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Forebrain muscarinic control of micturition reflex in rats
Neuropharmacology 41 (2001) 629–638 www.elsevier.com/locate/neuropharm
Forebrain muscarinic control of micturition reflex in rats O. Yokoyama *, N. Ootsuka, K. Komatsu, K. Kodama, S. Yotsuyanagi, S. Niikura, Y. Nagasaka, Y. Nakada, S. Kanie, M. Namiki Department of Urology, Kanazawa University School of Medicine, Kanazawa, 920-8641, Ishikawa, Japan Received 12 February 2001; received in revised form 2 July 2001; accepted 5 July 2001
Abstract Functional contribution of the cholinergic pathway between the frontal cortex and basal nucleus of Meynert to micturition reflex was investigated. Male Wistar rats were subjected to bilateral lesion of the basal forebrain by ibotenic acid (IA) injection (7.5 µg/rat on each side) (BF rats). Phosphate buffered saline (PBS) was injected into control rats (sham operated rats; SO rats). Cystometrograms were obtained from conscious BF and SO rats 7–10 days after IA/PBS injection. Bladder capacity (BC) of BF rats was significantly smaller than that of SO rats (approximately 43.7%) and was accompanied by decrease in choline-acetyltransferase activity in the frontal cortices. Oxotremorine M, a muscarinic receptor agonist, increased BC in BF rats, while pirenzepine, an M1 muscarinic receptor antagonist, counteracted the effect of the oxotremorine M-induced increase in BC. Injection of oxotremorine M into the dorsal pontine tegmentum (DPT) reduced BC in BF and SO rats, while injection of pirenzepine had no effect on cystometrograms. These findings indicate that the M1 muscarinic receptor plays a part in the forebrain inhibitory mechanisms involved in the micturition reflex and that muscarinic receptor in the DPT contributes to excitatory control of micturition reflex. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pons; Forebrain; Bladder overactivity; Urination disorders; Acetylcholine; Rat
1. Introduction There are two major diffuse modulatory cholinergic systems in the brain, one of which is called the basal forebrain complex (Butcher, 1995). The basal nucleus of Meynert provides most of the cholinergic innervation of the neocortex. The function of the cells in the basal forebrain complex remains mostly unknown, but interest in this region has been fuelled by the discovery that these are among the first cells to die off during the course of Alzheimer’s disease, which is characterized by a progressive and profound loss of cognitive functions (Davies and Maloney, 1976). Furthermore, a functional disturbance of the cholinergic pathway between the frontal cortex and the basal nucleus of Meynert has been reported to occur after middle cerebral artery occlusion (Kataoka et al., 1991). Bladder overactivity due to neural circuitry damage in
* Corresponding author. Tel.: +81-76-265-2393; fax: +81-76-2226726. E-mail address: [email protected] (O. Yokoyama).
the forebrain such as seen in brain ischemia and Alzheimer’s disease causes a sense of urgency with or without urge incontinence (Resnick and Baumann, 1988; Griffiths et al., 1994). This overactivity may be attributed to cortical cholinergic loss. It has been also suggested that damage of the inhibitory influence of the cortical cholinergic system enhances the micturition reflex in a rat model with bladder overactivity following middle cerebral artery occlusion (Nakada et al., 2000). The second diffuse cholinergic system (the cholinergic complex) consist of acetylcholine (ACh)-utilizing cells in the pons and midbrain tegmentum (Butcher, 1995). The brainstem cholinergic system has been implicated in regulating micturition reflex. Injection of carbachol, a muscarinic cholinergic agonist, into the locus coeruleus alpha induced micturition reflex and suppressed urethral sphincter activity in the decerebrate cat, producing the same effect as electrical stimulation at the same site (Sugaya et al., 1987). These reports indicate that central cholinergic systems play an important role in the regulation of the micturition reflex. Antimuscarinic drugs are generally used for the treatment of urinary frequency and incontinence, but,
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their influence on the micturition pathways in the brain has not been clarified. In the study presented here, rats with ibotenic lesions of the nuclei basalis magnocellularis (NBM) in the basal forebrain were used to examine the contribution of central cholinergic systems to the micturition reflex and bladder overactivity caused by forebrain lesions.
