Mediation of Micturition Reflex by Central Norepinephrine from the Locus Coeruleus in the Cat

0022-5347 /90/1434-0840$02.00/0 THE JOURNAL OF UROLOGY Copyright© 1990 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Vol. 143, April

Printed in U.S.A.

MEDIATION OF MICTURITION REFLEX BY CENTRAL NOREPINEPHRINE FROM THE LOCUS COERULEUS IN THE CAT NAOKI YOSHIMURA,* MASASHI SASA, OSAMU YOSHIDA AND SHUJI TAKAORI From the Departments of Urology and Pharmacology, Faculty of Medicine, Kyoto University, Kyoto, Japan

ABSTRACT

We examined whether norepinephrine originating in the locus coeruleus mediates the micturition reflex in anesthetized cats. 6-Hydroxydopamine, a catecholamine neurotoxin, injected bilaterally into the locus coeruleus markedly decreased catecholamine fluorescence in the lesioned area and induced urinary retention after 72 to 84 hr. At this time, there was no or only slight contraction of the urinary bladder induced by its distension, while the contraction was noted before the treatment. However, phenylephrine, an ai-receptor agonist, applied intrathecally in 6-hydroxydopaminetreated animals induced moderate bladder contraction. In sham-operated animals, the bladder contraction on its distension was inhibited by intrathecally applied prazosin, an a 1 -receptor antagonist. Thus, in the micturition reflex, norepinephrine derived from the locus coeruleus acts on the a 1 -adrenergic receptors in the sacral cord, and induces urinary bladder contraction via activation of the sacral parasympathetic preganglionic neurons. (J. Ural., 143: 840-843, 1990) The sacral parasympathetic center innervating the urinary bladder is controlled via a supraspinal pathway originating in the dorsolateral pontine tegmentum including the locus coeruleus (LC), which is mainly composed of norepinephrine-containing cells. 1- 5 Barrington6 found that the higher micturition center is located in the dorsolateral pontine tegmentum. Later, Tang7 reported that the brain stem transection at the intercollicular level caused hyperactive micturition reflex, whereas the subcollicular decerebration completely abolished the micturition reflex. In addition, electrical destruction of the LC region reportedly resulted in urinary retention. 8 Therefore, the region including the LC is considered to have facilitatory action on the micturition reflex, while there is an area to induce an inhibition of the reflex above the intercollicular level. Moreover, it is well known that, during the phase of spinal shock in patients with spinal cord injury, the urinary bladder is areflexic, resulting in urinary retention, which is considered to be due to the loss of facilitatory influences from the supraspinal micturition center. 9 However, there is disagreement regarding whether or not the LC itself is the higher micturition center controlling the micturition reflex. We have recently found that norepinephrine derived from the LC induces contraction of the urinary bladder through a 1 -adrenergic receptors in the sacral cord in cats. 10 Therefore, we examined the contribution of norepinephrine originating in the LC to the micturition reflex, using cats in which noradrenergic input to the sacral cord was destroyed by 6-hydroxydopamine (6-0HDA), a catecholamine neurotoxin, injected into the LC. MATERIALS AND METHODS

Thirteen adult cats weighing 2.5 to 3.8 kg. were used. Under ether anesthesia, a cannula was inserted into the right femoral vein. The intravesical pressure was recorded via a catheter (Fr. 6) inserted into the urinary bladder transurethrally. Physiological saline was continuously perfused at the rate of 0.5 ml./min. through a needle inserted percutaneously into the bladder dome to induce contraction of the urinary bladder. After fixation of the head of the animal on a stereotaxic apparatus, the ether anesthesia was replaced with a-chloralose (30 mg./kg., i.v.), and supplemental doses of a-chloralose (each 10 to 20 mg./kg., Accepted for publication November 17, 1989. *Requests for reprints: Dept. of Urology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Supported by a Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 63570092).

