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Effect of Chemical Stimulation of the Medial Frontal Lobe on the Micturition Reflex in Rats
Effect of Chemical Stimulation of the Medial Frontal Lobe on the Micturition Reflex in Rats Saori Nishijima, Kimio Sugaya,* Katsumi Kadekawa, Katsuhiro Ashitomi and Hideyuki Yamamoto From the Southern Knights’ Laboratory, L.L.P. and Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus (HY), Okinawa, Japan
Abbreviations and Acronyms MFL ⫽ medial frontal lobe PMC ⫽ pontine micturition center RPRF ⫽ rostral pontine reticular formation Submitted for publication May 31, 2011. Study received approval from the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus. * Correspondence: Southern Knights’ Laboratory, L.L.P., 2-7-7-301 Takahara, Okinawa, 9042171, Japan (telephone: ⫹81-98-989-7739; FAX: ⫹81-98-989-7739; e-mail: [email protected]).
Purpose: We assessed the influence of the medial frontal lobe on micturition after chemical stimulation. We also examined the relation between the medial frontal lobe and the rostral pontine reticular formation, which has a strong inhibitory effect on micturition. Materials and Methods: A total of 35 female rats underwent continuous cystometry. Bladder activity changes were examined after physiological saline, glutamate, the glutamate receptor antagonist MK-801, noradrenaline or the adrenergic ␣-1 receptor antagonist naftopidil was injected in the medial frontal lobe. When glutamate was injected in the medial frontal lobe, MK-801 was also injected in the rostral pontine reticular formation. Results: Glutamate injection in the medial frontal lobe prolonged the interval between bladder contractions while injection of the glutamate antagonist MK-801 shortened the interval. Glutamate injection in the medial frontal lobe just after MK-801 injection in the ipsilateral rostral pontine reticular formation also prolonged the interval between bladder contractions. However, after prior injection of MK-801 in the bilateral rostral pontine reticular formation glutamate injection in the medial frontal lobe did not influence cystometric parameters. Noradrenaline injection in the medial frontal lobe shortened the interval between bladder contractions while injection of its antagonist naftopidil prolonged the interval. Conclusions: Medial frontal lobe neurons excited by glutamate inhibited the micturition reflex via activation of the rostral pontine reticular formation by glutamatergic projection while medial frontal lobe neurons excited by noradrenaline facilitated the micturition reflex. Thus, the medial frontal lobe may be an important integration center for the initiation of micturition and urine storage mechanisms. Key Words: urinary bladder, frontal lobe, urination, glutamic acid, reflex
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MICTURITION is primarily a function of the autonomic nervous system mediated by the spinobulbospinal reflex pathway, which passes through a coordination center located in the rostral pons known as the PMC.1 This reflex pathway is modulated by a higher center in the cerebral cortex that is involved in voluntary control of micturition. There are some reports of the relationship between central nervous sys-
tem damage and urinary disorders. For example, middle cerebral artery occlusion induces urinary frequency in rats.2,3 Unilateral injection of 6-hydroxydopamine, a neurotoxin commonly used to cause lesions of dopaminergic pathways, in the medial forebrain bundle also causes bladder dysfunction.4 Decerebration above the level of the rostral pons decreases the bladder volume at which the micturition reflex is evoked in cats.5
0022-5347/12/1873-1116/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 187, 1116-1120, March 2012 Printed in U.S.A. DOI:10.1016/j.juro.2011.10.128
AND
RESEARCH, INC.
EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
Urinary frequency and urgency are common in patients with cerebrovascular disease and disturbed micturition is more often associated with lesions of the frontal lobe than those of the occipital lobe.6,7 Based on these reports the overall effect of brain centers is thought to be inhibition of the micturition reflex. However, it is unclear how important a role the forebrain has in micturition or urine storage. In the pons 2 centers regulate micturition besides the PMC. Electrical stimulation of the region ventrolateral to the PMC enhances external urethral sphincter activity and inhibits bladder contraction.8 –10 Thus, this region is called the pontine urine storage center. Another pontine region that controls lower urinary tract function is found ventromedial to the locus coeruleus complex. Electrical stimulation or injection of the cholinomimetic agent carbachol in this region inhibits bladder contraction in cats and rats.11–13 This region corresponds to the RPRF, also known as the nucleus reticularis pontis oralis in cats.11,12 The RPRF has a strong inhibitory effect on micturition since electrical stimulation of the PMC cannot evoke micturition after carbachol injection in the RPRF.14 In rats glutamate injection in the RPRF also strongly inhibits bladder contraction13 while injection of the glutamate receptor antagonist MK-801 facilitates bladder contraction.15 We assessed the influence of the MFL on micturition after stimulation by injection of excitatory agents and their antagonists. We also examined the relation between the MFL and the RPRF.