2. Methods Eighty-four male Wistar rats, weighing 240–260 g (mean=253 g), were used in this study. They were housed at a constant temperature (23±2°C) and humidity (50–60%) under a regular 12 h light/dark schedule (light on 7:00 a.m.–7:00 p.m.). Tap water and standard rat chow were freely available. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. All efforts were made to minimise animal suffering, to reduce the number of animals used, and to utilise alternatives to in vivo techniques, if available. 2.1. Induction of ibotenic lesions in the basal forebrain
The bladder end of the catheter (size 4; inside diameter: 0.8 mm, outside diameter: 1.3 mm; Kunii Co, Ltd, Tokyo, Japan) was passed through a small incision at the apex of the bladder dome. The other end of the catheter was tunnelled subcutaneously and exited through the skin at the back of the animal. After the abdominal skin had been sutured, the rats were placed in a restraining cage and allowed to recover from anesthesia. The cystometry catheter was connected to a pump (TE-311; Terumo Co. Ltd, Tokyo, Japan) for the continuous infusion of saline and to a pressure transducer (TP-200T; Nihon-Kohden Co. Ltd, Tokyo, Japan) by means of a polyethylene T-tube. Two hours after the implantation of the catheter, control cystometric recordings were performed without anesthesia by infusing physiological saline at room temperature into the bladder at a rate of 0.04 ml/min. Saline voided from the urethral meatus was collected and measured to determine the voided volume. By evacuating the bladder through the CMG catheter, the residual volume could be measured after the micturition reflex. Three CMG parameters (bladder capacity, voided volume and bladder contraction pressure) were measured for each cystometry. Bladder capacity was defined as the sum of the voided and residual volumes. 2.3. Choline acetyltransferase activity determination
Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg) and operated on with the methods described by Dubois et al. (1985). The rats were positioned in a stereotaxic frame (ST-7; Narishige Co. Ltd, Tokyo, Japan), the incisor bar was set at ⫺3.3 mm (Paxinos and Watson, 1986), and a scalp incision was made over the sagital suture. The coordinates of the cannula placements were 2.4 mm laterally to the midline and 1.4 mm caudally to the bregma as specified by Paxinos and Watson (1986). A 0.3 mm stainless steel cannula was lowered 6.8 mm ventrally from the dura. Ibotenic acid (Sigma Chemical Co., St. Louis, USA) was infused bilaterally through the cannula connected via Teflon tubing to a Hamilton microsyringe. Ibotenic acid was dissolved in phosphate buffered saline (PBS; Sigma) at a concentration of 10 µg/µl, and infused in a volume of 0.75 µl (on each side) for 5 min (BF rats). Sham operated (SO) rats injected with PBS served as control. The infusion cannula was removed 7 min after the end of the injection. Postoperatively, the rats were allowed free access to food and water. 2.2. Cystometrography (CMG) in conscious rats Cystometrograms (CMG) were obtained 7–10 days after ibotenic acid/PBS injection. Implantation of the CMG catheter into the bladder was performed as previously described (Yokoyama et al., 1997). Briefly, rats were anesthetized with halothane (2%), and the bladder was exposed through a midline incision in the abdomen.
After CMG recording, ChAT activities in the frontal cortices were assayed to assess the damage to cholinergic neuronal projections from the basal forebrain to the frontal cortices. The rats were killed by decapitation, the brain quickly removed, and the cortical areas dissected. The samples were homogenized in 20 vol of 10 mM ethylene-diaminetetra-acetic acid (EDTA) buffer (pH 7.4) and 0.2% (v/v) TritonX-100. ChAT activity was calculated by measuring the conversion of 1·[14C] acetyl-coenzyme A to [14C] acetylcholine (Fonnum, 1975). Incubation time lasted 15 min at 37°C. ChAT activity was expressed as pmol/mg protein/min. 2.4. Intracerebroventricular administration of drugs The implantation of an injection tube into the right lateral ventricle was performed immediately after implantation of the CMG catheter and under continued halothane anesthesia. The rats were positioned in a stereotaxic frame, a scalp incision was made over the sagital suture, and a hole (diameter about 1.0 mm) was drilled in the right parietal bone to expose the dural surface 1.0 mm lateral and 0 mm anterior from the bregma (Paxinos and Watson, 1986). A sterile stainless steel canula (inside diameter: 0.3 mm, outside diameter: 0.6 mm, length 10.5 mm) was lowered 53 mm ventrally from the skull surface with the end of a micromanipulator. By using a small screw placed in the skull as an anchor, the canula was fixed to the skull with dental acrylic cement.
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Drug administration was intracerebroventricular (5 µl/rat) while the rats were conscious.
distilled water and diluted with aCSF to adjust the concentration.
2.5. Administration of drugs into the dorsolateral pontine tegmentum or 4th ventricle
2.8. Data analysis
Two h after implantation of the CMG catheter, rats were anesthetized with urethane (1 g/kg, i.p.) and positioned in a stereotaxic frame. A bilateral small craniotomy was performed to insert a stainless steel canula (inside diameter: 0.1 mm, outside diameter: 0.3 mm) into the dorsal pontine tegmentum (DPT). The coordinates of the canula placement were 1.3 mm laterally to the midline, 87 mm caudally to the bregma and 6.8 mm ventrally from the skull surface as specified by Paxinos and Watson (1986). Drugs were injected into the DPT bilaterally. Drugs were administered into the 4th ventricle at the midline, 8.3 mm caudally to the bregma and 5.6 mm ventrally from the skull surface, via the same size canula. 2.6. Evaluation of the effects of drugs After control cystometric recordings, the effects of increasing doses (0.0015–15 pmol/rat, i.c.v.) or a single dose (0.015, 1.5, and 15 pmol/rat, i.c.v.) of oxotremorine M (a nonselective muscarinic acetylcholine receptor (mAChR) agonist), or the vehicle (artificial cerebrospinal fluid (aCSF); 138.6 nM NaCl, 3.35 nM KCl, 1.26 nM CaCl2, 1.16 nM MgCl2, 11.9 nM NaHCO3, pH 7.07.2) on the micturition reflex were investigated in conscious SO or BF rats. Increasing doses of the drug were administered cumulatively at 60-min intervals. The effects of a single dose of pirenzepine (a selective M1 mAChR antagonist) were also examined in conscious SO rats (1, 10, and 100 nmol/rat, i.c.v.) or BF (0.1, 1, and 10 nmol/rat, i.c.v.). To examine the interactions between oxotremorine M and pirenzepine, the former (0.015 pmol/rat, i.c.v.) was administered 5 min after the administration of pirenzepine (1 nmol/rat, i.c.v.) in BF rats. Effects of 4th-ventricle or DPT administration of oxotremorine M or pirenzepine on the micturition reflex were investigated in SO and BF rats under urethane anesthesia. Oxotremorine M or pirenzepine was administered into the 4th ventricle (1 µl/rat) or bilaterally to the DPT (1 µl to each side).
Data are expressed as means±SEM. Statistical comparisons were performed by means of one-way or twoway repeated measures analysis of variance (ANOVA) with subsequent individual comparisons conducted with the aid of Fisher’s PLSD test. A level of P⬍0.05 was considered statistically significant.
