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Influence of glutamatergic projections to the rostral pontine reticular formation on micturition in rats
Life Sciences 85 (2009) 732–736
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Influence of glutamatergic projections to the rostral pontine reticular formation on micturition in rats Hidekatsu Naka a, Saori Nishijima a,b, Katsumi Kadekawa a,b, Kimio Sugaya a,b,⁎, Seiichi Saito a a b
Division of Urology, Department of Organ-oriented Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan Southern Knights' Laboratory LLP, Okinawa, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2009 Accepted 28 September 2009 Keywords: Rostral pontine reticular formation Glutamate MK-801 Micturition reflex pathway
a b s t r a c t Aims: We examined the effect of injecting glutamate or a glutamate receptor antagonist into the rostral pontine reticular formation (RPRF) on the micturition reflex in anesthetized rats and conscious rats. Main method: Forty-eight female rats were divided into an isovolumetric cystometry group and a continuous cystometry group. Under urethane anesthesia or while conscious, physiological saline, glutamate, or MK-801 (a glutamate receptor antagonist) was injected into the RPRF, and then the changes of bladder activity were examined. Key findings: There was no significant change of bladder activity after injection of physiological saline. In anesthetized rats, the injection of either glutamate or MK-801 into the RPRF transiently inhibited bladder contractions. There was a complete recovery of bladder activity 10–20 min after glutamate or MK-801 injection and there were no significant changes of cystometry parameters after the recovery of bladder contractions. In conscious rats, injection of glutamate into the RPRF prolonged the interval between bladder contractions and decreased the baseline bladder pressure. On the other hand, injection of MK-801 into the RPRF caused numerous small bladder contractions, some of which were accompanied by a leakage of a small amount of fluid from around the urethral catheter. Significance: RPRF neurons receive glutamatergic projections, possibly from the forebrain, and the RPRF inhibits the micturition reflex pathway. RPRF neurons are also regulated by inhibitory interneurons, which receive glutamatergic projections as well. Therefore, the RPRF plays an important role in the regulation of urine storage. © 2009 Elsevier Inc. All rights reserved.
Introduction The micturition reflex is mediated by the autonomic nervous system, but the actual release of urine is regulated by voluntary neural mechanisms (Sugaya et al. 2005). The micturition reflex is a bladder-tobladder contraction reflex and the reflex center is located in the rostral pontine tegmentum (pontine micturition center: PMC), where electrical or chemical stimulation evokes micturition in cats (Sugaya et al. 1987a). In addition, there are two centers in the pons that inhibit micturition. Electrical stimulation of the region ventrolateral to the PMC enhances external urethral sphincter (EUS) activity and inhibits bladder contraction (Kuru 1965; Holstage et al. 1986; Nishizawa et al. 1987), so this region is called the pontine urine storage center (PUSC). Another pontine region that controls lower urinary tract function is located ventromedial to the locus coeruleus complex. Electrical stimulation or injection of carbachol (a cholinomimetic agent) into this region inhibits bladder contraction in cats and rats (Sugaya et al. 1987b; Kimura et al. 1995; Nishijima et al. 2005), ⁎ Corresponding author. Southern Knights' Laboratory LLP, 2-7-7-301 Takahara, Okinawa, 904-2171, Japan. Tel.: +81 90 4998 7459; fax: +81 98 936 9225. E-mail address: [email protected] (K. Sugaya). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.09.014
and this region corresponds to the rostral pontine reticular formation (RPRF), which is also known as the nucleus reticularis pontis oralis (PoO) in cats. The RPRF has a strong inhibitory effect on micturition, because electrical stimulation of the PMC cannot evoke micturition after injection of carbachol into the RPRF (Sugaya et al. 2006). We previously reported that glycine (the most important inhibitory amino acid neurotransmitter in the central nervous system) showed an increase in the lumbosacral cord when injection of carbachol or flavoxate into the RPRF inhibited bladder contraction (Nishijima et al. 2003; Sugaya et al. 2005), which suggested that descending projections from the RPRF inhibit the micturition reflex via glycinergic neurons in the lumbosacral cord. Generally, it is thought that micturition occurs after the release of inhibitory projections from the forebrain (cerebrum) to the PMC. However, there has been no evidence to support this hypothesis, and another possibility is that micturition is evoked by the release of excitatory projections from the forebrain to the RPRF. We previously found that the cerebral level of glutamate (the most important excitatory amino acid neurotransmitter in the central nervous system) was significantly decreased in rats with urinary frequency after cerebral infarction (Nishijima et al. 2003). Therefore, we hypothesized that urinary frequency after cerebral infarction might be induced by a
H. Naka et al. / Life Sciences 85 (2009) 732–736
weakening of the inhibitory influence of the RPRF on the micturition reflex pathway due to a decrease of excitatory glutamatergic projections from the forebrain to the RPRF. Accordingly, we performed the present study to examine the effects of injecting glutamate or a glutamate receptor antagonist into the RPRF on the micturition reflex in rats.
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connected via polyethylene tubing to an infusion pump and the bladder was filled with physiological saline at a rate of 0.05 ml/min. After bladder contractions had been stable for at least 30 min, 0.5 μl of physiological saline, 3 or 30 μM glutamate, or 3 or 30 μM MK-801 dissolved in 0.5 μl of physiological saline was injected into the RPRF through the catheter (n =5–6 each). Cystometry was continued for at least 60min after injection and the changes of bladder activity were recorded.
Materials and methods Statistical analysis Animal model Forty-eight female Sprague–Dawley rats weighing 193–226 g were used in this study. The protocol of the study was approved by the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus.