MATERIALS AND METHODS Animals A total of 35 female Sprague-Dawley® rats weighing 215 to 231 gm were used in this study. The study protocol was approved by the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus.
Continuous Cystometry in Conscious Rats Rats were anesthetized with 2% isoflurane. A PE-50 polyethylene catheter (Clay Adams, Parsippany, New Jersey) was inserted transurethrally in the bladder. Three small holes were made in the skull rostral (bregma 3.5 mm, right 0.8 mm) and caudal (bregma ⫺9.5 mm, right and left 1.0 mm). PE-10 polyethylene catheters (2 cm) were inserted to inject drugs in the MFL or the RPRF according to published coordinates.16 The MFL in this study was thought to correspond to the anterior cingulate gyrus in cats. For continuous cystometry each rat was placed in a NAIGAI-CFK-1P restraining cage (NMS, Tokyo, Japan) and allowed to recover from anesthesia. Cystometry was performed at least 1 hour after the rat was placed in the cage. The bladder catheter was connected to an infusion pump via polyethylene tubing. The bladder was filled with physiological saline at 0.05 ml per minute. After bladder contractions were stable for at least 30 minutes 1 l physiological saline, glutamate, the glutamate receptor antagonist MK-801 (NMDA [N-
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methyl D-aspartate] antagonist), noradrenaline or the adrenergic ␣-1D receptor antagonist naftopidil (10 M in 3 to 7 rats each) was injected in the MFL. When glutamate was injected in the MFL, MK-801 was also injected in the ipsilateral or bilateral RPRF since glutamate projections to the RPRF neuron inhibit micturition and this effect is blocked by MK-801.15 Maximum bladder contraction pressure, the interval between bladder contraction and baseline bladder pressure were measured for 3 to 8 contractions after injecting each agent.
Statistical Analysis Results are shown as the mean ⫾ SEM. The Student t test for paired and unpaired data was used for statistical analysis with p ⬍0.05 considered statistically significant.
RESULTS Before injecting each active drug physiological saline was injected in the MFL and the RPRF in 3 rats. There were no significant bladder contraction differences before vs after saline injection. Glutamate injection in the MFL in 7 rats significantly prolonged the interval between bladder contractions by 39% (p ⬍0.001, fig. 1, A). However, injection of the glutamate antagonist MK-801 in the MFL in 7 rats significantly shortened the interval between bladder contractions by 39% (p ⫽ 0.028, see table and fig. 1, B). There was no significant change in maximum contraction pressure or baseline bladder pressure after injecting these agents. When glutamate was injected in the MFL in 4 rats just after MK-801 injection in the ipsilateral RPRF, the interval between bladder contractions was also significantly prolonged by 28% (p ⫽ 0.010). Since MK801 injection in the RPRF shortens the interval between bladder contractions15 and the RPRF receives projection from the bilateral MFL in cats,17,18 we also tried injecting MK-801 in the bilateral RPRF. After prior injection of MK-801 in the bilateral RPRF glutamate injection in the MFL in 6 rats did not alter cystometric parameters (see table and fig. 2). When noradrenaline was injected in the MFL in 5 rats, bladder baseline pressure became irregular and many nonvoiding bladder contractions were recorded. The interval between voiding bladder contractions was significantly shortened by 47% after noradrenaline injection in the MFL (p ⫽ 0.006, fig. 3, A). On the other hand, injection of its antagonist naftopidil in the MFL in 6 rats significantly prolonged the interval between bladder contractions by 47% (p ⬍0.001, see table and fig. 3, B). There was no significant change in maximum bladder contraction pressure or baseline bladder pressure after injecting these agents.
DISCUSSION Injection of glutamate or its antagonist MK-801 in the MFL prolonged and shortened the interval be-
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EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
Figure 1. Continuous cystometry before and after injection in MFL. There were no significant changes in maximum bladder contraction pressure or baseline bladder pressure after bladder contraction recovery. A, 10 M glutamate significantly prolonged interval between bladder contractions. B, 10 M MK-801 significantly shortened interval between bladder contractions.
