [13,17]. Additionally, a series of physiological studies has shown that SP can exert a "neuromodulatory" effect on membrane ionic conductance of principal ganglionic neurons. This effect resembles a non-cholinergic slow excitatory postsynaptic potential (EPSP) in these neurons, also resembles pre- or post-ganglionic nerve root stimulation, and is abolished by capsaicin pretreatment [7, 18, 19, 21, 26]. Thus, a mechanism exists for a subspinal reflex arc involving afferent SP fibers and PGNs. Evidence from several previous studies has suggested that somatic SP sensory fibers are involved in pain transmission [15, 16, 22, 24, 25]. I f S P fibers have a similar function in visceral pathways, it would suggest that ganglionic SP FIGURE AT RIGHT FIG. 6. Schematic drawing of SP-containing primary afferents passing through the inferior mesenteric ganglion (IMG) and their possible synaptology. Enkephalin (Enk)-containing preganglionic neurons from the intermediolateral column of the lumbar spinal cord are also included. A SP-containing afferent collateral forms synaptic contact with the principal ganglionic neuron, and both afferents have several varicosities. It is possible that the preganglionic endings establish contact with the axon collateral or with the main afferent fibers from the SP-containing primary sensory neurons.
FIG. 6.
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W I L K 1 N El" A L .
fibers or their collaterals m a y exert a direct modulatory effect on gut activity in r e s p o n s e to visceral irritation. This n e u r o m o d u l a t o r y f e e d b a c k effect would o c c u r prior to afferent synaptic contact within the spinal cord. The f e e d b a c k o f SP afferent collaterals within the I M G may be modulated by enkephalinergic efferents. C o m b i n e d retrograde tracing and i m m u n o f l u o r e s c e n c e has r e v e a l e d that at least s o m e of the enkephalin-containing fibers in the I M G originate in the sympathetic preganglionic intermediolateral cell column of the spinal cord [6]. An i m m u n o e l e c t r o n - m i c r o s c o p i c a l analysis o f SP-I nerve fibers in the m e s e n t e r i c ganglion has indicated that axo-axonic contacts may o c c u r b e t w e e n enkephalin and SP terminals [17]. O n e physiological study by Konishi et al. [18] demonstrated that SP-mediated slow E P S P s in the inferior mesenteric ganglion are subject to enkephalinergic presynaptic inhibition. Figure 6 illustrates possible synaptic relationships of SP-containing afferents and afferent collaterals in the I M G . F u r t h e r electrophysiological, anatomical and ultrastructural investigations o f possible interactions b e t w e e n SP-ergic and
enkephalinergic systems in the sympathetic ganglia are necessary in order to completely understand the interaction bet w e e n visceral nociception and motility. Although the findings in the present study indicate that all of the SP-I nerve fibers in the mesenteric ganglia represent visceral afferents, it is yet possible that a divergence of function exists regarding synaptic contacts within the ganglia. E l e c t r o n m i c r o s c o p i c a l l y , K o n d o and Yui [17] distinguished clearly b e t w e e n SP terminals that contact principal ganglionic neurons and SP varicosities that engage in axoaxonic contacts with other presumed peptidergic or enkephalinergic varicosities. As illustrated in Fig. 6, it is unclear whether or not axo-axonic synaptic activity can affect axo-somatic activity of SP terminals. This information would be very useful in understanding the inhibitory action o f enkephalin on SP-induced slow EPSPs in the mesenteric ganglia. The relationships of the different types of SP terminals to each other, to the ganglion cells, and to enkephalinand other peptide-containing fibers represent areas for future research.
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