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Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord
Brain Research, 418 (1987) 371-376 Elsevier
371
BRE 22441
Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord Gfibor Jancs61 and Carlo Alberto Maggi 2 1Department of Physiology, University Medical School, Szeged (Hungary) and 2pharmacology Department, Research Laboratories, A. Menarini Pharmaceuticals, Florence (Italy) (Accepted 12 May 1987)
Key words: Capsaicin; Primary sensory afferent; Urinary bladder; Spinal cord; Horseradish peroxidase; Selective degeneration
The capsaicin-sensitive afferent innervation of the urinary bladder and the central nervous system distribution of urinary bladder afferents have been studied in the rat. Capsaicin-sensitive primary sensory neurones supplying the urinary bladder have been found in two groups of spinal ganglia located in the Th13-L 2 and L6-S1segments. Capsaicin-sensitive primary sensory afferents from the bladder terminate within Rexed's laminae I, V and X, and in the dorsal gray commissure of the lumbosacral spinal cord. In addition, the results point to a possible vagal sensory innervation of the urinary bladder.
Administration of the potent sensory neurotoxin capsaicin to newborn animals results in a selective destruction of a morphologically well characterized population of primary sensory neurones 11'12'14'16.Animals treated at birth with capsaicin display no gross abnormalities but show characteristic disturbances of several somatic and visceral sensory mechanisms as adults (see e.g. refs. 2, 4, 7, 13, 14 and 17). Indeed, hypertrophy of the urinary bladder and urine retention have been reported to occur after neonatal capsaicin treatment and have been attributed to a loss of substance P-containing afferent nerve fibres innervating the urinary bladder 29'3°. The available experimental evidence indicates that capsaicin-sensitive peptide-containing afferent nerves, by forming the afferent arch of the micturition reflex pathway, play a fundamental role in the nervous control of the urinary bladder 9,18'2°. Recent studies by Maggi et alfl ° pointed to the possibility that some capsaicin-sensitive bladder afferents involved in the initiation of vesicovesical micturition reflexes may terminate at supraspinal levels. The decrease in the number of peptide-containing
primary sensory ganglion cells supplying the rat urinary bladder after neonatal capsaicin treatment is well established 29,3°. However, the central projections of capsaicin-sensitive primary sensory neurones innervating the urinary bladder have not been studied. Therefore, the present experiments were disigned to reveal the distribution of capsaicin-sensitive bladder afferents within the spinal cord and to investigate, by using a pharmaco-morphological approach, the possibility of a termination of these particular afferents at more rostral levels of the neuraxis. Adult male S p r a g u e - D a w l e y CFY rats weighing 300-350 g were used in these experiments. One group of animals was injected with capsaicin or its vehicle (10% ethanol, 10% Tween 80 in saline) at a dose of 50 mg/kg on the second day of life 14. A n o t h e r group of animals was injected with a 100-mg/kg dose of capsaicin or with the vehicle as adults. The retrograde and transganglionic transport of horseradish peroxidase conjugated with wheat germ agglutinin ( W G A - H R P ) was used to demonstrate the cell bodies and the central projections of primary
Correspondence: G. Jancs6. Present address: Department of Physiology, The Medical School, University of Bristol, University Walk, Bristol BS8 1TD, U.K. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
372 sensory neurones innervating the urinary bladder. In neonatally treated animals the urinary bladder was exposed through a midline incision of the abdominal wall and carefully isolated by using cotton wool moistened with warm isotonic saline. Injections of a 1% solution of W G A - H R P (Sigma) in distilled water were given into the wall of the bladder at 3 different sites in a total volume of 7.5 #1, whereafter the exposed area was thoroughly rinsed with saline and the wound closed. After a survival time of 72 h the rats were perfused transcardially with an aldehyde-containing fixative according to the procedure of Rosene and Mesulam 27. The spinal sensory ganglia from Thl2-S 3 on both sides, the nodose ganglia, the brainstem and the spinal cord were removed and stored in 0.1 M phosphate buffer containing 10% sucrose. Frozen sections of 15 and 50 # m thickness were cut from the sensory ganglia and the brainstem and spinal cord, respectively, and processed for the