tween bladder contractions, respectively, while injection of noradrenaline or its antagonist naftopidil in the MFL shortened and prolonged the interval between bladder contractions, respectively. Thus, neurons in the MFL that are excited by glutamate or noradrenaline may inhibit or facilitate the micturition reflex, respectively. Generally it is thought that micturition occurs after the release of inhibitory projections from the forebrain (cerebrum) to the PMC. This theory is supported by reports that precollicular decerebrate rats and patients with cerebral infarction show urinary frequency.5–7 However, there is also the possibility that micturition is evoked by the suppression of excitatory projections from forebrain to RPRF. The RPRF, also known as the nucleus reticularis pontis oralis in cats, is located ventromedial to the locus coeruleus complex.11,12 Injection of the cholinomimetic agent carbachol or glutamate in this region strongly inhibits bladder contraction in cats and rats11,13 while MK-801 injection facilitates bladder contraction in rats.15 When bladder activity was inhibited by glutamate injection in the RPRF, the glycine level in the lumbosacral cord was noted to increase.13 On the other hand, rats with cerebral
infarction showed urinary frequency while the glutamate level in the cerebrum and the glycine level in the lumbosacral cord were significantly decreased compared with control values.3 Glycine is a major inhibitory neurotransmitter in the central nervous system that inhibits the micturition reflex at the level of the lumbosacral cord.3,13,19 In our study glutamate injection in the MFL inhibited bladder contraction. This effect was blocked by prior MK-801 injection in the bilateral RPRF but not in the ipsilateral RPRF. The RPRF receives projections from the bilateral MFL in cats17,18 and the same neuronal projections between MFL and RPRF may exist in the rat brain. Consequently interception of the ipsilateral RPRF pathway might not be enough to block the inhibitory effect of glutamate injection in the MFL on the micturition reflex. These results suggest that activation of MFL neurons expressing the NMDA glutamate receptor excites bilateral RPRF neurons that use glutamate as a neurotransmitter. Results also suggest that descending RPRF neurons inhibit the micturition reflex by activating inhibitory glycinergic neurons in the lumbosacral cord. In other words, micturition may pos-
Change in bladder activity after injection of glutamate vs MK-801 and noradrenaline vs naftopidil in MFL, and effect of co-administration of glutamate in MFL and MK-801 in ipsilateral or bilateral RPRF Mean ⫾ SEM MFL
Interval (mins): Before After p Value Max contraction pressure (cm H2O): Before After Baseline bladder pressure (cm H2O): Before After
Glutamate
MK-801
Noradrenaline
10.5 ⫾ 2.1 14.6 ⫾ 2.2 ⬍0.001
11.7 ⫾ 2.2 7.1 ⫾ 0.9 0.028
16.9 ⫾ 2.0 8.9 ⫾ 2.1 0.006
42.2 ⫾ 5.0 41.8 ⫾ 3.3
45.1 ⫾ 4.2 44.2 ⫾ 4.8
7.7 ⫾ 0.7 7.5 ⫾ 0.5
7.6 ⫾ 0.5 7.6 ⫾ 0.4
Mean ⫾ SEM MFL Glutamate Naftopidil
Ipsilat RPRF MK-801
Bilat RPRF MK-801
12.8 ⫾ 1.9 18.8 ⫾ 2.0 ⬍0.001
7.8 ⫾ 1.6 10.0 ⫾ 1.7 0.010
8.8 ⫾ 1.4 8.2 ⫾ 1.3 —
47.1 ⫾ 5.5 41.4 ⫾ 2.9
44.5 ⫾ 4.5 44.4 ⫾ 4.4
51.7 ⫾ 4.8 50.3 ⫾ 3.9
47.9 ⫾ 4.0 48.3 ⫾ 3.8
7.6 ⫾ 0.5 7.5 ⫾ 0.5
7.6 ⫾ 0.5 7.1 ⫾ 0.8
8.7 ⫾ 1.1 9.2 ⫾ 1.4
7.7 ⫾ 0.7 7.7 ⫾ 0.7
EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
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Figure 2. Effect of co-administration of glutamate with MK-801 in MFL. A, before injection. B, glutamate in MFL. C, co-administration of glutamate in MFL and MK-801 in ipsilateral RPRF prolonged interval between bladder contractions compared with before injection. D, co-administration of glutamate in MFL and MK-801 in bilateral RPRF did not prolong interval between bladder contractions.
sibly be evoked by the suppression of excitatory projections from forebrain to RPRF. Chemical stimulation of the PMC alters the interval between bladder contractions and alters bladder contraction pressure in cats20 while chemical stimulation of the RPRF only influences the interval between bladder contractions in cats and rats.13,21
Since injection of agents in the MFL only had an effect on the interval between bladder contractions in our study, the MFL may be more closely related to the RPRF than to the PMC. Naftopidil is an adrenergic ␣-1D receptor antagonist used for benign prostatic hyperplasia.22 It decreases smooth muscle tone in the urethra and in-
Figure 3. Continuous cystometry before and after injection in MFL. There was no significant change in maximum bladder contraction pressure or baseline bladder pressure after injection. A, noradrenaline induced detrusor overactivity, ie irregular bladder baseline pressure and many nonvoiding bladder contractions, and significantly shortened interval between voiding bladder contractions. B, adrenergic receptor antagonist naftopidil significantly prolonged interval between bladder contractions.