Results are reported as the mean ± standard deviation (SD). Student's t-tests for paired and unpaired data were used for statistical analysis, and P < 0.05 was considered to indicate statistical significance. Results
Isovolumetric cystometry under anesthesia Isovolumetric cystometry under anesthesia Isovolumetric cystometry was performed in 22 rats. The animals were anesthetized with urethane (0.8 g/kg subcutaneously and 0.4 g/ kg intraperitoneally), and a polyethylene catheter (PE50, Clay Adamus, USA) was inserted transurethrally into the bladder. The urethra was ligated around the catheter near the external urethral meatus to create an isovolumetric bladder, while the ureters were transected and the proximal cut ends were left open. Then a small hole was made in the skull (bregma −9.5 mm, R 1.0 mm) for injection of drugs into the RPRF according to the published coordinates (Swanson 1992). The bladder catheter was connected by polyethylene tubing to an infusion pump and a pressure transducer via a three-way stopcock. The tubing was filled with physiological saline, and the bladder pressure was displayed on a chart recorder (San-ei Electric Co., Ltd., Nagano, Japan). The bladder was filled with physiological saline (0.05 ml/min) to above the threshold volume in order to induce rhythmic isovolumetric contractions. After bladder contractions had been stable for at least 30min, 0.5 μl of physiological saline, 0.3 or 3 μM glutamate, or 3 μM MK801 (N-methyl D-aspartate (NMDA), a glutamatergic receptor antagonist) dissolved in 0.5 μl of physiological saline was injected into the RPRF using a microsyringe (n= 5–9 each). Isovolumetric cystometry was continued for at least 30 min after injection and the changes of bladder activity were recorded. Continuous cystometry in conscious rats Continuous cystometry was performed in 26 other rats. The animals were anesthetized with 2% isoflurane, and a polyethylene catheter (PE50) was inserted transurethrally into the bladder. Then a small hole was made in the skull (bregma −9.5 mm, R 1.0 mm), and a short (2 cm) polyethylene catheter (PE10, Clay Adamus, USA) was inserted for injection of drugs into the RPRF (Swanson 1992). This catheter was fixed to the skull with adhesive. For conscious cystometry, each rat was placed in a restraining cage (NAIGAI-CFK-1P, NMS, Tokyo, Japan) and was allowed to recover from anesthesia for about 30 min. Cystometry was performed at least 1 h after the animal had been placed in the cage. The bladder catheter was
Before injection of each drug under anesthesia, physiological saline was injected into the RPRF, with no significant differences of bladder conditions being detected between before and after injection. When glutamate was injected into the RPRF, bladder contractions were transiently abolished and the duration of their inhibition was dosedependent (9.0 ± 9.5 min at 0.3 μM, 12.1 ± 10.3 min at 3 μM) (Table 1 and Fig. 1A). Injection of MK-801 at 3 μM also transiently abolished bladder contractions, and its effect was more rapid and stronger than that of glutamate, with inhibition persisting for 16.4 ± 13.1 min (Table 1 and Fig. 1B). However, bladder contractions recovered completely by 10–20 min after the injection of either glutamate or MK-801 and there were no significant changes of any of the cystometry parameters after the recovery of bladder contractions (Table 1). Continuous cystometry in conscious rats In order to avoid the influence of anesthesia on the micturition reflex, we also performed continuous cystometry in conscious rats. Before the injection of each active drug, physiological saline was injected into the RPRF, but there were no significant differences of bladder contractions between before and after its injection. When 0.3 μM glutamate (this dose showed an inhibitory effect in control rats (N = 2) undergoing isovolumetric cystometry) or 3 μM glutamate (N = 6) was injected into the RPRF, none of the bladder contraction parameters showed any changes. However, injection of 30 μM glutamate (N = 5) prolonged the interval between bladder contractions by 22% and decreased the baseline bladder pressure by 10% (Table 2 and Fig. 2). These inhibitory effects of glutamate persisted for over 30 min. On the other hand, injection of 3 μM MK-801 (N = 7) or 30 μM MK-801 (N = 6) caused numerous small bladder contractions with an amplitude over 5 cmH2O, some of which were accompanied by a leakage of a small amount of fluid from around the urethral catheter (Fig. 3). Therefore, we calculated the cystometry parameters after drug injection by only assessing bladder contractions that
Table 1 Changes of bladder contractions after injection of drugs into the RPRF in rats under urethane anesthesia. Drugs
Glutamate MK-801
Amount
0.3 μM 3 μM 3 μM
Rat number
Inhibitory time (min)
9 8 5
9.0 ± 9.5 12.1 ± 10.3 16.4 ± 13.1
MCP (cmH2O)
Interval (min)
Baseline (cmH2O)
Before
Aftera
Before
Aftera
Before
Aftera
1.6 ± 0.3 1.5 ± 0.2 1.3 ± 0.2
1.4 ± 0.3 1.5 ± 0.3 1.3 ± 0.2
42.9 ± 11.1 43.9 ± 13.5 40.7 ± 6.4
43.9 ± 12.8 43.7 ± 14.4 41.1 ± 6.1
18.0 ± 7.2 17.4 ± 8.1 16.1 ± 8.1
17.0 ± 7.5 17.9 ± 9.2 16.1 ± 8.2
Interval: interval between bladder contractions, MCP: maximum bladder contraction pressure, and baseline: baseline bladder pressure. Bladder contractions transiently disappeared after injection of glutamate or MK-801. However, bladder contractions recovered completely at 10–20 min after injection and there were no significant changes of cystometry parameters after recovery. a Bladder activity after drug injection was calculated by the average for 30 min after bladder contraction recovered.