840

i.v.) were added as required. Then, craniotomy was performed for insertion of a microsyringe (inner diameter: 150 µm.) into the bilateral LC. Five animals each received 30 µg. of 6-0HDA (Sigma) dissolved in 15 µl. of 0.9% saline solution containing one mg./ml. ascorbic acid slowly injected through a cannula inserted into the LC (P: 2.0, L: 2.0, H: -2.0) 11 for 15 min. using a microinjecter (IM-1, Narishige, Japan). In five sham-operated cats, the same volume of vehicle without 6-0HDA was injected into the bilateral LC in the same manner. Immediately after surgery, animals were injected with penicillin G (100,000 U). After completion of application of 6-0HDA, the animals were transferred to a cage, being allowed to move freely and take water and food. Under a-chloralose anesthesia (30 mg.jkg., i.v.), the head of the animal was fixed in the stereotaxic instrument and contraction of the urinary bladder induced by continuous infusion of saline into the bladder was recorded before and 72 to 84 hr. after injection in both 6-0HDA-treated and sham-operated animals. In the experiments after the treatment, a polyethylene tube (Fr. 2) was inserted from a slit in the dura mater at C2-3 to the intrathecal space at the level of L5-6 spine, at which 813 segments were located. The cannula was confirmed to be located at the level below L6-7 segments after termination of each experiment. All wound edges and pressure points were locally anesthetized with 8% lidocaine spray throughout the experiments. Body temperature was maintained at 37 .0 to 37 .5C with a heating pad. The electrocardiogram (II lead) was continuously monitored during the experiments. Drugs applied into the intrathecal space were as follows: phenylephrine hydrochloride (Waka, Japan) and prazosin hydrochloride (Taito-Pfizer, Japan). A volume of 0.1 ml. containing the doses of each drug was intrathecally injected through a cannula introduced in the area of 81-3 segments. Statistical significance was determined by Student's t test. After cessation of the experiments, the LC lesion was confirmed by formaldehyde-induced fluorescence technique. 12 Briefly, the animal was perfused through the heart with the "FAGLUPAGA" solution containing 4% paraformaldehyde, 0.5% glutaraldehyde, 0.2% picric acid and 2% glyoxylic acid. The brain was then removed, stored in the "FAGLUPAGAS" solution containing 15% sucrose overnight, and cut into serial frontal sections at 20 µm. thickness. Amine fluorescence in the region of the LC was examined with a fluorescence microscope (Nikon, Japan) using a wide band violet excitation filter. In

841

MICTURITION REFLEX

CENTRAL NOREPI:NEPHRINE

addition, two animals, which did not undergo surgery, were processed as controls for confirmation of the lesion. Further details of the procedure have been described previously. 10 • " 3• 14

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FIG. 3. Effect of6-hydroxydopamine (6-0HDA) locally injected into locus coeruleus and intrathecal application of phenylephrine upon contraction of urinary bladder. A, mean contraction pressure of urinary bladder. B, mean threshold bladder volume to induce contraction of urinary bladder. Data were obtained 72 hr. after 6-0HDA injection. Vertical bars: standard errors. Number of animals: 5. # p<0.01; compared with before value. * p<0.01; compared with no treatment in 60HDA-lesioned animals.

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in the cell bodies and fluorescent varicosities in the region of the bilateral LC of 6-0HDA-treated animals compared with control animals (fig. 1). Bladder motility. Contraction of the urinary bladder was induced continuous infusion of saline in all animals before the injection of 6-0HDA (fig. 2A), when the bladder was distended by the mean volume of 10.0 ± 2.1 ml. (mean ± S.E.M.; n=5). The maximum intravesical pressure on contraction was 44.7 ± 3.6 cm. water (n=5) (fig. 3). However 72 hr. after the injection of 6-0HDA, the urinary bladder was markedly distended, and over 60 ml. of urine was obtained by catheterization in all five animals, which suggests that urinary retention had developed. Contraction of the urinary bladder was not elicited by the infusion of up to 40 ml. of saline in two out of five animals treated with 6-0HDA. In the remaining three animals, only slight contraction was induced by the infusion of over 20 ml. of saline (fig. 2B). When the bladder contraction was not induced by infusion of over 40 ml., the minimum volume to produce contraction was regarded as 40 ml. Under these circumstances, the maximum intravesical pressure on contraction and the mean minimum volume of infusion into the bladder to induce contraction in the 6-0HDA-treated group were 6.4 ± 3.0 cm. water and 32.2 ± 2.5 ml., respectively