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EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
hibits the micturition reflex at the lumbosacral cord level.23,24 In our study noradrenaline injection in the MFL induced detrusor overactivity (irregular bladder baseline pressure, many nonvoiding bladder contractions and urinary frequency) while the micturition reflex was inhibited by naftopidil. Results suggest that the excitation of MFL neurons that express adrenergic receptors facilitates the micturition reflex, which is the opposite effect of that seen after glutamate injection in the MFL. Accordingly MFL neurons excited by glutamate may differ from those excited by noradrenaline. Urinary frequency and urgency are common in patients with cerebrovascular disease while disturbed micturition is more common in patients with lesions of the frontal lobe than in those with lesions of the occipital lobe.6,7 However, partial decerebration involving the MFL increases the threshold volume that induces micturition in rats.21 Also, regions
facilitating or inhibiting the micturition reflex as a result of electrical stimulation coexist in the MFL, including the anterior cingulate gyrus in cats.25 This area of the cat brain contains neurons that show increased or decreased activity during the micturition reflex.26 Thus, the MFL may be an important integration center involved in the initiation of micturition and urine storage that voluntarily regulates the micturition reflex depending on circumstances.
CONCLUSIONS MFL neurons excited by glutamate inhibited the micturition reflex via activation of the RPRF by glutamatergic projections while MFL neurons excited by noradrenaline facilitated the micturition reflex. Glutamate and noradrenaline had no influence on bladder contraction pressure. Thus, MFL may be an important integration center for micturition initiation and for urine storage mechanisms.
REFERENCES 1. Sugaya K, Nishijima S, Miyazato M et al: Central nervous control of micturition and urine storage. J Smooth Muscle Res 2005; 41: 117.
10. Nishizawa O, Sugaya K, Noto H et al: Pontine urine storage center in the dog. Tohoku J Exp Med 1987; 153: 77.
2. Yokoyama O, Yoshiyama M, Namiki M et al: Changes in dopaminergic and glutamatergic excitatory mechanisms of micturition reflex after middle cerebral artery occlusion in conscious rats. Exp Neurol 2002; 173: 129.
11. Sugaya K, Mori S, Nishizawa O et al: Chemical stimulations of the pontine micturition center and the nucleus reticularis pontis oralis. Neurourol Urodyn 1987; 6: 143.
3. Nishijima S, Sugaya K, Miyazato M et al: Relationship between bladder activity and amino acid levels in the central nervous system in rats with cerebral infarction. Biomed Res 2003; 24: 173. 4. Soler R, Füllhase C, Santos C et al: Development of bladder dysfunction in a rat model of dopaminergic brain lesion. Neurourol Urodyn 2011; 30: 188. 5. Tang PC: Levels of brain stem and diencephalon controlling micturition reflex. J Neurophysiol 1955; 18: 583. 6. Steers WD: Physiology and pharmacology of the bladder and urethra. In: Campbell’s Urology, 7th ed. Edited by PC Walsh, AB Retik, ED Vaughan Jr et al. Philadelphia: WB Saunders 1998; pp 870–915. 7. Sakakibara R, Hattori T, Yasuda K et al: Micturitional disturbance after acute hemispheric stroke: analysis of the lesion site by CT and MRI. J Neurol Sci 1996; 137: 47. 8. Kuru M: Nervous control of micturition. Physiol Rev 1965; 45: 425. 9. Holstege G, Griffiths D, de Wall H et al: Anatomical and physiological observations on supraspinal control of bladder and urethral sphincter muscles in the cat. J Comp Neurol 1986; 250: 449.