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Fig. 1. Isovolumetric cystometry before and after injection of glutamate (A) or MK-801 (B) into the RPRF under urethane anesthesia. (A) Injection of glutamate (0.3 μM) into the RPRF abolished bladder contractions after 2–3 min. There were no significant changes of cystometry parameters after the recovery of bladder contractions. (B) Injection of MK-801 (3 μM) into the RPRF immediately abolished bladder contractions. There were also no significant changes of cystometry parameters after the recovery of bladder contractions.
reached at least two thirds of the maximum contraction pressure before injection. As a result, injection of MK-801 into the RPRF shortened the interval between bladder contractions by 32% at 3 μM and by 48% at 30 μM (Table 2), with this excitatory effect persisting for about 60 min. There were no changes of the other cystometry parameters after injection of MK-801. We also examined whether the inhibitory effect of glutamate on bladder activity via the RPRF recovered after injection of MK-801 in conscious rats (N = 4). First, injection of 30 μM glutamate into the RPRF prolonged the interval between bladder contractions (Fig. 4B). After 30 min, injection of 30 μM MK-801 into the RPRF led to a marked increase of bladder contractions (Fig. 4C) compared with either before or after injection of glutamate (Fig. 4A). Discussion The present study showed that injection of glutamate into the RPRF of conscious rats prolonged the interval between bladder contractions and decreased the baseline bladder pressure, while injection of MK-801 induced numerous small bladder contractions accompanied by a slight leakage of a small amount of fluid from around the urethral catheter. These results suggest that glutamate activated descending RPRF neurons via NMDA glutamatergic receptors, after which the descending neurons inhibited the micturition reflex. In rats under urethane anesthesia, on the other hand, injection of either glutamate or MK801 into the RPRF inhibited bladder contraction. Therefore, glutamatergic projections to the RPRF might activate both descending neurons from the RPRF and inhibitory interneurons within the RPRF that project to the descending neurons (Fig. 5). In cats, it has been reported that injection of carbachol into the nucleus reticularis pontis oralis increases the discharge rate of nucleus reticularis gigantocellularis reticulospinal neurons and decreases
soleus muscle contraction via the activation of segmental inhibitory interneurons projecting to motoneurons (Takakusaki et al. 1994). In addition, injection of a cholinomimetic agent into the RPRF induces muscle atonia and increases the spinal glycine level in cats (Kodama et al. 2003). Our previous study showed that injection of carbachol or flavoxate into the RPRF inhibited bladder contractions and increased the spinal cord glycine level in rats (Nishijima et al. 2005). Glycine has been identified as an important inhibitory neurotransmitter in the central nervous system, and intrathecal injection of glycine inhibits bladder contraction (Miyazato et al. 2003). Therefore, it has been suggested that the segmental inhibitory interneurons projecting to motoneurons in the lumbosacral cord are glycinergic neurons, and that activation of descending RPRF neurons inhibits the micturition reflex via stimulation of spinal glycinergic neurons (Sugaya et al. 2005). In the present study, injection of glutamate into the RPRF of conscious rats also inhibited bladder contraction, while the injection of an NMDA glutamatergic receptor antagonist (MK-801) promoted bladder contraction. Therefore, RPRF neurons might receive glutamatergic projections via NMDA glutamatergic receptors, and the inhibition of micturition by injection of glutamate into the RPRF might also involve spinal glycinergic neurons. In rats under urethane anesthesia, injection of glutamate into the RPRF inhibited bladder contraction, suggesting that glutamate activated RPRF neurons projecting to spinal glycinergic neurons. However, injection of MK-801 into the RPRF of anesthetized rats inhibited bladder contraction. These results suggest that RPRF neurons might also be innervated by inhibitory interneurons which have NMDA receptors. Differing effects of MK-801 on micturition in the conscious state and under urethane anesthesia have also been reported before. For example, intravenous administration of MK-801 to conscious rats with cerebral infarction resulted in a slight decrease of bladder capacity, while bladder capacity was significantly increased
Table 2 Changes of bladder contractions after injection of drugs into the RPRF in conscious rats. Drugs
Glutamate MK-801
Amount
3 μM 30 μM 3 μM 30 μM
Rat number 6 5 7 6
MCP (cmH2O)
Interval (min)
Baseline (cmH2O)
Before
After
Before
After
Before
After
4.3 ± 1.8 5.2 ± 1.5 5.6 ± 2.8 4.8 ± 1.6
5.1 ± 1.3 6.4 ± 2.2⁎ 3.3 ± 1.3⁎ 2.1 ± 1.0⁎
34.7 ± 4.3 35.2 ± 6.5 39.8 ± 8.3 39.3 ± 5.5
34.2 ± 6.4 35.8 ± 5.7 37.1 ± 11.2 43.8 ± 7.4
8.5 ± 2.2 8.7 ± 3.1 6.5 ± 0.9 8.0 ± 1.8
8.7 ± 2.8 7.7 ± 2.2⁎ 6.6 ± 0.9 7.7 ± 1.1
Interval: interval between bladder contractions, MCP: maximum bladder contraction pressure, baseline: baseline bladder pressure. The interval between bladder contractions was prolonged and the baseline bladder pressure was decreased after injection of glutamate, while the interval between bladder contractions was shortened after injection of MK-801. ⁎ P < 0.05.
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Fig. 2. Continuous cystometry before (A) and after (B) injection of glutamate into the RPRF of conscious rats. Injection of glutamate into the RPRF prolonged the interval between bladder contractions (B) compared with that before injection (A).
Fig. 3. Continuous cystometry before (A) and after (B) injection of MK-801 into the RPRF in conscious rats. Injection of MK-801 into the RPRF caused numerous small bladder contractions, some of which were accompanied by a minor leakage (B).
without a decrease of the contraction pressure when MK-801 was given under urethane anesthesia (Yokoyama et al. 2002). In addition, intravenous administration of MK-801 did not alter reflex bladder activity in unanesthetized decerebrate rats, but suppressed reflex bladder contraction in intact rats under urethane anesthesia (Yoshiyama et al. 1994). Our previous study showed that intrathecal
injection of glutamate increased the glycine level in the lumbosacral cord, while intrathecal injection of MK-801 decreased glutamate and glycine levels in the lumbosacral cord of both intact rats and rats with spinal cord injury, suggesting that glutamatergic neurons have stimulatory projections to both glutamatergic and glycinergic neurons in the lumbosacral cord (Ashitomi et al. 2006). These results indicate
Fig. 4. Continuous cystometry before (A) and after injection of glutamate (B) or MK-801 (C) into the RPRF in conscious rats. Injection of 30 uM glutamate into the RPRF prolonged the interval between bladder contractions (B) compared with that before injection (A), while the interval was restored by injection of MK-801 (C).