demonstration of H R P enzyme activity using tetramethylbenzidine (Sigma) as a chromogen 22. Sensory ganglion ceils showing heavy perikaryal labelling and/or a prominent nucleus with distinct cytoplasmic staining were counted under bright-field illumination at an objective magnification of x40. Sections of the brainstem and spinal cord were viewed and photographed under dark-field illumination. Camera lucida drawings were made by using a Leitz Dialux microscope equipped with a drawing tube. Animals treated with capsaicin or its vehicle as adults were anaesthetized and perfused through the left heart ventricle with 10% formalin 7 - 9 h after the injection. Serial frozen sections of the lumbosacral
spinal cord and the brainstem were processed for the demonstration of axon terminal degeneration according to Eager's silver impregnation technique °. In control animals injection of W G A - H R P into the wall of the urinary bladder resulted in the labelling of two segmentally different groups of sensory neurones located in the Thl3-1_.2 and L~-S~ spinal ganglia. The numbers and segmental distribution of retrogradely labelled dorsal root ganglion neurones are shown in Table I. These findings corroborate previous observations x,24,29,3° and are consistent with the notion that both the lumbar (hypogastric) and sacral (pelvic) plexuses contribute to the sensory innervation of the urinary bladder 1. As it is shown in Table I. neonatal capsaicin treatment resulted in a dramatic reduction in the number of retrogradely labelled cells in these sensory ganglia although the percentage distribution of the labelled neurones remained essentially unchanged. These results are consistent with previous findings showing that the fibre population of the rat pelvic nerve ts dominated by unmyelinated axons 1° and that neonatal administration of capsalcin at a dose similar to that used in the present study results in an almost complete loss of unmyelinated dorsal root afferent fibres 25. In addition to spinal sensory ganglia a few labelled cells were consistently revealed in the nodose ganglia of both control and capsaicin-treated rats. Their number varied between 10 and 15 per ganglion and was apparently unaffected by capsaicin treatment. Although innervation of the urinary bladder by vagal afferent fibres has not been demonstrated before, this is not entirely unexpected in the light of recent
TABLE I Numbers and segmental distribution o f labelled dorsal root ganglion cells after injection o f W G A - H R P into the wall o f the urinary bladder o f control and capsaicin-treated rats Th12
Th13
Lt
L2
L3
L4
L5
Lo
,5"I
$2
0 0 0 0
49 5 7 87
71 64 23 330
56 110 72 248
0 9 l 0
(I I1 0 0
0 0 0 0
247 311 202 468
555 492 717 689
0 0 0 0
0 0 0 0
0 0 4 7
10 3 13 11
7 3 13 7
0 0 0 0
0 0 0 0
0 0 0 0
12 47 3 8
99 7l 162 114
0 0 0 0
Control 1
2 3 4 Capsaicin 1 2 3 4
373 observations showing a substantial contribution of vagal afferent fibres to the sensory innervation of the rat uropoietic system s. The low number of retrogradely labelled nodose ganglion cells may be related to the small actual amount of tracer injected in the present experiments rather than to the number of nodose ganglion cells innervating the bladder. In studies aimed at the accurate estimation of the contribution of the vagus to the afferent innervation of the kidney, significantly higher amounts of the tracer were injected into the organ s. The restricted viscerotopic localization of the labelled dorsal root ganglion neurones demonstrated in the present study makes a non-specific labelling of nodose ganglion cells unlikely. In control animals distinct transganglionic labelling of urinary bladder afferents was observed in the L6-S 1 spinal cord segments. Primary afferents entering the cord through the L 6 and $l spinal dorsal roots distributed to the Lissauer zone and labelled the entire mediolateral extent of the tract. Afferent collaterals from the Lissauer tract entered the marginal zone or reached the deeper layers of the grey matter by way of two major projections. Few afferent fibres reached the dorsal grey commissure through the medial collateral pathway. The great majority of primary afferent collaterals left Lissauer tract via a lateral collateral pathway formed by one or more prominent fascicles running between the longitudinally oriented myelinated fibre bundles of the spinal reticular formation towards the intermediolateral grey. Upon entering the grey matter afferent collaterals gave rise to the following main terminal arborization territories: fibres coursing medially apparently terminated in the dorsal grey commissure and within the dorsalmost region of lamina X. Ventrally and ventrolaterally directed fibres extended into the lateral part of laminae V and VI and into the region of the sacral parasympathetic nucleus (Fig. 1A,B). Fibre bundles forming the lateral collateral pathway emerged from Lissauer tract with a more or less regular periodicity. In our material these bundles measuring about 40 #m were spaced approximately 150-300 ktm apart as measured in parasagittal sections. The pattern of spinal termination of capsaicinsensitive bladder afferents described here closely resembles that reported for pelvic nerve afferents in the rat 24 and further corroborates the view that the