12. Kimura Y, Ukai Y, Kimura K et al: Inhibitory influence from the nucleus reticularis pontis oralis on the micturition reflex induced by electrical stimulation of the pontine micturition center in cats. Neurosci Lett 1995; 195: 214. 13. Nishijima S, Sugaya K, Miyazato M et al: Activation of the rostral pontine reticular formation increases the spinal glycine level and inhibits bladder contraction in rats. J Urol 2005; 173: 1812. 14. Sugaya K, Nishijima S, Miyazato M et al: Inhibitory effect of the nucleus reticularis pontis oralis on the pontine micturition center and pontine urine storage center in decerebrate cats. Biomed Res 2006; 27: 211. 15. Naka H, Nishijima S, Kadekawa K et al: Influence of glutamatergic projections to the rostral pontine reticular formation on micturition in rats. Life Sci 2009; 85: 732. 16. Swanson LW: Brain Maps: Structure of the Rat Brain. New York: Elsevier Science Publishers 1992. 17. Kohama T: Neuroanatomical studies on the urine storage facilitatory areas in the cat brain. Part I. Input neuronal structures to the nucleus locus subcoaruleus and the nucleus reticularis pontis oralis. Nippon Hinyokika Gakkaishi 1992; 83: 1469. 18. Kohama T: Neuroanatomical studies on the urine storage facilitatory areas in the cat brain. Part II. Output neuronal structure from the nucleus locus
subcoeruleus and the nucleus reticularis pontis oralis. Nippon Hinyokika Gakkaishi 1992; 83: 1478. 19. Miyazato M, Sasatomi K, Hiragata S et al: GABA receptor activation in the lumbosacral spinal cord decreases detrusor overactivity in spinal cord injured rats. J Urol 2008; 179: 1178. 20. Sugaya K: The effects of chemical stimulations of the pontine micturition center in decerebrate cats. Nippon Hinyokika Gakkai Zasshi 1988; 79: 1364. 21. Sugaya K, Nishizawa O and Tsuchida S: Brain diseases and micturitional disorders—animal experiments. Nippon Shinkeiinseiboukou Gakkaishi 1994; 5: 5. 22. Takei R, Ikegaki I, Shibata K et al: Naftopidil, a novel alpha1-adrenoceptor antagonist, displays selective inhibition of canine prostatic pressure and high affinity binding to cloned human alpha1adrenoceptors. Jpn J Pharmacol 1999; 79: 447. 23. McConnell JD: Epidemiology, etiology, pathophysiology, and diagnosis of benign prostatic hyperplasia In: Campbell’s Urology, 7th ed. Edited by PC Walsh, AB Retik, ED Vaughan Jr et al. Philadelphia: WB Saunders 1998; pp 1429 –1452. 24. Sugaya K, Nishijima S, Miyazato M et al: Effects of intrathecal injection of tamsulosin and naftopidil, alpha-1A and ⫺1D adrenergic receptor antagonists, on bladder activity in rats. Neurosci Lett 2002; 328: 74. 25. Gjone R and Setekleiv J: Excitatory and inhibitory bladder responses to stimulation of the cerebral cortex in the cat. Acta Physiol Scand 1963; 59: 337. 26. Yamamoto T, Sakakibara R, Nakazawa K et al: Neuronal activities of forebrain structures with respect to bladder contraction in cats. Neurosci Lett 2010; 473: 42.
Abbreviations and Acronyms MFL ⫽ medial frontal lobe PMC ⫽ pontine micturition center RPRF ⫽ rostral pontine reticular formation Submitted for publication May 31, 2011. Study received approval from the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus. * Correspondence: Southern Knights’ Laboratory, L.L.P., 2-7-7-301 Takahara, Okinawa, 9042171, Japan (telephone: ⫹81-98-989-7739; FAX: ⫹81-98-989-7739; e-mail: [email protected]).