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to RPRF neurons due to cerebral infarction or injection of MK-801 induces frequency. Therefore, overactivity of the bladder associated with cerebral infarction may not be caused by a decrease in the activity of inhibitory projections from the forebrain to the PMC (Nitti and Blaivas 2007), but may be due to a decrease in glutamatergic projections from the forebrain to RPRF neurons. Conclusion RPRF neurons may receive glutamatergic projections from the forebrain that are urethane-sensitive, and RPRF neurons seem to activate lumbosacral neurons which inhibit the micturition reflex. In addition, RPRF neurons are inhibited by inhibitory interneurons that receive glutamatergic projections. Accordingly, the RPRF which is the pontine micturition inhibitory area (Sugaya et al. 2005) plays an important role in the regulation of urine storage. Acknowledgment This study was supported by a Grant-in-Aid for Scientific Research (2079115) from the Japan Society for the Promotion of Science. Fig. 5. Schema of glutamatergic projections to the RPRF. In both conscious rats and rats under urethane anesthesia, injection of glutamate into the RPRF activates descending RPRF neurons (R) and inhibitory interneurons (I) via NMDA receptors (blue triangles) in the RPRF, but glutamate predominantly acts on the descending RPRF neurons. As a result, activated descending RPRF neurons facilitate spinal inhibitory interneurons, which inhibit the micturition reflex pathway and block the micturition. In conscious rats, MK-801 blocks the NMDA receptor, but its effect on glutamatergic projections to descending RPRF neurons from the glutamate neurons (G) in the forebrain is predominant over that on glutamatergic projections to inhibitory interneurons in the RPRF. Thus, injected MK-801 inhibits the descending RPRF neurons and the micturition reflex is facilitated. In rats under urethane anesthesia, however, glutamatergic projections from the forebrain are weakened because this pathway is urethanesensitive, so that glutamatergic projections to the RPRF predominantly target the inhibitory interneurons. Under these circumstances, injected MK-801 mainly acts on the inhibitory interneurons, so that the descending RPRF neurons are facilitated and micturition is inhibited.
that NMDA receptors play an important role in both facilitatory and inhibitory central neural control of voiding, and that there is a significant interaction between urethane anesthesia and NMDAdependent glutamatergic transmission. It is possible that the activity of direct glutamatergic projections to RPRF neurons is weaker under urethane anesthesia, while the activity of glutamatergic projections to inhibitory interneurons innervating RPRF neurons is not affected. Thus, the glutamatergic facilitatory pathway to RPRF neurons is urethane-sensitive, while injection of MK-801 under urethane anesthesia has an antagonistic effect on the NMDA receptors of inhibitory interneurons projecting to RPRF neurons. In the present study, glutamate had an inhibitory effect at much lower doses in anesthetized animals than in conscious animals. In conscious animals, neurons may receive various inputs and the output of a particular class of neurons may reflect the overall balance of these inputs. On the other hand, there may be fewer inputs in anesthetized animals, so that even a small stimulus (such as a low dose of glutamate) can influence bladder activity. However, the details of the mechanisms involved are currently unknown. According to our previous study, the glutamate level in the cerebrum and the glycine level in the spinal cord were both decreased in rats with cerebral infarction and urinary frequency (Nishijima et al. 2003). In the present study, injection of MK-801 into the RPRF of conscious rats induced frequency. These results suggest that glutamatergic projections from the forebrain (cerebrum) to RPRF neurons inhibit the micturition reflex, and that a decrease of activity in glutamatergic projections from the forebrain
References Ashitomi K, Sugaya K, Miyazato M, Nishijima S, Ogawa Y. Intrathecal glutamate promotes glycinergic neuronal activity and inhibits the micturition reflex in urethaneanesthetized rats. International Journal of Urology 13 (12), 1519–1524, 2006. Holstege G, Griffiths D, de Wall H, Dalm E. Anatomical and physiological observations on supraspinal control of bladder and urethral sphincter muscles in the cat. Journal of Comparative Neurology 250 (4), 449–461, 1986. Kimura Y, Ukai Y, Kimura K, Sugaya K, Nishizawa O. Inhibitory influence from the nucleus reticularis pontis oralis on the micturition reflex induced by electrical stimulation of the pontine micturition center in cats. Neuroscience Letters 195 (3), 214–216, 1995. Kodama T, Lai YY, Siegel JM. Changes in inhibitory amino acid release linked to pontineinduced atonia: an in vivo microdialysis study. Journal of Neuroscience 23 (4), 1548–1554, 2003. Kuru M. Nervous control of micturition. Physiological Reviews 45, 425–494 4, 1965. Miyazato M, Sugaya K, Nishijima S, Ashitomi K, Hatano T, Ogawa Y. Inhibitory effect of intrathecal glycine on the micturition reflex in normal and spinal cord injury rats. Experimental Neurology 183 (1), 232–240, 2003. Nishijima S, Sugaya K, Miyazato M, Ashitomi K, Morozumi M, Ogawa Y. Relationship between bladder activity and amino acid levels in the central nervous system in rats with cerebral infarction. Biomedical Research 24 (3), 173–180, 2003. Nishijima S, Sugaya K, Miyazato M, Shimabukuro S, Morozumi M, Ogawa Y. Activation of the rostral pontine reticular formation increases the spinal glycine level and inhibits bladder contraction in rats. Journal of Urology 173 (5), 1812–1816, 2005. Nishizawa O, Sugaya K, Noto H, Harada T, Tsuchida S. Pontine urine storage center in the dog. Tohoku journal of Experimental Medicine 153 (1), 77–78, 1987. Nitti VW, Blaivas JG. Urinary Incontinence: Epidemiology, Pathophysiology, Evaluation, and Management Overview. In: Wein AJ, Kavoussi, LR, Novick AC, Partin AW, Peters CA, (Eds), Campbell–Walsh Urology, 9th edition vol. 3, Saunders Elsevier, Philadelphia pp. 2046–2078, 2007. Sugaya K, Matsuyama K, Takakusaki K, Mori S. Electrical and chemical stimulation of the pontine micturition center. Neuroscience Letters 80 (2), 197–201, 1987a. Sugaya K, Mori S, Nishizawa O, Noto H, Tsuchida S. Chemical stimulations of the pontine micturition center and the nucleus reticularis pontis oralis. Neurourology and Urodynamics 6, 143–144, 1987b. Sugaya K, Nishijima S, Miyazato M, Ogawa Y. Central nervous control of micturition and urine