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(fig. 3). In the sham-operated animals, contraction of the urinary bladder induced by its distension was similar to that before the sham-operation (fig. 4A and B): the mean pressure of the bladder on contraction was 48.8 ± 3.5 and 43.6 ± 5.6 cm. water, and the mean minimum volume to induce contraction was 11.3 ± 2.4 and 10.9 ± 3.6 ml. before and after the operation, respectively. There was no remarkable change in the electrocardiogram monitored before and after 6-0HDA treatment. lntrathecal application of drugs. In 6-0HDA-treated animals, intrathecally applied phenylephrine dose-dependently induced moderate contraction of the urinary bladder on its distension. The enhanced contraction was seen 10 min. after injection, and lasted for 120 min. (fig. 2C and D). With intrathecal application of phenylephrine at doses of 0.5 and 1 mg., the mean pressure of the bladder on contraction was significantly (p <0.01) increased to 19.2 ± 3.6 and 34.3 ± 4.8 cm. water (n=5), respectively (fig. 3A). In addition, phenylephrine at doses of 0.5 and 1 mg significantly (p <0.01) reduced the mean threshold volume for inducing bladder contraction from 32.2 ± 2.5 ml. (n=5) to 21.0 ± 2.9 ml. and 12. 7 ± 2.1 ml., respectively (fig. 3B). In five sham-operated animals, the bladder contraction induced by distension was blocked by intrathecal application of prazosin (100 ,ug.). Partial recovery was observed 120 min. after the application (fig. 4C and D).

in the present study were not chronically catheterized after 60HDA treatment, it is possible that the urinary retention observed after 6-0HDA treatment was due to the damage of bladder afferents mediating the micturition reflex by acute overdistension of the urinary bladder. However, since micturition reflex was potentiated in the 6-0HDA-treated animals after intrathecal injection of phenylephrine, a 1-adrenergic receptor agonist, the bladder afferents were considered to have remained intact after 6-0HDA treatment. Therefore, the results obtained with 6-0HDA are in line with our previous findings that contraction of the urinary bladder induced by LC stimulation was diminished in the animal given reserpine and increased again with subsequent injection of L-dopa, a precursor of norepinephrine. 10 The bladder contraction in the shamoperated animals was blocked by prazosin, an a 1-adrenergic receptor antagonist. These results further support our conclusion that norepinephrine originating in the LC neurons is involved in the micturition reflex; that is, input from the urinary bladder activates the LC neurons, and then norepinephrine released from the LC excites the parasympathetic preganglionic neurons located in the sacral cord via ai-adrenergic receptors, thereby inducing contraction of the urinary bladder. This conclusion is also supported by other electrophysiological evidence 15 as well as histochemical and autoradiographical findings of noradrenergic innervation from the LC of the sacral intermediolateral cell column 16· 17 and existence of a 1-adrenergic receptors in the sacral cord18· respectively. Elam et al. demonstrated that LC neurons are activated by distension of the urinary bladder. 15 N oradrenergic termination in the sacral cord was histochemically localized in the ventral horn and the intermediolateral cell column, 17 and a HRP study revealed that sacral parasympathetic neurons originate in the sacral intermediolateral cell column. 19 In addition, the distribution of a 1adrenergic binding sites is considered to correspond to the density of the noradrenergic innervations. 18 Therefore, it is likely that intrathecally applied phenylephrine acted on the sacral intermediolateral cell column, where a 1-adrenergic receptors are located, and then excited sacral parasympathetic neurons to induce contraction of the urinary bladder, although the possibility can not be excluded that the drug simultaneously stimulated the sacral ventral horn, where noradrenergic terminals and a1 -adrenergic receptors are present. The small contraction obtained by increasing the volume of saline in the urinary bladder in the LC-lesioned animals is considered to be due to release of norepinephrine from the remainder of the norepinephrine-containing cells, which are outside the LC, such as in the parabrachial nucleus in the cat13 and/or the non-noradrenergic effects from a part of the dorsolateral pontine tegmentum such as the nucleus tegmentalis laterodorsalis and reticular formation. 20- 22 However, this small bladder contraction in 6-0HDA-treated animals might also be due to intrinsic smooth muscle activity. Since phenylephrine intrathecally injected into LC-lesioned animals enhanced the contraction of the urinary bladder on its distension with a much smaller volume, norepinephrine appears to play an important role in the micturition reflex. REFERENCES 1. Bradley, W. E. and Sundin, T.: The physiology and pharmacology