3. Results 3.1. Cystometrographically observed influences of ibotenic lesions in the basal forebrain in conscious rats During the 7–10 day post-operative period, BF rats displayed aphagia and ataxia, and needed 5 days to completely recover their preoperative body weight. There was no difference in body weight between BF and SO rats when CMGs were obtained 7–10 days after ibotenic acid/PBS injection. Bilateral lesions of the nuclei basalis caused by ibotenic acid changed bladder capacity as monitored by CMG in conscious rats (Fig. 1). Bladder capacity in BF rats was 0.66±0.05 ml and significantly smaller than that in SO rats (1.51±0.11 ml)[P⬍0.01, Fig. 1(a)]. Residual volumes in BF and SO rats were very small and no significant difference between the groups, so that the micturition volume was nearly equivalent to bladder capacity [Fig. 1(b)]. Bladder contraction pressure in BF rats did not differ from that in SO rats [Fig. 1(c)]. 3.2. Choline acetyltransferase activity Regional brain ChAT activity was assayed at the end of the experiments. A significant decrease in ChAT activity in the frontal cortex was recognized in BF rats, in comparison to that in SO rats or control (untreated) rats (Fig. 2). ChAT activity in BF rats was approximately 74.2% of that in SO rats and 73.4% of that in control rats. 3.3. Effects of oxotremorine M and pirenzepine on micturition reflex in conscious rats
2.7. Drugs Drugs used in this study comprised oxotremorine M, N,N,N-Trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn1-ammonium iodide (Research Biochemical International, Natick, USA) and pirenzepine, 5,11-Dihydro11-[(4-methyl-1-piperazinyl)acetyl]-6H-pyrido[2,3-b][1,4] bonzodiazepin-6-one dihydrochloride (Research Biochemical International). These drugs were dissolved in
To examine the contribution of central muscarinic systems to the micturition reflex, oxotremorine M or pirenzepine was administered into the right lateral ventricle. Increasing doses of oxotremorine M (0.0015-15 pmol/rat, i.c.v.) produced a biphasic change in bladder capacity in BF rats (Fig. 3). Low doses of oxotremorine M (0.015, 0.15 pmol/rat) elicited significant increases in bladder capacity in BF rats compared with vehicle
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Fig. 1. Effects of basal forebrain lesions on (a) bladder capacity, (b) residual volume and (c) bladder contraction pressure. SO: sham operated rats, in which phosphate buffered saline was injected into the basal forebrain. BF: basal forebrain lesion rats, in which ibotenic acid was injected into the basal forebrain. Columns represent means±SEM; n=9 per group. **Indicate value of P⬍0.01 for comparison of bladder capacity between SO and BF rats.
administration (P⬍0.01, P⬍0.05, respectively). However, a larger dose (15 pmol/rat) significantly reduced bladder capacity (P⬍0.01). In SO rats low doses of oxotremorine M did not change bladder capacity, while a larger dose (15 pmol/rat) significantly reduced bladder capacity (P⬍0.01). The amplitude of bladder contraction was not altered in either SO or BF rats after administration of oxotremorine M (data not shown). A single dose of oxotremorine M (0.015, 1.5 pmol/rat, i.c.v.) did not change bladder capacity in SO rats compared with vehicle administration, while a larger dose (15 pmol/rat, i.c.v.) significantly reduced bladder capacity, which reached its minimum value (41.6%
reduction from preadministration capacity) within 15 min [P⬍0.01, Fig. 4(a)]. In contrast in BF rats, a single dose of oxotremorine M (0.015, 1.5, and 15 pmol/rat, i.c.v.) produced a significant increase in bladder capacity, which reached its maximum value (37.9% increase over preadministration capacity for 0.015 pmol/rat) within 15 min after administration [Fig. 4(b)]. No reduction in bladder capacity was recognized even at a dose of 15 pmol of oxotremorine M. None of the oxotremorine M doses produced a significant change in the amplitude of bladder contraction or residual volume in either SO or BF rats (data not shown). A single dose of pirenzepine (100 nmol/rat) elicited a
Fig. 2. Choline acetyltransferase (ChAT) activity in three groups, normal: untreated rats; SO: sham operated rats, in which phosphate buffered saline was injected into the basal forebrain; BF: basal forebrain lesion rats, in which ibotenic acid was injected into the basal forebrain. Columns represent means±SEM; n=9 per group. **Indicates value of P⬍0.01 for comparison of ChAT between normal and BF rats, and SO and BF rats.
Fig. 3. Log dose–response curves showing the effects expressed as percentage changes of increasing doses of oxotremorine M on bladder capacity in sham operated (SO) rats (vehicle, 䊊 with dotted line; oxotremorine M, 䊊 with solid line) and basal forebrain lesion (BF) rats (vehicle, 쐌 with dotted line; oxotremorine M, 쐌 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. BF rats (vehicle); ##P⬍0.01 vs, SO rats (vehicle).
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5(d)] and increased residual volume [Fig. 5(f)]. Lower doses of pirenzepine (0.1, 1 nmol/rat) did not alter bladder capacity, amplitude of bladder contraction, or residual volume. 3.4. Antagonistic effects of pirenzepine on oxotremorine M-induced increase in bladder capacity in conscious rats To study the interactions between oxotremorine M and pirenzepine, we administered a fixed dose of pirenzepine (1 nmol/rat) to BF rat. This dose was selected because it did not cause any significant changes in CMG parameters (Fig. 5). For experiments designed to identify the pharmacological interaction between oxotremorine M and pirenzepine, a single dose of oxotremorine M (0.015 pmol/rat, i.c.v.) was injected 5 min after i.c.v, administration of pirenzepine (1 nmol/rat) (Fig. 6). Intracerebroventricular administration of oxotremorine M after that of aCSF (vehicle–oxotremorine M group) resulted in a statistically significant increase in bladder capacity (P⬍0.01) when compared with aCSF administration (vehicle–vehicle group). In contrast, administration of pirenzepine completely nullified the oxotremorine M-induced increase in bladder capacity (pirenzepine–oxotremorine M group). There was no significant difference in percentage changes in bladder capacity between the pirenzepine–oxotremorine M and pirenzepine–vehicle groups.