substantia gelatinosa is essentially deprived of a prominent visceral afferent input 3'5'2426. Although transganglionic labelling of primary afferents was consistently obtained in control animals after the injection of W G A - H R P into the urinary bladder wall, a similar labelling could not be demonstrated in any of the rats treated with capsaicin at birth. The most likely explanation of this finding is that the amount of the tracer taken up and transported by the few remaining sensory neurones supplying the urinary bladder was insufficient to provide a reliable labelling of their central processes. The results also imply that the great majority of afferent nerves innervating the urinary bladder belong to capsaicin-sensitive primary sensory neurones. These findings provide an anatomical basis to the observation that reflex micturition is almost abolished in rats treated with capsaicin at birth Is. However, bladder voiding can be elicited in response to transvesical saline filling in a certain percentage of these animals providing cystometrograms are obtained in the conscious state (Conte and Maggi, unpublished data). This supports the idea that sensory input for 'conscious' micturition may be at least in part, capsaicin-resistant 19. The projection of capsaicin-sensitive primary afferents to the region of the sacral parasympathetic nucleus is particularly interesting: recent experiments in spinal rats indicate that capsaicin-sensitive afferents provide synaptic input for initiation of a spinal vesicovesical excitatory reflex 21. On the other hand, primary afferents terminating in lamina V may provide synaptic input to second order sensory neurones projecting from this area to supraspinal centres 23. In accord with our previous reports 15, systemic injection of capsaicin to adult rats resulted in a massive, rapid degeneration of primary sensory afferents terminating within the superficial laminae of the spinal cord dorsal horn. In addition, less marked but distinct axon terminal degeneration was observed in lamina V and within the dorsal grey commissure of the spinal cord at L6-$1 segmental levels (Fig. 1C-E). Available experimental evidence indicates that degeneration in lamina II and to some extent in lamina I may be attributed to the destruction of somatic afferents 3'5"26. It is, therefore, reasonable to assume that degenerating axon terminals observed in laminae I
374
B
E
"°
0
Fig. 1. A: dark-field photomicrograph illustrating the distribution of WGA-HRP-labelIed urinary bladder afferents in the L6 spinal cord segment of a control rat × 92. B: reconstruction by camera lucida drawing of 4 transverse sections of the L~ spinal cord segment showing the characteristic distribution of labelled urinary bladder afferents in control animals C and D: degenerating axon terminals in laminae I and II (C) and in lamina V (D) of the L6 spinal cord segment of a capsaicin treated rat. Eager's silver impregnation technique × 368. E: spinal termination fields of capsaicin-sensitive primary afferents based on camera lucida drawings of silver-stained sections of the L6spinal cord segment of an adult rat treated with capsaicin 7 h before killing.
and V and in the dorsal grey commissure overlapping with areas of termination of urinary bladder afferents represent the central projection areas of capsaicinsensitive primary sensory n e u r o n e s innervating pelvic viscera, including the urinary bladder.
In agreement with previous findings systemic injection of capsaicin produced extensive axon terminal degeneration in the brainstem within the nuclei of the solitary tract and the trigemmal nuclear complex 15. Neither capsaicin-induced degeneration, nor
375 HRP-labelled neuronal elements could be demon-
ment on this innervation. The pattern of spinal termi-
strated in the dorsolateral pontine t e g m e n t u m , a re-
nation of capsaicin-sensitive primary sensory after-
gion specifically implicated in the supraspinal control of micturition28.
ents from the rat bladder has also been described.