storage. Journal of Smooth Muscle Research 41 (3), 117–132, 2005. Sugaya K, Nishijima S, Miyazato M, Oda M, Ogawa Y. Inhibitory effect of the nucleus reticularis pontis oralis on the pontine micturition center and pontine urine storage center in decerebrate cats. Biomedical Research 27 (5), 211–217, 2006. Swanson LW. Brain Maps: Structure of the Rat Brain, First ed. Elsevier Science Publishers, San Deigo, 1992. Takakusaki K, Shimoda N, Matsuyama K, Mori S. Discharge properties of medullary reticulospinal neurons during postural changes induced by intrapontine injections of carbachol, atropine and serotonine, and their functional linkages to hindlimb motoneurons in cats. Experimental Brain Research 99 (3), 361–374, 1994. Yokoyama O, Yoshiyama M, Namiki M, de Groat WC. Changes in dopaminergic and glutamatergic excitatory mechanisms of micturition reflex after middle cerebral artery occlusion in conscious rats. Experimental Neurology 173 (1), 129–135, 2002. Yoshiyama M, Roppolo JR, De Groat WC. Alteration by urethane of glutamatergic control of micturition. European Journal of Pharmacology 264 (3), 417–425, 1994.
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Influence of glutamatergic projections to the rostral pontine reticular formation on micturition in rats Hidekatsu Naka a, Saori Nishijima a,b, Katsumi Kadekawa a,b, Kimio Sugaya a,b,⁎, Seiichi Saito a a b
Division of Urology, Department of Organ-oriented Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan Southern Knights' Laboratory LLP, Okinawa, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2009 Accepted 28 September 2009 Keywords: Rostral pontine reticular formation Glutamate MK-801 Micturition reflex pathway
a b s t r a c t Aims: We examined the effect of injecting glutamate or a glutamate receptor antagonist into the rostral pontine reticular formation (RPRF) on the micturition reflex in anesthetized rats and conscious rats. Main method: Forty-eight female rats were divided into an isovolumetric cystometry group and a continuous cystometry group. Under urethane anesthesia or while conscious, physiological saline, glutamate, or MK-801 (a glutamate receptor antagonist) was injected into the RPRF, and then the changes of bladder activity were examined. Key findings: There was no significant change of bladder activity after injection of physiological saline. In anesthetized rats, the injection of either glutamate or MK-801 into the RPRF transiently inhibited bladder contractions. There was a complete recovery of bladder activity 10–20 min after glutamate or MK-801 injection and there were no significant changes of cystometry parameters after the recovery of bladder contractions. In conscious rats, injection of glutamate into the RPRF prolonged the interval between bladder contractions and decreased the baseline bladder pressure. On the other hand, injection of MK-801 into the RPRF caused numerous small bladder contractions, some of which were accompanied by a leakage of a small amount of fluid from around the urethral catheter. Significance: RPRF neurons receive glutamatergic projections, possibly from the forebrain, and the RPRF inhibits the micturition reflex pathway. RPRF neurons are also regulated by inhibitory interneurons, which receive glutamatergic projections as well. Therefore, the RPRF plays an important role in the regulation of urine storage. © 2009 Elsevier Inc. All rights reserved.
Introduction The micturition reflex is mediated by the autonomic nervous system, but the actual release of urine is regulated by voluntary neural mechanisms (Sugaya et al. 2005). The micturition reflex is a bladder-tobladder contraction reflex and the reflex center is located in the rostral pontine tegmentum (pontine micturition center: PMC), where electrical or chemical stimulation evokes micturition in cats (Sugaya et al. 1987a). In addition, there are two centers in the pons that inhibit micturition. Electrical stimulation of the region ventrolateral to the PMC enhances external urethral sphincter (EUS) activity and inhibits bladder contraction (Kuru 1965; Holstage et al. 1986; Nishizawa et al. 1987), so this region is called the pontine urine storage center (PUSC). Another pontine region that controls lower urinary tract function is located ventromedial to the locus coeruleus complex. Electrical stimulation or injection of carbachol (a cholinomimetic agent) into this region inhibits bladder contraction in cats and rats (Sugaya et al. 1987b; Kimura et al. 1995; Nishijima et al. 2005), ⁎ Corresponding author. Southern Knights' Laboratory LLP, 2-7-7-301 Takahara, Okinawa, 904-2171, Japan. Tel.: +81 90 4998 7459; fax: +81 98 936 9225. E-mail address: [email protected] (K. Sugaya). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.09.014
and this region corresponds to the rostral pontine reticular formation (RPRF), which is also known as the nucleus reticularis pontis oralis (PoO) in cats. The RPRF has a strong inhibitory effect on micturition, because electrical stimulation of the PMC cannot evoke micturition after injection of carbachol into the RPRF (Sugaya et al. 2006). We previously reported that glycine (the most important inhibitory amino acid neurotransmitter in the central nervous system) showed an increase in the lumbosacral cord when injection of carbachol or flavoxate into the RPRF inhibited bladder contraction (Nishijima et al. 2003; Sugaya et al. 2005), which suggested that descending projections from the RPRF inhibit the micturition reflex via glycinergic neurons in the lumbosacral cord. Generally, it is thought that micturition occurs after the release of inhibitory projections from the forebrain (cerebrum) to the PMC. However, there has been no evidence to support this hypothesis, and another possibility is that micturition is evoked by the release of excitatory projections from the forebrain to the RPRF. We previously found that the cerebral level of glutamate (the most important excitatory amino acid neurotransmitter in the central nervous system) was significantly decreased in rats with urinary frequency after cerebral infarction (Nishijima et al. 2003). Therefore, we hypothesized that urinary frequency after cerebral infarction might be induced by a
H. Naka et al. / Life Sciences 85 (2009) 732–736
weakening of the inhibitory influence of the RPRF on the micturition reflex pathway due to a decrease of excitatory glutamatergic projections from the forebrain to the RPRF. Accordingly, we performed the present study to examine the effects of injecting glutamate or a glutamate receptor antagonist into the RPRF on the micturition reflex in rats.