DISCUSSION

When 6-0HDA was locally injected into the bilateral LC, there was a marked reduction of catecholamine fluorescence in the LC areas. In 6-0HDA-treated animals, the finding of urinary retention was in accordance with that in rats following electric lesioning in the LC. 8 In addition, the threshold volume for inducing bladder contraction was much greater and the contraction was much smaller than those before treatment with 6-0HDA and in the sham-operated animals. Since the animals

of urinary tract dysfunction. Clin. Neuropharmacol., 5: 131, 1982. 2. De Groat, W. C.: Nervous control of the urinary bladder of the cat. Brain Res., 87: 201, 1975. 3. Holstege, G., Griffiths, D., De Wall, H. and Dalm, E.: Anatomical and physiological observations on supraspinal control of bladder and urethral sphincter muscles in the cat. J. Comp. Neurol., 250: 449, 1986. 4. Kuru, M. and Yamamoto, H.: Fiber connections of the pontine detrusor nucleus (Barrington). J. Comp. Neurol., 123: 161, 1964.

CEI\JTRAL l_\,TOREPit~EPHRINE IN IV!ICTURITIOJ:J REFLEX 5. Nishizawa, 0., Sugaya, K., Noto, H., Harada, T. and Tsuchida, S.: Pontine micturition center in the dog. J. UroL, 140: 872, 1988. 6. Barrington, F. J. F.: The effect of lesions of the hind- and midbrain on micturition in the cat. Quart. J. Physiol., 15: 81, 1925. 7. Tang, P. C.: Levels of brain stem and diencephalon controlling micturition reflex. J. Neurophysiol., 18: 583, 1955. 8. Osumi, Y., Oishi, R., Fujiwara, H. and Takaori, S.: Hyperdipsia induced by bilateral destruction of the locus coeruleus in rats. Brain Res., 86: 419, 1975. 9. Fam, B. A., Sarkarti, M. and Yalla, S. V.: Spinal cord injury. In: Neurourology and Urodynamics. Edited by S. V. Yalla, E. J. McGuire, A. Elbadawi and J. G. Blaivas. New York: Macmillan, chapt. 18, pp 291-295, 1988. 10. Yoshimura, N., Sasa, M., Ohno, Y., Yoshida, 0. and Takaori, S.: Contraction of urinary bladder by central norepinephrine originating in the locus coeruleus. J. Urol., 139: 423, 1988. 11. Berman, A. L.: The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates. Madison: University of Wisconsin Press, 1968. 12. Imai, H., Kimura, H. and Maeda, T.: A stable and simple method of "FAGLUPAGAS fixation" for catecholamines for routine examination. Acta Histochem. Cytochem., 15: 798, 1982. 13. Sasa, M., Munekiyo, K., Ikeda, H. and Takaori, S.: Noradrenalinemediated inhibition by locus coeruleus of spinal trigeminal neurons. Brain Res., 80: 443, 1974. 14. Sasa, M., Ohno, Y., Ito, J., Kashii, S., Utsumi, S. and Takaori, S.: Beta-receptor involvement in locus coeruleus-induced inhibition

15.

16.

17.

18.

19.

20. 21.

22.

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of spinal trigeminal nucleus neuror;.s: n1icroiontophoretic and HRP studies. Brain Res., 377: 337, 1986. Elam, M., Thoren, P. and Svensson, T. H.: Locus coeruleus neurons and sympathetic nerves: activation by visceral afferents. Brain Res., 375: 117, 1986. Westlund, K. N., Bowker, R. M., Ziegler, M. G. and Coulter, J. D.: Descending noradrenergic projections and their spinal terminations. Prog. Brain Res., 57: 219, 1982. Westlund, K. N., Bowker, R. M., Ziegler, M. G. and Coulter, J. D.: Noradrenergic projections to the spinal cord of the rat. Brain Res., 263: 15, 1983. Giron Jr, L. T., McCann, S. A. and Crist-Orlando, S. G.: Pharmacological characterization and regional distribution of a-noradrenergic binding sites of rat spinal cord. Eur. J. Pharmacol., 115: 285, 1985. Nadelhaft, I., De Groat, W. C. and Morgan, C.: Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horesradish peroxidase. J. Comp. Neural., 193: 265, 1980. Lowey, A. D., Saper, C. B. and Baker, R. P.: Descending projections from the pontine micturition center. Brain Res., 172: 533, 1979. 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: 27, 1978. McMahon, S. B. and Spillane, K.: Brain stem influences on the parasympathetic supply to the urinary bladder of the cat. Brain Res., 234: 237, 1982.