Fig. 4. Time course of changes in bladder capacity after intracerebroventricular administration of oxotremorine M in (a) sham operated rats and (b) basal forebrain (BF) rats. Three doses of oxotremorine M were administered to SO rats (vehicle, 䊊 with dotted line; 0.015 pmol, 䊊 with solid line; 1.5 pmol, 왕 with solid line; 15 pmol, 䊐 with solid line) and BF rats (vehicle, 쐌 with dotted line; 0.015 pmol, 쐌 with solid line; 1.5 pmol, 왖 with solid line; 15 pmol, 䊏 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. SO rats (vehicle) or BF rats (vehicle).
small but significant increase in bladder capacity in SO rats compared with vehicle administration [43.7% increase over preadministration capacity, Fig. 5(a)]. This dose of pirenzepine also reduced the amplitude of bladder contraction [Fig. 5(c)] and increased residual volume [Fig. 5(e)]. Lower doses of pirenzepine (1, 10 nmol/rat) did not alter bladder capacity, amplitude of bladder contraction, or residual volume, In BF rats 10 nmol of pirenzepine, 10 times less than that used for SO rats, produced a significant increase in bladder capacity compared with vehicle administration [189.4% increase over preadministration capacity, Fig. 5(b)]. This dose produced a reduction in the amplitude of bladder contraction [Fig.
3.5. Effects of 4th ventricle or DPT administration of oxotremorine M or pirenzepine in anesthetized rats To examine the contribution of muscarinic systems in the pons to the micturition reflex, oxotremorine M or pirenzepine was administered into the 4th ventricle or DPT. Oxotremorine M (15 pmol), when injected into the 4th ventricle in urethane anesthetized rats, produced a transient enhancement of the micturition reflex in SO and BF rats within 1 minute after the administration [Fig. 7(b)]. This enhancing effect, which lasted a few min, was followed by inhibition of the micturition reflex in BF rats. No increase in residual volume was seen. Administration of the vehicle (aCSF) into the 4th ventricle had no effect on the micturition reflex. Bilateral administration of oxotremorine M (1.5 pmol) into the DPT, enhanced the micturition reflex without causing any change in residual volume [Fig. 7(a)]. Pirenzepine (100 nmol, injected into the 4th ventricle), produced an inhibitory effect on the micturition reflex accompanied by an increase in residual volume [Fig. 8(b)], but when it was administered (10 nmol) into the DPT, it did not change the micturition reflex [Fig. 8(a)].
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Fig. 5. Time course of changes in (a and b) bladder capacity, (c and d) bladder contraction pressure, and (e and f) residual volume after intracerebroventricular administration of pirenzepine in (a, c, and e) sham operated (SO) rats and (b, d, and f) basal forebrain (BF) rats. Three doses of pirenzepine were administered to SO rats (vehicle, 䊊 with dotted line; 1 nmol, 䊊 with solid line; 10 nmol, 왕 with solid line; 100 nmol, 䊐 with solid line) and BF rats (vehicle, 쐌 with dotted line; 0.1 nmol, 䉬 with solid line; 1 nmol, 쐌 with solid line; 10 nmol, 왖 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. SO rats (vehicle) or BF rats (vehicle).
4. Discussion Anatomical and electrophysiological studies have revealed that activation of bladder parasympathetic nerves during micturition is mediated by a spinobulbospinal pathway passing through a micturition center (i.e., the potine micturition center, PMC) in the DPT (Morrison, 1987). The on–off switch of the micturition reflex is believed to be located in the PMC, which receives inhibitory or excitatory input from the forebrain (de Groat et al., 1993). Anatomical tracing studies in
animals using the transneuronal transport of a pseudorabies virus have revealed multiple sites in the cerebral cortex, diencephalon and the brainstem that are likely to be the source of the modulatory mechanisms in micturition reflex (Nadelhaft et al., 1992; Vizzard et al., 1995). Lesions of the cerebral cortex resulting from tumors, aneurysms, or vascular disease appear to enhance the micturition reflex at least in part by removing the cortical inhibitory control of the micturition center in the brain stem. Various neurotransmitters have been implicated in the supraspinal control of micturition in animals (de
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Fig. 6. Time course of changes in bladder capacity after intracerebroventricular administration of oxotremorine M (0.015 pmol/rat) in basal forebrain lesion (BF) rats pretreated with pirenzepine (1 nmol/ rat, i.c.v.). Data were compared among 4 groups (vehicle–vehicle, 쐌 with dotted line; pirenzepine–vehicle, 쐌 with solid line; vehicle– oxotremorine M, 왖 with solid line; pirenzepine–oxotremorine M, 䊏 with solid line). Values represent means±SEM; n=7 per group. **P⬍0.01, *P⬍0.05 vs. BF rats (pirenzepine–oxotremorine M, 䊏 with solid line).