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connected via polyethylene tubing to an infusion pump and the bladder was filled with physiological saline at a rate of 0.05 ml/min. After bladder contractions had been stable for at least 30 min, 0.5 μl of physiological saline, 3 or 30 μM glutamate, or 3 or 30 μM MK-801 dissolved in 0.5 μl of physiological saline was injected into the RPRF through the catheter (n =5–6 each). Cystometry was continued for at least 60min after injection and the changes of bladder activity were recorded.
Materials and methods Statistical analysis Animal model Forty-eight female Sprague–Dawley rats weighing 193–226 g were used in this study. The protocol of the study was approved by the Institute for Animal Experiments, Faculty of Medicine, University of the Ryukyus.
Results are reported as the mean ± standard deviation (SD). Student's t-tests for paired and unpaired data were used for statistical analysis, and P < 0.05 was considered to indicate statistical significance. Results
Isovolumetric cystometry under anesthesia Isovolumetric cystometry under anesthesia Isovolumetric cystometry was performed in 22 rats. The animals were anesthetized with urethane (0.8 g/kg subcutaneously and 0.4 g/ kg intraperitoneally), and a polyethylene catheter (PE50, Clay Adamus, USA) was inserted transurethrally into the bladder. The urethra was ligated around the catheter near the external urethral meatus to create an isovolumetric bladder, while the ureters were transected and the proximal cut ends were left open. Then a small hole was made in the skull (bregma −9.5 mm, R 1.0 mm) for injection of drugs into the RPRF according to the published coordinates (Swanson 1992). The bladder catheter was connected by polyethylene tubing to an infusion pump and a pressure transducer via a three-way stopcock. The tubing was filled with physiological saline, and the bladder pressure was displayed on a chart recorder (San-ei Electric Co., Ltd., Nagano, Japan). The bladder was filled with physiological saline (0.05 ml/min) to above the threshold volume in order to induce rhythmic isovolumetric contractions. After bladder contractions had been stable for at least 30min, 0.5 μl of physiological saline, 0.3 or 3 μM glutamate, or 3 μM MK801 (N-methyl D-aspartate (NMDA), a glutamatergic receptor antagonist) dissolved in 0.5 μl of physiological saline was injected into the RPRF using a microsyringe (n= 5–9 each). Isovolumetric cystometry was continued for at least 30 min after injection and the changes of bladder activity were recorded. Continuous cystometry in conscious rats Continuous cystometry was performed in 26 other rats. The animals were anesthetized with 2% isoflurane, and a polyethylene catheter (PE50) was inserted transurethrally into the bladder. Then a small hole was made in the skull (bregma −9.5 mm, R 1.0 mm), and a short (2 cm) polyethylene catheter (PE10, Clay Adamus, USA) was inserted for injection of drugs into the RPRF (Swanson 1992). This catheter was fixed to the skull with adhesive. For conscious cystometry, each rat was placed in a restraining cage (NAIGAI-CFK-1P, NMS, Tokyo, Japan) and was allowed to recover from anesthesia for about 30 min. Cystometry was performed at least 1 h after the animal had been placed in the cage. The bladder catheter was
Before injection of each drug under anesthesia, physiological saline was injected into the RPRF, with no significant differences of bladder conditions being detected between before and after injection. When glutamate was injected into the RPRF, bladder contractions were transiently abolished and the duration of their inhibition was dosedependent (9.0 ± 9.5 min at 0.3 μM, 12.1 ± 10.3 min at 3 μM) (Table 1 and Fig. 1A). Injection of MK-801 at 3 μM also transiently abolished bladder contractions, and its effect was more rapid and stronger than that of glutamate, with inhibition persisting for 16.4 ± 13.1 min (Table 1 and Fig. 1B). However, bladder contractions recovered completely by 10–20 min after the injection of either glutamate or MK-801 and there were no significant changes of any of the cystometry parameters after the recovery of bladder contractions (Table 1). Continuous cystometry in conscious rats In order to avoid the influence of anesthesia on the micturition reflex, we also performed continuous cystometry in conscious rats. Before the injection of each active drug, physiological saline was injected into the RPRF, but there were no significant differences of bladder contractions between before and after its injection. When 0.3 μM glutamate (this dose showed an inhibitory effect in control rats (N = 2) undergoing isovolumetric cystometry) or 3 μM glutamate (N = 6) was injected into the RPRF, none of the bladder contraction parameters showed any changes. However, injection of 30 μM glutamate (N = 5) prolonged the interval between bladder contractions by 22% and decreased the baseline bladder pressure by 10% (Table 2 and Fig. 2). These inhibitory effects of glutamate persisted for over 30 min. On the other hand, injection of 3 μM MK-801 (N = 7) or 30 μM MK-801 (N = 6) caused numerous small bladder contractions with an amplitude over 5 cmH2O, some of which were accompanied by a leakage of a small amount of fluid from around the urethral catheter (Fig. 3). Therefore, we calculated the cystometry parameters after drug injection by only assessing bladder contractions that
Table 1 Changes of bladder contractions after injection of drugs into the RPRF in rats under urethane anesthesia. Drugs
Glutamate MK-801
Amount
0.3 μM 3 μM 3 μM
Rat number
Inhibitory time (min)
9 8 5
9.0 ± 9.5 12.1 ± 10.3 16.4 ± 13.1
MCP (cmH2O)
Interval (min)
Baseline (cmH2O)
Before
Aftera
Before
Aftera
Before
Aftera
1.6 ± 0.3 1.5 ± 0.2 1.3 ± 0.2
1.4 ± 0.3 1.5 ± 0.3 1.3 ± 0.2
42.9 ± 11.1 43.9 ± 13.5 40.7 ± 6.4
43.9 ± 12.8 43.7 ± 14.4 41.1 ± 6.1
18.0 ± 7.2 17.4 ± 8.1 16.1 ± 8.1
17.0 ± 7.5 17.9 ± 9.2 16.1 ± 8.2
Interval: interval between bladder contractions, MCP: maximum bladder contraction pressure, and baseline: baseline bladder pressure. Bladder contractions transiently disappeared after injection of glutamate or MK-801. However, bladder contractions recovered completely at 10–20 min after injection and there were no significant changes of cystometry parameters after recovery. a Bladder activity after drug injection was calculated by the average for 30 min after bladder contraction recovered.