Purpose: We assessed the influence of the medial frontal lobe on micturition after chemical stimulation. We also examined the relation between the medial frontal lobe and the rostral pontine reticular formation, which has a strong inhibitory effect on micturition. Materials and Methods: A total of 35 female rats underwent continuous cystometry. Bladder activity changes were examined after physiological saline, glutamate, the glutamate receptor antagonist MK-801, noradrenaline or the adrenergic ␣-1 receptor antagonist naftopidil was injected in the medial frontal lobe. When glutamate was injected in the medial frontal lobe, MK-801 was also injected in the rostral pontine reticular formation. Results: Glutamate injection in the medial frontal lobe prolonged the interval between bladder contractions while injection of the glutamate antagonist MK-801 shortened the interval. Glutamate injection in the medial frontal lobe just after MK-801 injection in the ipsilateral rostral pontine reticular formation also prolonged the interval between bladder contractions. However, after prior injection of MK-801 in the bilateral rostral pontine reticular formation glutamate injection in the medial frontal lobe did not influence cystometric parameters. Noradrenaline injection in the medial frontal lobe shortened the interval between bladder contractions while injection of its antagonist naftopidil prolonged the interval. Conclusions: Medial frontal lobe neurons excited by glutamate inhibited the micturition reflex via activation of the rostral pontine reticular formation by glutamatergic projection while medial frontal lobe neurons excited by noradrenaline facilitated the micturition reflex. Thus, the medial frontal lobe may be an important integration center for the initiation of micturition and urine storage mechanisms. Key Words: urinary bladder, frontal lobe, urination, glutamic acid, reflex
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MICTURITION is primarily a function of the autonomic nervous system mediated by the spinobulbospinal reflex pathway, which passes through a coordination center located in the rostral pons known as the PMC.1 This reflex pathway is modulated by a higher center in the cerebral cortex that is involved in voluntary control of micturition. There are some reports of the relationship between central nervous sys-
tem damage and urinary disorders. For example, middle cerebral artery occlusion induces urinary frequency in rats.2,3 Unilateral injection of 6-hydroxydopamine, a neurotoxin commonly used to cause lesions of dopaminergic pathways, in the medial forebrain bundle also causes bladder dysfunction.4 Decerebration above the level of the rostral pons decreases the bladder volume at which the micturition reflex is evoked in cats.5
0022-5347/12/1873-1116/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 187, 1116-1120, March 2012 Printed in U.S.A. DOI:10.1016/j.juro.2011.10.128
AND
RESEARCH, INC.
EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
Urinary frequency and urgency are common in patients with cerebrovascular disease and disturbed micturition is more often associated with lesions of the frontal lobe than those of the occipital lobe.6,7 Based on these reports the overall effect of brain centers is thought to be inhibition of the micturition reflex. However, it is unclear how important a role the forebrain has in micturition or urine storage. In the pons 2 centers regulate micturition besides the PMC. Electrical stimulation of the region ventrolateral to the PMC enhances external urethral sphincter activity and inhibits bladder contraction.8 –10 Thus, this region is called the pontine urine storage center. Another pontine region that controls lower urinary tract function is found ventromedial to the locus coeruleus complex. Electrical stimulation or injection of the cholinomimetic agent carbachol in this region inhibits bladder contraction in cats and rats.11–13 This region corresponds to the RPRF, also known as the nucleus reticularis pontis oralis in cats.11,12 The RPRF has a strong inhibitory effect on micturition since electrical stimulation of the PMC cannot evoke micturition after carbachol injection in the RPRF.14 In rats glutamate injection in the RPRF also strongly inhibits bladder contraction13 while injection of the glutamate receptor antagonist MK-801 facilitates bladder contraction.15 We assessed the influence of the MFL on micturition after stimulation by injection of excitatory agents and their antagonists. We also examined the relation between the MFL and the RPRF.
MATERIALS AND METHODS Animals A total of 35 female Sprague-Dawley® rats weighing 215 to 231 gm were used in this study. The study protocol was approved by the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus.
Continuous Cystometry in Conscious Rats Rats were anesthetized with 2% isoflurane. A PE-50 polyethylene catheter (Clay Adams, Parsippany, New Jersey) was inserted transurethrally in the bladder. Three small holes were made in the skull rostral (bregma 3.5 mm, right 0.8 mm) and caudal (bregma ⫺9.5 mm, right and left 1.0 mm). PE-10 polyethylene catheters (2 cm) were inserted to inject drugs in the MFL or the RPRF according to published coordinates.16 The MFL in this study was thought to correspond to the anterior cingulate gyrus in cats. For continuous cystometry each rat was placed in a NAIGAI-CFK-1P restraining cage (NMS, Tokyo, Japan) and allowed to recover from anesthesia. Cystometry was performed at least 1 hour after the rat was placed in the cage. The bladder catheter was connected to an infusion pump via polyethylene tubing. The bladder was filled with physiological saline at 0.05 ml per minute. After bladder contractions were stable for at least 30 minutes 1 l physiological saline, glutamate, the glutamate receptor antagonist MK-801 (NMDA [N-
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methyl D-aspartate] antagonist), noradrenaline or the adrenergic ␣-1D receptor antagonist naftopidil (10 M in 3 to 7 rats each) was injected in the MFL. When glutamate was injected in the MFL, MK-801 was also injected in the ipsilateral or bilateral RPRF since glutamate projections to the RPRF neuron inhibit micturition and this effect is blocked by MK-801.15 Maximum bladder contraction pressure, the interval between bladder contraction and baseline bladder pressure were measured for 3 to 8 contractions after injecting each agent.
Statistical Analysis Results are shown as the mean ⫾ SEM. The Student t test for paired and unpaired data was used for statistical analysis with p ⬍0.05 considered statistically significant.