Since H R P labelling could not be observed in medullary or pontine structures after the injection of the
urinary bladder) input in the area of the dorsal grey commissure is particularly interesting in view of the
tracer into the wall of the urinary bladder, the results
proposed role of this region for the integration of somatovisceral afferentations and co-ordination of the excretive functions of pelvic viscera Is. In addition,
failed to provide evidence for the notion that capsai-
The convergence of somatic and visceral (from the
cin-sensitive primary sensory neurones innervating the bladder project to supraspinal regions 2°. It is to
the results draw attention to a possible innervation of
be emphasized however, that the existence of such
the urinary bladder by sensory fibres of vagal origin.
projections cannot be excluded on the basis of the present observations. Indeed, a medullary projection of renal afferent fibres could only be demonstrated by using double labelling with fluorescent dyes but not by transganglionic labelling with H R P 31.
This work was supported by a grant ( O T K A 4/1104/86) from the H u n g a r i a n A c a d e m y of Sciences and by a grant (VES 46287) from IMI, Rome. The
In conclusion, the present study confirms previous
authors wish to thank Dr. F. Cervero and Dr. S.N. Lawson for critical reading of the manuscript. We
reports on the n u m b e r and segmental distribution of primary sensory neurones innervating the urinary bladder and on the effect of neonatal capsaicin treat-
thank K. Mohficsi and H. Szab6 for skillful technical assistance, P. Robbins for photographic work and Sue Maskell for typing the manuscript.
1 Applebaum, A.E., Vance, W.H. and Coggeshall, R.E., Segmental localization of sensory cells that innervate the bladder, J. Comp. Neurol., 192 (1980) 203-209. 2 Buck, S.H. and Burks, T.F., The neurophysiology of capsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. 3 Cervero, F. and Connell, L.A., Distribution of somatic and visceral primary afferent fibres within the thoracic spinal cord of the cat, J. Comp. Neurol., 230 (1984) 88-98. 4 Cervero, F. and Plenderleith, M.B., Spinal cord sensory systems after neonatal capsaicin, Acta Physiol. Hung., in press. 5 De Groat, W.C., Spinal cord projections and neuropeptides in visceral afferent neurons. In F. Cervero and J.F.B. Morrison (Eds.), Visceral Sensation, Progress in Brain Research, Vol. 67, Elsevier, Amsterdam, 1986, pp. 165-187. 6 Eager, R.P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 7 Fitzgerald, M., Capsaicin and sensory neurones - - a review, Pain, 15 (1983) 109-130. 8 Gattone II, V.H., Marfurt, C.F. and Dallie, S., Extrinsic innervation of the rat kidney: a retrograde tracing study, Am. J. Physiol., 250 (1986) F189-F196. 9 Holzer-Petsche, U. and Lembeck, F., Systemic capsaicin treatment impairs the micturition reflex in the rat, Br. J. Pharmacol., 83 (1984) 935-941. 10 Hulsebosch, C.E. and Coggeshall, R.E., An analysis of the axon populations in the nerves to the pelvic viscera in the rat, J. Comp. Neurol., 211 (1982) 1-10. 11 Jancs6, G., H6kfelt, T., Lundberg, J.M., Kirfily, E., Halfisz, N., Nilsson, G., Terenius, L., Rehfeld, J., Steinbusch, H., Verhofstad, A., Elde, R., Said, S. and Brown, M., Immunohistochemical studies on the effect of capsaicin
on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine, J. Neuroeytol., 10 (1981) 963-980. 12 Jancs6, G., Kir~ly, E. and Jancs6-Gfibor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones, Nature (London), 270 (1977) 741-743. 13 Jancs6, G., Kir~ily,E. and Jancs6-G~ibor, A., Chemosensitive pain fibres and inflammation, Int. J. Tissue React., 2 (1981) 57-66. 14 Jancs6, G. and Kirfily, E., Sensory neurotoxins: chemically induced selective destruction of primary sensory neurons, Brain Research, 210 (1981) 83-89. 15 Jancs6, G., Kir~ily,E., Jo6, F., Such, G. and Nagy, A., Selective degeneration by capsaicin of a subpopulation of primary sensory neurons in the adult rat, Neurosci. Lett., 59 (1985) 209-214. 