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Fig. 1. Isovolumetric cystometry before and after injection of glutamate (A) or MK-801 (B) into the RPRF under urethane anesthesia. (A) Injection of glutamate (0.3 μM) into the RPRF abolished bladder contractions after 2–3 min. There were no significant changes of cystometry parameters after the recovery of bladder contractions. (B) Injection of MK-801 (3 μM) into the RPRF immediately abolished bladder contractions. There were also no significant changes of cystometry parameters after the recovery of bladder contractions.
reached at least two thirds of the maximum contraction pressure before injection. As a result, injection of MK-801 into the RPRF shortened the interval between bladder contractions by 32% at 3 μM and by 48% at 30 μM (Table 2), with this excitatory effect persisting for about 60 min. There were no changes of the other cystometry parameters after injection of MK-801. We also examined whether the inhibitory effect of glutamate on bladder activity via the RPRF recovered after injection of MK-801 in conscious rats (N = 4). First, injection of 30 μM glutamate into the RPRF prolonged the interval between bladder contractions (Fig. 4B). After 30 min, injection of 30 μM MK-801 into the RPRF led to a marked increase of bladder contractions (Fig. 4C) compared with either before or after injection of glutamate (Fig. 4A). Discussion The present study showed that injection of glutamate into the RPRF of conscious rats prolonged the interval between bladder contractions and decreased the baseline bladder pressure, while injection of MK-801 induced numerous small bladder contractions accompanied by a slight leakage of a small amount of fluid from around the urethral catheter. These results suggest that glutamate activated descending RPRF neurons via NMDA glutamatergic receptors, after which the descending neurons inhibited the micturition reflex. In rats under urethane anesthesia, on the other hand, injection of either glutamate or MK801 into the RPRF inhibited bladder contraction. Therefore, glutamatergic projections to the RPRF might activate both descending neurons from the RPRF and inhibitory interneurons within the RPRF that project to the descending neurons (Fig. 5). In cats, it has been reported that injection of carbachol into the nucleus reticularis pontis oralis increases the discharge rate of nucleus reticularis gigantocellularis reticulospinal neurons and decreases
soleus muscle contraction via the activation of segmental inhibitory interneurons projecting to motoneurons (Takakusaki et al. 1994). In addition, injection of a cholinomimetic agent into the RPRF induces muscle atonia and increases the spinal glycine level in cats (Kodama et al. 2003). Our previous study showed that injection of carbachol or flavoxate into the RPRF inhibited bladder contractions and increased the spinal cord glycine level in rats (Nishijima et al. 2005). Glycine has been identified as an important inhibitory neurotransmitter in the central nervous system, and intrathecal injection of glycine inhibits bladder contraction (Miyazato et al. 2003). Therefore, it has been suggested that the segmental inhibitory interneurons projecting to motoneurons in the lumbosacral cord are glycinergic neurons, and that activation of descending RPRF neurons inhibits the micturition reflex via stimulation of spinal glycinergic neurons (Sugaya et al. 2005). In the present study, injection of glutamate into the RPRF of conscious rats also inhibited bladder contraction, while the injection of an NMDA glutamatergic receptor antagonist (MK-801) promoted bladder contraction. Therefore, RPRF neurons might receive glutamatergic projections via NMDA glutamatergic receptors, and the inhibition of micturition by injection of glutamate into the RPRF might also involve spinal glycinergic neurons. In rats under urethane anesthesia, injection of glutamate into the RPRF inhibited bladder contraction, suggesting that glutamate activated RPRF neurons projecting to spinal glycinergic neurons. However, injection of MK-801 into the RPRF of anesthetized rats inhibited bladder contraction. These results suggest that RPRF neurons might also be innervated by inhibitory interneurons which have NMDA receptors. Differing effects of MK-801 on micturition in the conscious state and under urethane anesthesia have also been reported before. For example, intravenous administration of MK-801 to conscious rats with cerebral infarction resulted in a slight decrease of bladder capacity, while bladder capacity was significantly increased
Table 2 Changes of bladder contractions after injection of drugs into the RPRF in conscious rats. Drugs
Glutamate MK-801
Amount
3 μM 30 μM 3 μM 30 μM
Rat number 6 5 7 6
MCP (cmH2O)
Interval (min)
Baseline (cmH2O)
Before
After
Before
After
Before
After
4.3 ± 1.8 5.2 ± 1.5 5.6 ± 2.8 4.8 ± 1.6
5.1 ± 1.3 6.4 ± 2.2⁎ 3.3 ± 1.3⁎ 2.1 ± 1.0⁎
34.7 ± 4.3 35.2 ± 6.5 39.8 ± 8.3 39.3 ± 5.5
34.2 ± 6.4 35.8 ± 5.7 37.1 ± 11.2 43.8 ± 7.4
8.5 ± 2.2 8.7 ± 3.1 6.5 ± 0.9 8.0 ± 1.8
8.7 ± 2.8 7.7 ± 2.2⁎ 6.6 ± 0.9 7.7 ± 1.1
Interval: interval between bladder contractions, MCP: maximum bladder contraction pressure, baseline: baseline bladder pressure. The interval between bladder contractions was prolonged and the baseline bladder pressure was decreased after injection of glutamate, while the interval between bladder contractions was shortened after injection of MK-801. ⁎ P < 0.05.