RESULTS Before injecting each active drug physiological saline was injected in the MFL and the RPRF in 3 rats. There were no significant bladder contraction differences before vs after saline injection. Glutamate injection in the MFL in 7 rats significantly prolonged the interval between bladder contractions by 39% (p ⬍0.001, fig. 1, A). However, injection of the glutamate antagonist MK-801 in the MFL in 7 rats significantly shortened the interval between bladder contractions by 39% (p ⫽ 0.028, see table and fig. 1, B). There was no significant change in maximum contraction pressure or baseline bladder pressure after injecting these agents. When glutamate was injected in the MFL in 4 rats just after MK-801 injection in the ipsilateral RPRF, the interval between bladder contractions was also significantly prolonged by 28% (p ⫽ 0.010). Since MK801 injection in the RPRF shortens the interval between bladder contractions15 and the RPRF receives projection from the bilateral MFL in cats,17,18 we also tried injecting MK-801 in the bilateral RPRF. After prior injection of MK-801 in the bilateral RPRF glutamate injection in the MFL in 6 rats did not alter cystometric parameters (see table and fig. 2). When noradrenaline was injected in the MFL in 5 rats, bladder baseline pressure became irregular and many nonvoiding bladder contractions were recorded. The interval between voiding bladder contractions was significantly shortened by 47% after noradrenaline injection in the MFL (p ⫽ 0.006, fig. 3, A). On the other hand, injection of its antagonist naftopidil in the MFL in 6 rats significantly prolonged the interval between bladder contractions by 47% (p ⬍0.001, see table and fig. 3, B). There was no significant change in maximum bladder contraction pressure or baseline bladder pressure after injecting these agents.
DISCUSSION Injection of glutamate or its antagonist MK-801 in the MFL prolonged and shortened the interval be-
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EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
Figure 1. Continuous cystometry before and after injection in MFL. There were no significant changes in maximum bladder contraction pressure or baseline bladder pressure after bladder contraction recovery. A, 10 M glutamate significantly prolonged interval between bladder contractions. B, 10 M MK-801 significantly shortened interval between bladder contractions.
tween bladder contractions, respectively, while injection of noradrenaline or its antagonist naftopidil in the MFL shortened and prolonged the interval between bladder contractions, respectively. Thus, neurons in the MFL that are excited by glutamate or noradrenaline may inhibit or facilitate the micturition reflex, respectively. Generally it is thought that micturition occurs after the release of inhibitory projections from the forebrain (cerebrum) to the PMC. This theory is supported by reports that precollicular decerebrate rats and patients with cerebral infarction show urinary frequency.5–7 However, there is also the possibility that micturition is evoked by the suppression of excitatory projections from forebrain to RPRF. The RPRF, also known as the nucleus reticularis pontis oralis in cats, is located ventromedial to the locus coeruleus complex.11,12 Injection of the cholinomimetic agent carbachol or glutamate in this region strongly inhibits bladder contraction in cats and rats11,13 while MK-801 injection facilitates bladder contraction in rats.15 When bladder activity was inhibited by glutamate injection in the RPRF, the glycine level in the lumbosacral cord was noted to increase.13 On the other hand, rats with cerebral
infarction showed urinary frequency while the glutamate level in the cerebrum and the glycine level in the lumbosacral cord were significantly decreased compared with control values.3 Glycine is a major inhibitory neurotransmitter in the central nervous system that inhibits the micturition reflex at the level of the lumbosacral cord.3,13,19 In our study glutamate injection in the MFL inhibited bladder contraction. This effect was blocked by prior MK-801 injection in the bilateral RPRF but not in the ipsilateral RPRF. The RPRF receives projections from the bilateral MFL in cats17,18 and the same neuronal projections between MFL and RPRF may exist in the rat brain. Consequently interception of the ipsilateral RPRF pathway might not be enough to block the inhibitory effect of glutamate injection in the MFL on the micturition reflex. These results suggest that activation of MFL neurons expressing the NMDA glutamate receptor excites bilateral RPRF neurons that use glutamate as a neurotransmitter. Results also suggest that descending RPRF neurons inhibit the micturition reflex by activating inhibitory glycinergic neurons in the lumbosacral cord. In other words, micturition may pos-