Groat et al., 1993), but no reports have dealt with cortical diencephalic neurotransmitter systems exerting an inhibitory control on the PMC except for this and one other study (Yoshimura et al., 1993). Dopaminergic pathways from the substantia nigra to the basal ganglia have an inhibitory effect on the micturition reflex via activation of D1 receptors. The inhibitory action of dopamine is indicated by the changes in voiding function in patients with Parkinson’s disease and in a monkey
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with Parkinsonism induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine. The present pharmacological studies have implicated central muscarinic mechanisms as playing important roles in both the inhibitory and excitatory control of the micturition reflex. Ibotenic lesions in the basal forebrain, inducing severe neuronal damage to the nucleus basalis magnocellularis, were found to reduce bladder capacity and ChAT activity in the frontal cortices. These results indicate that the cholinergic pathway between the frontal cortex and the basal nucleus contributes to the inhibitory control of the micturition reflex. Impairment of this regulatory system is likely to cause neurogenic bladder dysfunction in patients with Alzheimer type senile dementia (Resnick and Baumann, 1988). No increase in residual volume or reduction in bladder contraction pressure was observed in our rat model, which means that the forebrain cholinergic pathway appears to raise the bladder threshold of the micturition switching circuit. Furthermore, impairment of this regulatory pathway is largely responsible for the development of the overactive bladder induced by cerebral infarction (Nakada et al., 2000). The quantitative analysis of mAChR and subtype mRNA in the acute phase of a middle cerebral artery occlusion model demonstrated a tendency toward a reduction in mRNA levels in the ischemic cortex (Kuji et al., 1997). Even a transient cerebral ischemia causes a reduction in ChAT activity, ACh contents, and release in chronic phase (Iwasaki et al., 1996; Kakihara et al., 1984). Intracerebroventricular administration of oxotremorine M, a nonselective mAChR agonist, produced primarily an inhibitory effect on the micturition reflex in BF rats, although some enhancement effects were also noted. Oxotremorine M did not necessarily make up for the total reduction in bladder capacity resulting from BF lesions, which indicates that dysfunction of the central
Fig. 7. Effects of administration of oxotremorine M into (a) the dorsal pontine tegmentum (DPT, 1.5 pmol/rat) or (b) the 4th ventricle (15 pmol/rat) on cystometrogram and electromyographic (EMG) finding of the external urethral sphincter in urethane anesthetized rats. ECP, bladder contraction pressure; RV, residual volume.
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Fig. 8. Effects of administration of pirenzepine into (a) the dorsal pontine tegmentum (DPT, 10 nmol/rat) or (b) the 4th ventricle (100 nmol/rat) on cystometrogram and electromyographic (EMG) finding of the external urethral sphincter in urethane anesthetized rats. BCP, bladder contraction pressure; RV, residual volume.
cholinergic system can not be counteracted only by supplementation with ACh. Other neurotransmitter systems relaying ACh neurons may also undergo changes in BF rats. In previous reports we suggested that NMDA (N-methyl-d-aspartate) glutamatergic or D1 dopaminergic mechanisms, which originate in the forebrain, contributed to inhibitory control of the micturition reflex (Yokoyama et al. 1999, 2000). Moreover, a recent study has shown that muscarinic activation inhibits the release of glutamate in the corticostriatal pathway (Niittykoski et al., 1999). Cholinergic neurons may thus influence the glutamatergic pathways that regulate the micturition reflex in the brain. Selective M1 mAChR antagonist pirenzepine, when administered at one-tenth of the concentration which had effect on the micturition reflex, counteracted the oxotremorine M-induced increase in bladder capacity in BF rats. This indicates that the M1 muscarinic receptors in the cerebral cortex have an inhibitory effect on the micturition reflex pathway. An autoradiographic study has provided the evidence of M1 mAChR over many forebrain structures including the cerebral cortex, while a very small fraction of the total number of receptors sites in the thalamus and brainstem (Mash and Potter, 1986). No critical studies have been published which focus on the functional role of the forebrain M1 muscarinic system in the micturition reflex, but it is impaired in BF models, thus resulting in enhancement of the micturition reflex. The excitatory effect of a large dose of oxotremorine M on the micturition reflex requires an explanation. A single dose (15 pmol/rat) of intracerebroventricular oxotremorine M reduced bladder capacity in SO rats, and cumulative doses (up to 15 pmol/rat) had the same effect in both SO and BF rats. Decerebration experiments have revealed that activation of cholinergic recep-
tors in the pontine-mesencephalic brain region with mAChR agonist oxotremorine induces bladder hyperactivity (Sille´ n et al., 1982), while focally injected carbachol into the DPT, which activates cholinoceptive neuronal cell, elicited the micturition reflex in an acute decerebrate cat (Sugaya et al., 1987). Activation of cholinoceptive neurons in the DPT presumably becomes a trigger for the recruitment of any one of a number of neuronal circuits involved in micturition. Our study demonstrated that chemical stimulation with oxotremorine M injected into the DPT facilitated the micturition reflex. Since M1 mAChR does not exist in the pons (Mash and Potter, 1986) and pirenzepine injected into the DPT had no effect on the micturition reflex, M2 mAChR might mediate this response. Injection into the 4th ventricle close to the DPT elicited primarily an excitatory response, which was always followed by an inhibitory response. These results suggest that muscarinic mechanisms in the DPT contribute to enhancement of the micturition reflex, and that the prolonged inhibitory effect of oxotremorine M injected into the 4th ventricle seems to be caused by its inhibitory influence on the PMC via M1 muscarinic receptors in the cerebral cortex. Therefore, the excitatory effect of a large dose of oxotremorine M injected into the lateral ventricle is likely to be the results of its effect on cholinoceptive neurons in the DPT. In BF rats, however, the difference in response between the cumulatively applied oxotremorine M and single dose of oxotremorine M is unexplainable. Another question that needs answering is what the mechanism underlies the action of selective M1 mAChR antagonist pirenzepine? In our study pirenzepine had an inhibitory effect on the micturition reflex when injected into the lateral ventricle, but none when injected into the DPT. As mentioned earlier M1 mAChR does not exist in the pons, so that the inhibitory influence of pirenzep-