16 Lawson, S.N. and Nickels, S.M., The use of morphometric techniques to analyse the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots, J. Physiol. (London), 303 (1980) 12P. 17 Lembeck, F. and Gamse, R., Substance P in peripheral sensory processes. In R. Porter and M. O'Connor (Eds.), Substance P in the Nervous System, Ciba Foundation Symposium, 91, Pitman, London, 1982, pp. 35-54. 18 Maggi, C.A. and Meli, A., The role of neuropeptides in the regulation of the micturition reflex, J. Auton. Pharmacol., 6 (1986) 133-162. 19 Maggi, C.A. and Meli, A., 'Conscious' and 'reflex' micturition: do they have a separate sensory input? Implications for clinical urodynamics, Neurourol. Urodyn., in press. 20 Maggi, C.A., Santicioli, P., Borsini, F., Giuliani, S. and Meli, A., The role of the capsaicin-sensitive innervation of the rat urinary bladder in the activation of micturition re-
376
21
22
23
24
25
flex, Naunyn-Schmiedeberg's Arch. Pharmacol., 332 (1986) 276-283. Maggi, C.A., Santicioli, P., Geppetti, P., Furio, M., FriUi, S., Conte, B., Fanciulacci, M., Guiliani, S. and Meli, A., The contribution of capsaicin-sensitive innervation to activation of the spinal vesico-vesical reflex in rats: relationship between substance P levels in the urinary bladder and the sensory-afferent function in capsaicin-sensitive sensory neurons, Brain Research, in press. Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochern. Cytochem., 26 (1978) 106-117. Milen, R.J., Foreman, R.G., Giesler, G.J. and Willis, W.D., Convergence of cutaneous and pelvic visceral nociceptive inputs onto primate spinothalamic neurons, Pain. 11 (1981) 163-183. Nadelhaft, I. and Booth, A.M., The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study, J. Comp. Neurol., 226 (1984) 238-245. Nagy, J.I., Iversen, L.L., Goedert, M., Chapman, D. and Hunt, S.P., Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat, J. Neurosci., 3 (1983) 399-406.
26 Neuhuber. W.L., Sandoz, P.A. and l-ryscak. T.. The central projections of primary afferent neurons of greater splanchnic and intercostal nerves in the rat. A horseradish peroxidase study, Anat. Embrvol.. 174 (1986) 123-t44. 27 Rosene. D.L.. and Mesulam. M.-M., Fixation variables in horseradish peroxidase neurochemistrv. I. The effects of fixation time and perfusion procedures upon enzyme activity. J. Histochem. Cytochem.. 26 [ 1978) 28-39. 28 Satoh. K.. Shimizu. N.. Tohyama. M and Maeda_ T.. Localization of the micturition reflex center at dorsolateral pontine tegmentum of the rat Neurosci. Lett. 8 ~1978~ 27-33 29 Sharkey, K.A.. Williams. R.(J.. Schultzberg, M. and Dockray, G.J., Sensory substance P innervation of the urinary bladder: possible site of action of capsaicin m causing urine retention in rats. Neuroscience, 3 ~1983) 861-868. 30 Su. H.C.. Wharton. J.. Polak. J.M.. Mutderry. P.K.. Ghatel. M.A.. Gibson, S.J., Terenghi, G . Morrison. J.F.B.. Ballesta. J and Bloom. S.R.. Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry, Neuroscience. 18 I1986) 727-747, 31 Wyss, J.M. and Donovan. M.K.. A direct projection from the kidney to the brainstem Brain Research. 298 (t984p 130-134.
371
BRE 22441
Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord Gfibor Jancs61 and Carlo Alberto Maggi 2 1Department of Physiology, University Medical School, Szeged (Hungary) and 2pharmacology Department, Research Laboratories, A. Menarini Pharmaceuticals, Florence (Italy) (Accepted 12 May 1987)
Key words: Capsaicin; Primary sensory afferent; Urinary bladder; Spinal cord; Horseradish peroxidase; Selective degeneration
The capsaicin-sensitive afferent innervation of the urinary bladder and the central nervous system distribution of urinary bladder afferents have been studied in the rat. Capsaicin-sensitive primary sensory neurones supplying the urinary bladder have been found in two groups of spinal ganglia located in the Th13-L 2 and L6-S1segments. Capsaicin-sensitive primary sensory afferents from the bladder terminate within Rexed's laminae I, V and X, and in the dorsal gray commissure of the lumbosacral spinal cord. In addition, the results point to a possible vagal sensory innervation of the urinary bladder.