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Fig. 2. Continuous cystometry before (A) and after (B) injection of glutamate into the RPRF of conscious rats. Injection of glutamate into the RPRF prolonged the interval between bladder contractions (B) compared with that before injection (A).
Fig. 3. Continuous cystometry before (A) and after (B) injection of MK-801 into the RPRF in conscious rats. Injection of MK-801 into the RPRF caused numerous small bladder contractions, some of which were accompanied by a minor leakage (B).
without a decrease of the contraction pressure when MK-801 was given under urethane anesthesia (Yokoyama et al. 2002). In addition, intravenous administration of MK-801 did not alter reflex bladder activity in unanesthetized decerebrate rats, but suppressed reflex bladder contraction in intact rats under urethane anesthesia (Yoshiyama et al. 1994). Our previous study showed that intrathecal
injection of glutamate increased the glycine level in the lumbosacral cord, while intrathecal injection of MK-801 decreased glutamate and glycine levels in the lumbosacral cord of both intact rats and rats with spinal cord injury, suggesting that glutamatergic neurons have stimulatory projections to both glutamatergic and glycinergic neurons in the lumbosacral cord (Ashitomi et al. 2006). These results indicate
Fig. 4. Continuous cystometry before (A) and after injection of glutamate (B) or MK-801 (C) into the RPRF in conscious rats. Injection of 30 uM glutamate into the RPRF prolonged the interval between bladder contractions (B) compared with that before injection (A), while the interval was restored by injection of MK-801 (C).
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to RPRF neurons due to cerebral infarction or injection of MK-801 induces frequency. Therefore, overactivity of the bladder associated with cerebral infarction may not be caused by a decrease in the activity of inhibitory projections from the forebrain to the PMC (Nitti and Blaivas 2007), but may be due to a decrease in glutamatergic projections from the forebrain to RPRF neurons. Conclusion RPRF neurons may receive glutamatergic projections from the forebrain that are urethane-sensitive, and RPRF neurons seem to activate lumbosacral neurons which inhibit the micturition reflex. In addition, RPRF neurons are inhibited by inhibitory interneurons that receive glutamatergic projections. Accordingly, the RPRF which is the pontine micturition inhibitory area (Sugaya et al. 2005) plays an important role in the regulation of urine storage. Acknowledgment This study was supported by a Grant-in-Aid for Scientific Research (2079115) from the Japan Society for the Promotion of Science. Fig. 5. Schema of glutamatergic projections to the RPRF. In both conscious rats and rats under urethane anesthesia, injection of glutamate into the RPRF activates descending RPRF neurons (R) and inhibitory interneurons (I) via NMDA receptors (blue triangles) in the RPRF, but glutamate predominantly acts on the descending RPRF neurons. As a result, activated descending RPRF neurons facilitate spinal inhibitory interneurons, which inhibit the micturition reflex pathway and block the micturition. In conscious rats, MK-801 blocks the NMDA receptor, but its effect on glutamatergic projections to descending RPRF neurons from the glutamate neurons (G) in the forebrain is predominant over that on glutamatergic projections to inhibitory interneurons in the RPRF. Thus, injected MK-801 inhibits the descending RPRF neurons and the micturition reflex is facilitated. In rats under urethane anesthesia, however, glutamatergic projections from the forebrain are weakened because this pathway is urethanesensitive, so that glutamatergic projections to the RPRF predominantly target the inhibitory interneurons. Under these circumstances, injected MK-801 mainly acts on the inhibitory interneurons, so that the descending RPRF neurons are facilitated and micturition is inhibited.
that NMDA receptors play an important role in both facilitatory and inhibitory central neural control of voiding, and that there is a significant interaction between urethane anesthesia and NMDAdependent glutamatergic transmission. It is possible that the activity of direct glutamatergic projections to RPRF neurons is weaker under urethane anesthesia, while the activity of glutamatergic projections to inhibitory interneurons innervating RPRF neurons is not affected. Thus, the glutamatergic facilitatory pathway to RPRF neurons is urethane-sensitive, while injection of MK-801 under urethane anesthesia has an antagonistic effect on the NMDA receptors of inhibitory interneurons projecting to RPRF neurons. In the present study, glutamate had an inhibitory effect at much lower doses in anesthetized animals than in conscious animals. In conscious animals, neurons may receive various inputs and the output of a particular class of neurons may reflect the overall balance of these inputs. On the other hand, there may be fewer inputs in anesthetized animals, so that even a small stimulus (such as a low dose of glutamate) can influence bladder activity. However, the details of the mechanisms involved are currently unknown. According to our previous study, the glutamate level in the cerebrum and the glycine level in the spinal cord were both decreased in rats with cerebral infarction and urinary frequency (Nishijima et al. 2003). In the present study, injection of MK-801 into the RPRF of conscious rats induced frequency. These results suggest that glutamatergic projections from the forebrain (cerebrum) to RPRF neurons inhibit the micturition reflex, and that a decrease of activity in glutamatergic projections from the forebrain
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