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ine might be due to its effect on the forebrain via M1 mAChR. The release of GABA from the cerebral cortical GABA neurons is reportedly modulated by presynaptic M1 mAChR (Hashimoto et al., 1994). Suppression of GABA release by carbachol can be counteracted by pirenzepine in the cerebral cortex, meaning that pirenzepine induces GABA release through M1 mAChR. Intracerebroventricular administration of GABAergic agents (muscimol and baclofen) produces dose-dependent inhibition of the micturition reflex at the supraspinal level (Kanie et al., 2000). Therefore, pirenzepine possibly suppresses the micturition reflex by means of activating GABAergic mechanisms in the cerebral cortex. The effects of pirenzepine on bladder capacity, bladder contraction pressure and residual volume were clearly different in SO and BF rats with BF rats being more sensitive and responding to one-tenth of the dose which had an effect on SO rats. This difference in sensitivity between SO and BF rats may reflect changes in GABAergic mechanisms in the cerebral cortex following BF lesions. Another possibility is that pirenzepine can act as a M2 mAChR antagonist at high doses. Pirenzepine preferentially binds to M1 mAChR, and also exhibit low affinity to M2 and M3 mAChR (Wess et al., 1991). An inhibitory effect of pirenzepine on the micturition reflex when applied to the lateral ventricle might be due to its affinity for M2 mAChR in the pons. Using an M2 mAChR antagonist, this problem will be solved in more detail in future experiments. In conclusion, M1 mAChR appears to be involved in forebrain inhibitory mechanisms in the micturition reflex. Our findings suggest that injury to the basal forebrain causes a change in M1 muscarinic inhibitory sytems, which results in enhancement of the micturition reflex. Therefore, M1 mAChR agonists can be expected to be beneficial for the treatment of the overactive bladder caused by Alzheimer type dementia.
References Butcher, L.L., 1995. Cholinergic neurons and networks. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, Sydney, pp. 1003–1015. Davies, P., Maloney, A.J.F., 1976. Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet ii, 1403. de Groat, W.C., Booth, A.M., Yoshimura, N., 1993. Neurophysiology of micturition and its modification in animal models of human disease. In: Maggi, C.A. (Ed.), The Autonomic Nervous System. Nervous Control of the Urogenital System. Harwood Academic, London, pp. 227–290. Dubois, B., Mayo, W., Agid, Y., Le Moal, M., Simon, H., 1985. Profound disturbances of spontaneous and learned behaviors in the rat. Brain Research 338, 249–258. Fonnum, F., 1975. A rapid radiochemical method for the determination of choline acetyltransferase. Journal of Neurochemistry 24, 407– 409. Griffiths, D.J., McCracken, P.N., Harrison, G.M., Gormley, E.A.,
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Moore, K., Hooper, R. et al., 1994. Cerebral aetiology of urinary urge incontinence in elderly people. Age Ageing 23, 246–250. Hashimoto, T., Shu, H., Kuriyama, K., 1994. Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex. Neurochemistry International 24, 389–394. Iwasaki, K., Kitamura, Y., Ohgami, Y., Mishima, K., Fujiwara, M., 1996. The disruption of spatial cognition and changes in brain amino acid, monoamine and acetylcholine in rats with transient cerebral ischemia. Brain Research 709, 163–172. Kakihara, M., Yamazaki, N., Nagaoka, A., 1984. Effects of idebenons (cv-2619) on the concentrations of acetylcholine and choline in various brain regions of rats with cerebral ischemia. Japanese Journal of Pharmacology 36, 357–363. Kanie, S., Yokoyama, O., Komatsu, K., Kodama, K., Yotsuyanagi, S., Niikura, S., Nagasaka, Y., Miyamoto, K., Namiki, M., 2000. GABAergic contribution to rat bladder hyperactivity after middle cerebral artery occlusion. American Journal of Physiology 279, R1230–R1238. Kataoka, K., Hayakawa, T., Kuroda, R., Yuguchi, T., Yamada, K., 1991. Cholinergic differentiation after focal cerebral infarct in rats. Stroke 22, 1291–1296. Kuji, I., Matsuda, H., Sumiya, H., Taki, J., Tsuji, S., Kinuya, K. et al., 1997. Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion. European Journal of Nuclear Medicine 24, 665–669. Mash, D.C., Potter, L.T., 1986. Autoradiographic localization of M1 and M2 muscarine receptors in the rat brain. Neuroscience 19, 551–564. Morrison, J.F.B., 1987. Bladder control: role of higher levels of the central nervous system. In: Torrens, M., Morrison, J.F.B. (Eds.), The Physiology of the Lower Urinary Tract. Springer-Verlag, Berlin, pp. 237–276. Nadelhaft, I., Vera, P.L., Card, J.P., Miselis, R.R., 1992. Central nervous system neurons labelled following the injection of pseudorabies virus into the rat urinary bladder. Neuoscience Letters 143, 271–274. Nakada, Y., Yokoyama, O., Komatsu, K., Kodama, K., Yotsuyanagi, S., Niikura, S. et al., 2000. Effects of aniracetam on bladder overactivity in rats with cerebral infarction. The Journal of Pharmacology and Experimental Therapeutics 293, 921–928. Niittykoski, M., Ruotsalainen, S., Haapalinna, A., Larson, J., Sirvio¨ , J., 1999. Activation of muscarinic M3-like receptors and b-adrenoceptors, but not M2-like muscarinic receptors or a-adrenoceptors, directly modulates corticostriatal neurotransmission in vitro. Neuroscience 90, 95–105. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, London. Resnick, N.M., Baumann, M.M., 1988. Incontinence in the nursing home patients. Clinical Geriatric Medicine 4, 549–570. Sille´ n, U., Rubenson, A., Hja¨ lma˚ s, K., 1982. Central cholinergic mechanisms in 1-dopa induced hyperactive urinary bladder of the rat. Urological Research 10, 239–243. Sugaya, K., Matsuyama, K., Takakusaki, K., Mori, S., 1987. Electrical and chemical stimulation of the pontine micturition center. Neuroscience Letters 80, 197–201. Yokoyama, O., Yoshiyama, M., Namiki, M., de Groat, W.C., 1997. The influence of anesthesia on bladder hyperactivity induced by middle cerebral artery occlusion in the rat. American Journal of Physiology 273, R1900–R1907. Yokoyama, O., Yoshiyama, M., Namiki, M., de Groat, W.C., 1999. Glutamatergic and dopaminergic contributions to rat bladder hyperactivity following cerebral artery occlusion. American Journal of Physiology 276, R935–R942. Yokoyama, O., Yoshiyama, M., Namiki, M., de Groat, W.C., 2000. Role of the forebrain in bladder overactivity following cerebral infarction in the rat. Experimental Neurology 163, 469–476. Yoshimura, N., Mizuta, E., Kuno, S., Sasa, M., Yoshida, O., 1993.