Administration of the potent sensory neurotoxin capsaicin to newborn animals results in a selective destruction of a morphologically well characterized population of primary sensory neurones 11'12'14'16.Animals treated at birth with capsaicin display no gross abnormalities but show characteristic disturbances of several somatic and visceral sensory mechanisms as adults (see e.g. refs. 2, 4, 7, 13, 14 and 17). Indeed, hypertrophy of the urinary bladder and urine retention have been reported to occur after neonatal capsaicin treatment and have been attributed to a loss of substance P-containing afferent nerve fibres innervating the urinary bladder 29'3°. The available experimental evidence indicates that capsaicin-sensitive peptide-containing afferent nerves, by forming the afferent arch of the micturition reflex pathway, play a fundamental role in the nervous control of the urinary bladder 9,18'2°. Recent studies by Maggi et alfl ° pointed to the possibility that some capsaicin-sensitive bladder afferents involved in the initiation of vesicovesical micturition reflexes may terminate at supraspinal levels. The decrease in the number of peptide-containing
primary sensory ganglion cells supplying the rat urinary bladder after neonatal capsaicin treatment is well established 29,3°. However, the central projections of capsaicin-sensitive primary sensory neurones innervating the urinary bladder have not been studied. Therefore, the present experiments were disigned to reveal the distribution of capsaicin-sensitive bladder afferents within the spinal cord and to investigate, by using a pharmaco-morphological approach, the possibility of a termination of these particular afferents at more rostral levels of the neuraxis. Adult male S p r a g u e - D a w l e y CFY rats weighing 300-350 g were used in these experiments. One group of animals was injected with capsaicin or its vehicle (10% ethanol, 10% Tween 80 in saline) at a dose of 50 mg/kg on the second day of life 14. A n o t h e r group of animals was injected with a 100-mg/kg dose of capsaicin or with the vehicle as adults. The retrograde and transganglionic transport of horseradish peroxidase conjugated with wheat germ agglutinin ( W G A - H R P ) was used to demonstrate the cell bodies and the central projections of primary
Correspondence: G. Jancs6. Present address: Department of Physiology, The Medical School, University of Bristol, University Walk, Bristol BS8 1TD, U.K. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
372 sensory neurones innervating the urinary bladder. In neonatally treated animals the urinary bladder was exposed through a midline incision of the abdominal wall and carefully isolated by using cotton wool moistened with warm isotonic saline. Injections of a 1% solution of W G A - H R P (Sigma) in distilled water were given into the wall of the bladder at 3 different sites in a total volume of 7.5 #1, whereafter the exposed area was thoroughly rinsed with saline and the wound closed. After a survival time of 72 h the rats were perfused transcardially with an aldehyde-containing fixative according to the procedure of Rosene and Mesulam 27. The spinal sensory ganglia from Thl2-S 3 on both sides, the nodose ganglia, the brainstem and the spinal cord were removed and stored in 0.1 M phosphate buffer containing 10% sucrose. Frozen sections of 15 and 50 # m thickness were cut from the sensory ganglia and the brainstem and spinal cord, respectively, and processed for the demonstration of H R P enzyme activity using tetramethylbenzidine (Sigma) as a chromogen 22. Sensory ganglion ceils showing heavy perikaryal labelling and/or a prominent nucleus with distinct cytoplasmic staining were counted under bright-field illumination at an objective magnification of x40. Sections of the brainstem and spinal cord were viewed and photographed under dark-field illumination. Camera lucida drawings were made by using a Leitz Dialux microscope equipped with a drawing tube. Animals treated with capsaicin or its vehicle as adults were anaesthetized and perfused through the left heart ventricle with 10% formalin 7 - 9 h after the injection. Serial frozen sections of the lumbosacral
