distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord

Brain Research, 418 (1987) 371-376 371 Elsevier

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-S 1 segments. 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. An-

imals 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 reten-

tion 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 experi-

mental 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-sensi-

tive 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 uri-

nary bladder after neonatal capsaicin treatment is well established 29,3°. However, the central projec-

tions of capsaicin-sensitive primary sensory neurones

innervating the urinary bladder have not been stud-

ied. Therefore, the present experiments were dis-

igned to reveal the distribution of capsaicin-sensitive

bladder afferents within the spinal cord and to inves-

tigate, by using a pharmaco-morphologica l ap-

proach, the possibility of a termination of these par-

ticular afferents at more rostral levels of the neurax- is.

Adult male Sprague-Dawley 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. Another

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 wa-

ter were given into the wall of the bladder at 3 differ-

ent sites in a total volume of 7.5 #1, whereafter the ex-

posed 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-con-

taining 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 brain-

stem and the spinal cord were removed and stored in

0.1 M phosphate buffer containing 10% sucrose. Fro-

zen sections of 15 and 50 #m thickness were cut from

the sensory ganglia and the brainstem and spinal

cord, respectively, and processed for the demonstra-

tion of H R P enzyme activity using tetramethylbenzi- dine (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 photo-

graphed under dark-field illumination. Camera luci-

da drawings were made by using a Leitz Dialux mi-

croscope 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 label-

ling 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 innerva-

tion 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 dis-

tribution of the labelled neurones remained essen-

tially 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 re-

sults 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 L t L 2 L 3 L 4 L 5 L o ,5" I $2

0 49 71 56 0 (I 0 247 555 0 0 5 64 110 9 I1 0 311 492 0 0 7 23 72 l 0 0 202 717 0 0 87 330 248 0 0 0 468 689 0

Control 1 2 3 4

Capsaicin 1 2 3 4

0 0 10 7 0 0 0 12 99 0 0 0 3 3 0 0 0 47 7 l 0 0 4 13 13 0 0 0 3 162 0 0 7 11 7 0 0 0 8 114 0

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 viscero- topic localization of the labelled dorsal root ganglion neurones demonstrated in the present study makes a non-specific labelling of nodose ganglion cells unlike-

ly. In control animals distinct transganglionic label-

ling 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 margi- nal 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 lon- gitudinally oriented myelinated fibre bundles of the

spinal reticular formation towards the intermediolat- eral 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. Ven- trally 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 capsaicin- sensitive bladder afferents described here closely re- sembles that reported for pelvic nerve afferents in

the rat 24 and further corroborates the view that the

373

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 demon-

strated 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 trans- ported 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 observa- tion 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 neu-

rones projecting from this area to supraspinal centres 23.

In accord with our previous reports 15, systemic in-

jection of capsaicin to adult rats resulted in a mas- sive, 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 L 6 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 L 6 spinal 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 terminat ion of urinary bladder afferents

represent the central project ion areas of capsaicin-

sensitive primary sensory neurones innervating pel-

vic viscera, including the urinary bladder.

In agreement with previous findings systemic in-

jection of capsaicin produced extensive axon termi-

nal degeneration in the brainstem within the nuclei of

the solitary tract and the trigemmal nuclear com-

plex 15. Neither capsaicin-induced degenerat ion, nor

375

HRP-labelled neuronal elements could be demon-

strated in the dorsolateral pontine tegmentum, a re-

gion specifically implicated in the supraspinal control

of micturition 28.

Since HRP labelling could not be observed in med-

ullary or pontine structures after the injection of the

tracer into the wall of the urinary bladder, the results

failed to provide evidence for the notion that capsai-

cin-sensitive primary sensory neurones innervating

the bladder project to supraspinal regions 2°. It is to

be emphasized however, that the existence of such

projections cannot be excluded on the basis of the

present observations. Indeed, a medullary projec-

tion of renal afferent fibres could only be demon-

strated by using double labelling with fluorescent

dyes but not by transganglionic labelling with HRP 31.

In conclusion, the present study confirms previous

reports on the number and segmental distribution of

primary sensory neurones innervating the urinary

bladder and on the effect of neonatal capsaicin treat-

ment on this innervation. The pattern of spinal termi-

nation of capsaicin-sensitive primary sensory after-

ents from the rat bladder has also been described.

The convergence of somatic and visceral (from the

urinary bladder) input in the area of the dorsal grey

commissure is particularly interesting in view of the

proposed role of this region for the integration of so-

matovisceral afferentations and co-ordination of the

excretive functions of pelvic viscera Is. In addition,

the results draw attention to a possible innervation of

the urinary bladder by sensory fibres of vagal origin.

This work was supported by a grant ( O TK A

4/1104/86) from the Hungar ian Academy of Sciences

and by a grant (VES 46287) from IMI, Rome. The

authors wish to thank Dr. F. Cervero and Dr. S.N.

Lawson for critical reading of the manuscript. We

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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distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord

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