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Forebrain muscarinic control of micturition reflex in rats O. Yokoyama *, N. Ootsuka, K. Komatsu, K. Kodama, S. Yotsuyanagi, S. Niikura, Y. Nagasaka, Y. Nakada, S. Kanie, M. Namiki Department of Urology, Kanazawa University School of Medicine, Kanazawa, 920-8641, Ishikawa, Japan Received 12 February 2001; received in revised form 2 July 2001; accepted 5 July 2001
Abstract Functional contribution of the cholinergic pathway between the frontal cortex and basal nucleus of Meynert to micturition reflex was investigated. Male Wistar rats were subjected to bilateral lesion of the basal forebrain by ibotenic acid (IA) injection (7.5 µg/rat on each side) (BF rats). Phosphate buffered saline (PBS) was injected into control rats (sham operated rats; SO rats). Cystometrograms were obtained from conscious BF and SO rats 7–10 days after IA/PBS injection. Bladder capacity (BC) of BF rats was significantly smaller than that of SO rats (approximately 43.7%) and was accompanied by decrease in choline-acetyltransferase activity in the frontal cortices. Oxotremorine M, a muscarinic receptor agonist, increased BC in BF rats, while pirenzepine, an M1 muscarinic receptor antagonist, counteracted the effect of the oxotremorine M-induced increase in BC. Injection of oxotremorine M into the dorsal pontine tegmentum (DPT) reduced BC in BF and SO rats, while injection of pirenzepine had no effect on cystometrograms. These findings indicate that the M1 muscarinic receptor plays a part in the forebrain inhibitory mechanisms involved in the micturition reflex and that muscarinic receptor in the DPT contributes to excitatory control of micturition reflex. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pons; Forebrain; Bladder overactivity; Urination disorders; Acetylcholine; Rat
1. Introduction There are two major diffuse modulatory cholinergic systems in the brain, one of which is called the basal forebrain complex (Butcher, 1995). The basal nucleus of Meynert provides most of the cholinergic innervation of the neocortex. The function of the cells in the basal forebrain complex remains mostly unknown, but interest in this region has been fuelled by the discovery that these are among the first cells to die off during the course of Alzheimer’s disease, which is characterized by a progressive and profound loss of cognitive functions (Davies and Maloney, 1976). Furthermore, a functional disturbance of the cholinergic pathway between the frontal cortex and the basal nucleus of Meynert has been reported to occur after middle cerebral artery occlusion (Kataoka et al., 1991). Bladder overactivity due to neural circuitry damage in
* Corresponding author. Tel.: +81-76-265-2393; fax: +81-76-2226726. E-mail address: [email protected] (O. Yokoyama).
the forebrain such as seen in brain ischemia and Alzheimer’s disease causes a sense of urgency with or without urge incontinence (Resnick and Baumann, 1988; Griffiths et al., 1994). This overactivity may be attributed to cortical cholinergic loss. It has been also suggested that damage of the inhibitory influence of the cortical cholinergic system enhances the micturition reflex in a rat model with bladder overactivity following middle cerebral artery occlusion (Nakada et al., 2000). The second diffuse cholinergic system (the cholinergic complex) consist of acetylcholine (ACh)-utilizing cells in the pons and midbrain tegmentum (Butcher, 1995). The brainstem cholinergic system has been implicated in regulating micturition reflex. Injection of carbachol, a muscarinic cholinergic agonist, into the locus coeruleus alpha induced micturition reflex and suppressed urethral sphincter activity in the decerebrate cat, producing the same effect as electrical stimulation at the same site (Sugaya et al., 1987). These reports indicate that central cholinergic systems play an important role in the regulation of the micturition reflex. Antimuscarinic drugs are generally used for the treatment of urinary frequency and incontinence, but,
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their influence on the micturition pathways in the brain has not been clarified. In the study presented here, rats with ibotenic lesions of the nuclei basalis magnocellularis (NBM) in the basal forebrain were used to examine the contribution of central cholinergic systems to the micturition reflex and bladder overactivity caused by forebrain lesions.