spinal cord and the brainstem were processed for the demonstration of axon terminal degeneration according to Eager's silver impregnation technique °. In control animals injection of W G A - H R P into the wall of the urinary bladder resulted in the labelling of two segmentally different groups of sensory neurones located in the Thl3-1_.2 and L~-S~ spinal ganglia. The numbers and segmental distribution of retrogradely labelled dorsal root ganglion neurones are shown in Table I. These findings corroborate previous observations x,24,29,3° and are consistent with the notion that both the lumbar (hypogastric) and sacral (pelvic) plexuses contribute to the sensory innervation of the urinary bladder 1. As it is shown in Table I. neonatal capsaicin treatment resulted in a dramatic reduction in the number of retrogradely labelled cells in these sensory ganglia although the percentage distribution of the labelled neurones remained essentially unchanged. These results are consistent with previous findings showing that the fibre population of the rat pelvic nerve ts dominated by unmyelinated axons 1° and that neonatal administration of capsalcin at a dose similar to that used in the present study results in an almost complete loss of unmyelinated dorsal root afferent fibres 25. In addition to spinal sensory ganglia a few labelled cells were consistently revealed in the nodose ganglia of both control and capsaicin-treated rats. Their number varied between 10 and 15 per ganglion and was apparently unaffected by capsaicin treatment. Although innervation of the urinary bladder by vagal afferent fibres has not been demonstrated before, this is not entirely unexpected in the light of recent
TABLE I Numbers and segmental distribution o f labelled dorsal root ganglion cells after injection o f W G A - H R P into the wall o f the urinary bladder o f control and capsaicin-treated rats Th12
Th13
Lt
L2
L3
L4
L5
Lo
,5"I
$2
0 0 0 0
49 5 7 87
71 64 23 330
56 110 72 248
0 9 l 0
(I I1 0 0
0 0 0 0
247 311 202 468
555 492 717 689
0 0 0 0
0 0 0 0
0 0 4 7
10 3 13 11
7 3 13 7
0 0 0 0
0 0 0 0
0 0 0 0
12 47 3 8
99 7l 162 114
0 0 0 0
Control 1
2 3 4 Capsaicin 1 2 3 4
373 observations showing a substantial contribution of vagal afferent fibres to the sensory innervation of the rat uropoietic system s. The low number of retrogradely labelled nodose ganglion cells may be related to the small actual amount of tracer injected in the present experiments rather than to the number of nodose ganglion cells innervating the bladder. In studies aimed at the accurate estimation of the contribution of the vagus to the afferent innervation of the kidney, significantly higher amounts of the tracer were injected into the organ s. The restricted viscerotopic localization of the labelled dorsal root ganglion neurones demonstrated in the present study makes a non-specific labelling of nodose ganglion cells unlikely. In control animals distinct transganglionic labelling of urinary bladder afferents was observed in the L6-S 1 spinal cord segments. Primary afferents entering the cord through the L 6 and $l spinal dorsal roots distributed to the Lissauer zone and labelled the entire mediolateral extent of the tract. Afferent collaterals from the Lissauer tract entered the marginal zone or reached the deeper layers of the grey matter by way of two major projections. Few afferent fibres reached the dorsal grey commissure through the medial collateral pathway. The great majority of primary afferent collaterals left Lissauer tract via a lateral collateral pathway formed by one or more prominent fascicles running between the longitudinally oriented myelinated fibre bundles of the spinal reticular formation towards the intermediolateral grey. Upon entering the grey matter afferent collaterals gave rise to the following main terminal arborization territories: fibres coursing medially apparently terminated in the dorsal grey commissure and within the dorsalmost region of lamina X. Ventrally and ventrolaterally directed fibres extended into the lateral part of laminae V and VI and into the region of the sacral parasympathetic nucleus (Fig. 1A,B). Fibre bundles forming the lateral collateral pathway emerged from Lissauer tract with a more or less regular periodicity. In our material these bundles measuring about 40 #m were spaced approximately 150-300 ktm apart as measured in parasagittal sections. The pattern of spinal termination of capsaicinsensitive bladder afferents described here closely resembles that reported for pelvic nerve afferents in the rat 24 and further corroborates the view that the
substantia gelatinosa is essentially deprived of a prominent visceral afferent input 3'5'2426. Although transganglionic labelling of primary afferents was consistently obtained in control animals after the injection of W G A - H R P into the urinary bladder wall, a similar labelling could not be demonstrated in any of the rats treated with capsaicin at birth. The most likely explanation of this finding is that the amount of the tracer taken up and transported by the few remaining sensory neurones supplying the urinary bladder was insufficient to provide a reliable labelling of their central processes. The results also imply that the great majority of afferent nerves innervating the urinary bladder belong to capsaicin-sensitive primary sensory neurones. These findings provide an anatomical basis to the observation that reflex micturition is almost abolished in rats treated with capsaicin at birth