Change in bladder activity after injection of glutamate vs MK-801 and noradrenaline vs naftopidil in MFL, and effect of co-administration of glutamate in MFL and MK-801 in ipsilateral or bilateral RPRF Mean ⫾ SEM MFL
Interval (mins): Before After p Value Max contraction pressure (cm H2O): Before After Baseline bladder pressure (cm H2O): Before After
Glutamate
MK-801
Noradrenaline
10.5 ⫾ 2.1 14.6 ⫾ 2.2 ⬍0.001
11.7 ⫾ 2.2 7.1 ⫾ 0.9 0.028
16.9 ⫾ 2.0 8.9 ⫾ 2.1 0.006
42.2 ⫾ 5.0 41.8 ⫾ 3.3
45.1 ⫾ 4.2 44.2 ⫾ 4.8
7.7 ⫾ 0.7 7.5 ⫾ 0.5
7.6 ⫾ 0.5 7.6 ⫾ 0.4
Mean ⫾ SEM MFL Glutamate Naftopidil
Ipsilat RPRF MK-801
Bilat RPRF MK-801
12.8 ⫾ 1.9 18.8 ⫾ 2.0 ⬍0.001
7.8 ⫾ 1.6 10.0 ⫾ 1.7 0.010
8.8 ⫾ 1.4 8.2 ⫾ 1.3 —
47.1 ⫾ 5.5 41.4 ⫾ 2.9
44.5 ⫾ 4.5 44.4 ⫾ 4.4
51.7 ⫾ 4.8 50.3 ⫾ 3.9
47.9 ⫾ 4.0 48.3 ⫾ 3.8
7.6 ⫾ 0.5 7.5 ⫾ 0.5
7.6 ⫾ 0.5 7.1 ⫾ 0.8
8.7 ⫾ 1.1 9.2 ⫾ 1.4
7.7 ⫾ 0.7 7.7 ⫾ 0.7
EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
1119
Figure 2. Effect of co-administration of glutamate with MK-801 in MFL. A, before injection. B, glutamate in MFL. C, co-administration of glutamate in MFL and MK-801 in ipsilateral RPRF prolonged interval between bladder contractions compared with before injection. D, co-administration of glutamate in MFL and MK-801 in bilateral RPRF did not prolong interval between bladder contractions.
sibly be evoked by the suppression of excitatory projections from forebrain to RPRF. Chemical stimulation of the PMC alters the interval between bladder contractions and alters bladder contraction pressure in cats20 while chemical stimulation of the RPRF only influences the interval between bladder contractions in cats and rats.13,21
Since injection of agents in the MFL only had an effect on the interval between bladder contractions in our study, the MFL may be more closely related to the RPRF than to the PMC. Naftopidil is an adrenergic ␣-1D receptor antagonist used for benign prostatic hyperplasia.22 It decreases smooth muscle tone in the urethra and in-
Figure 3. Continuous cystometry before and after injection in MFL. There was no significant change in maximum bladder contraction pressure or baseline bladder pressure after injection. A, noradrenaline induced detrusor overactivity, ie irregular bladder baseline pressure and many nonvoiding bladder contractions, and significantly shortened interval between voiding bladder contractions. B, adrenergic receptor antagonist naftopidil significantly prolonged interval between bladder contractions.
1120
EFFECT OF CHEMICAL STIMULATION OF MEDIAL FRONTAL LOBE ON MICTURITION REFLEX
hibits the micturition reflex at the lumbosacral cord level.23,24 In our study noradrenaline injection in the MFL induced detrusor overactivity (irregular bladder baseline pressure, many nonvoiding bladder contractions and urinary frequency) while the micturition reflex was inhibited by naftopidil. Results suggest that the excitation of MFL neurons that express adrenergic receptors facilitates the micturition reflex, which is the opposite effect of that seen after glutamate injection in the MFL. Accordingly MFL neurons excited by glutamate may differ from those excited by noradrenaline. Urinary frequency and urgency are common in patients with cerebrovascular disease while disturbed micturition is more common in patients with lesions of the frontal lobe than in those with lesions of the occipital lobe.6,7 However, partial decerebration involving the MFL increases the threshold volume that induces micturition in rats.21 Also, regions
facilitating or inhibiting the micturition reflex as a result of electrical stimulation coexist in the MFL, including the anterior cingulate gyrus in cats.25 This area of the cat brain contains neurons that show increased or decreased activity during the micturition reflex.26 Thus, the MFL may be an important integration center involved in the initiation of micturition and urine storage that voluntarily regulates the micturition reflex depending on circumstances.
CONCLUSIONS MFL neurons excited by glutamate inhibited the micturition reflex via activation of the RPRF by glutamatergic projections while MFL neurons excited by noradrenaline facilitated the micturition reflex. Glutamate and noradrenaline had no influence on bladder contraction pressure. Thus, MFL may be an important integration center for micturition initiation and for urine storage mechanisms.
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