Is. However, bladder voiding can be elicited in response to transvesical saline filling in a certain percentage of these animals providing cystometrograms are obtained in the conscious state (Conte and Maggi, unpublished data). This supports the idea that sensory input for 'conscious' micturition may be at least in part, capsaicin-resistant 19. The projection of capsaicin-sensitive primary afferents to the region of the sacral parasympathetic nucleus is particularly interesting: recent experiments in spinal rats indicate that capsaicin-sensitive afferents provide synaptic input for initiation of a spinal vesicovesical excitatory reflex 21. On the other hand, primary afferents terminating in lamina V may provide synaptic input to second order sensory neurones projecting from this area to supraspinal centres 23. In accord with our previous reports 15, systemic injection of capsaicin to adult rats resulted in a massive, rapid degeneration of primary sensory afferents terminating within the superficial laminae of the spinal cord dorsal horn. In addition, less marked but distinct axon terminal degeneration was observed in lamina V and within the dorsal grey commissure of the spinal cord at L6-$1 segmental levels (Fig. 1C-E). Available experimental evidence indicates that degeneration in lamina II and to some extent in lamina I may be attributed to the destruction of somatic afferents 3'5"26. It is, therefore, reasonable to assume that degenerating axon terminals observed in laminae I
374
B
E
"°
0
Fig. 1. A: dark-field photomicrograph illustrating the distribution of WGA-HRP-labelIed urinary bladder afferents in the L6 spinal cord segment of a control rat × 92. B: reconstruction by camera lucida drawing of 4 transverse sections of the L~ spinal cord segment showing the characteristic distribution of labelled urinary bladder afferents in control animals C and D: degenerating axon terminals in laminae I and II (C) and in lamina V (D) of the L6 spinal cord segment of a capsaicin treated rat. Eager's silver impregnation technique × 368. E: spinal termination fields of capsaicin-sensitive primary afferents based on camera lucida drawings of silver-stained sections of the L6spinal cord segment of an adult rat treated with capsaicin 7 h before killing.
and V and in the dorsal grey commissure overlapping with areas of termination of urinary bladder afferents represent the central projection areas of capsaicinsensitive primary sensory n e u r o n e s innervating pelvic viscera, including the urinary bladder.
In agreement with previous findings systemic injection of capsaicin produced extensive axon terminal degeneration in the brainstem within the nuclei of the solitary tract and the trigemmal nuclear complex 15. Neither capsaicin-induced degeneration, nor
375 HRP-labelled neuronal elements could be demon-
ment on this innervation. The pattern of spinal termi-
strated in the dorsolateral pontine t e g m e n t u m , a re-
nation of capsaicin-sensitive primary sensory after-
gion specifically implicated in the supraspinal control of micturition28.
ents from the rat bladder has also been described.
Since H R P labelling could not be observed in medullary or pontine structures after the injection of the
urinary bladder) input in the area of the dorsal grey commissure is particularly interesting in view of the
tracer into the wall of the urinary bladder, the results
proposed role of this region for the integration of somatovisceral afferentations and co-ordination of the excretive functions of pelvic viscera Is. In addition,
failed to provide evidence for the notion that capsai-
The convergence of somatic and visceral (from the
cin-sensitive primary sensory neurones innervating the bladder project to supraspinal regions 2°. It is to
the results draw attention to a possible innervation of
be emphasized however, that the existence of such
the urinary bladder by sensory fibres of vagal origin.
projections cannot be excluded on the basis of the present observations. Indeed, a medullary projection of renal afferent fibres could only be demonstrated by using double labelling with fluorescent dyes but not by transganglionic labelling with H R P 31.
This work was supported by a grant ( O T K A 4/1104/86) from the H u n g a r i a n A c a d e m y of Sciences and by a grant (VES 46287) from IMI, Rome. The
In conclusion, the present study confirms previous
authors wish to thank Dr. F. Cervero and Dr. S.N. Lawson for critical reading of the manuscript. We
reports on the n u m b e r and segmental distribution of primary sensory neurones innervating the urinary bladder and on the effect of neonatal capsaicin treat-
thank K. Mohficsi and H. Szab6 for skillful technical assistance, P. Robbins for photographic work and Sue Maskell for typing the manuscript.
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