2. Methods Eighty-four male Wistar rats, weighing 240–260 g (mean=253 g), were used in this study. They were housed at a constant temperature (23±2°C) and humidity (50–60%) under a regular 12 h light/dark schedule (light on 7:00 a.m.–7:00 p.m.). Tap water and standard rat chow were freely available. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. All efforts were made to minimise animal suffering, to reduce the number of animals used, and to utilise alternatives to in vivo techniques, if available. 2.1. Induction of ibotenic lesions in the basal forebrain
The bladder end of the catheter (size 4; inside diameter: 0.8 mm, outside diameter: 1.3 mm; Kunii Co, Ltd, Tokyo, Japan) was passed through a small incision at the apex of the bladder dome. The other end of the catheter was tunnelled subcutaneously and exited through the skin at the back of the animal. After the abdominal skin had been sutured, the rats were placed in a restraining cage and allowed to recover from anesthesia. The cystometry catheter was connected to a pump (TE-311; Terumo Co. Ltd, Tokyo, Japan) for the continuous infusion of saline and to a pressure transducer (TP-200T; Nihon-Kohden Co. Ltd, Tokyo, Japan) by means of a polyethylene T-tube. Two hours after the implantation of the catheter, control cystometric recordings were performed without anesthesia by infusing physiological saline at room temperature into the bladder at a rate of 0.04 ml/min. Saline voided from the urethral meatus was collected and measured to determine the voided volume. By evacuating the bladder through the CMG catheter, the residual volume could be measured after the micturition reflex. Three CMG parameters (bladder capacity, voided volume and bladder contraction pressure) were measured for each cystometry. Bladder capacity was defined as the sum of the voided and residual volumes. 2.3. Choline acetyltransferase activity determination
Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg) and operated on with the methods described by Dubois et al. (1985). The rats were positioned in a stereotaxic frame (ST-7; Narishige Co. Ltd, Tokyo, Japan), the incisor bar was set at ⫺3.3 mm (Paxinos and Watson, 1986), and a scalp incision was made over the sagital suture. The coordinates of the cannula placements were 2.4 mm laterally to the midline and 1.4 mm caudally to the bregma as specified by Paxinos and Watson (1986). A 0.3 mm stainless steel cannula was lowered 6.8 mm ventrally from the dura. Ibotenic acid (Sigma Chemical Co., St. Louis, USA) was infused bilaterally through the cannula connected via Teflon tubing to a Hamilton microsyringe. Ibotenic acid was dissolved in phosphate buffered saline (PBS; Sigma) at a concentration of 10 µg/µl, and infused in a volume of 0.75 µl (on each side) for 5 min (BF rats). Sham operated (SO) rats injected with PBS served as control. The infusion cannula was removed 7 min after the end of the injection. Postoperatively, the rats were allowed free access to food and water. 2.2. Cystometrography (CMG) in conscious rats Cystometrograms (CMG) were obtained 7–10 days after ibotenic acid/PBS injection. Implantation of the CMG catheter into the bladder was performed as previously described (Yokoyama et al., 1997). Briefly, rats were anesthetized with halothane (2%), and the bladder was exposed through a midline incision in the abdomen.
After CMG recording, ChAT activities in the frontal cortices were assayed to assess the damage to cholinergic neuronal projections from the basal forebrain to the frontal cortices. The rats were killed by decapitation, the brain quickly removed, and the cortical areas dissected. The samples were homogenized in 20 vol of 10 mM ethylene-diaminetetra-acetic acid (EDTA) buffer (pH 7.4) and 0.2% (v/v) TritonX-100. ChAT activity was calculated by measuring the conversion of 1·[14C] acetyl-coenzyme A to [14C] acetylcholine (Fonnum, 1975). Incubation time lasted 15 min at 37°C. ChAT activity was expressed as pmol/mg protein/min. 2.4. Intracerebroventricular administration of drugs The implantation of an injection tube into the right lateral ventricle was performed immediately after implantation of the CMG catheter and under continued halothane anesthesia. The rats were positioned in a stereotaxic frame, a scalp incision was made over the sagital suture, and a hole (diameter about 1.0 mm) was drilled in the right parietal bone to expose the dural surface 1.0 mm lateral and 0 mm anterior from the bregma (Paxinos and Watson, 1986). A sterile stainless steel canula (inside diameter: 0.3 mm, outside diameter: 0.6 mm, length 10.5 mm) was lowered 53 mm ventrally from the skull surface with the end of a micromanipulator. By using a small screw placed in the skull as an anchor, the canula was fixed to the skull with dental acrylic cement.
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Drug administration was intracerebroventricular (5 µl/rat) while the rats were conscious.
distilled water and diluted with aCSF to adjust the concentration.
2.5. Administration of drugs into the dorsolateral pontine tegmentum or 4th ventricle
2.8. Data analysis
Two h after implantation of the CMG catheter, rats were anesthetized with urethane (1 g/kg, i.p.) and positioned in a stereotaxic frame. A bilateral small craniotomy was performed to insert a stainless steel canula (inside diameter: 0.1 mm, outside diameter: 0.3 mm) into the dorsal pontine tegmentum (DPT). The coordinates of the canula placement were 1.3 mm laterally to the midline, 87 mm caudally to the bregma and 6.8 mm ventrally from the skull surface as specified by Paxinos and Watson (1986). Drugs were injected into the DPT bilaterally. Drugs were administered into the 4th ventricle at the midline, 8.3 mm caudally to the bregma and 5.6 mm ventrally from the skull surface, via the same size canula. 2.6. Evaluation of the effects of drugs After control cystometric recordings, the effects of increasing doses (0.0015–15 pmol/rat, i.c.v.) or a single dose (0.015, 1.5, and 15 pmol/rat, i.c.v.) of oxotremorine M (a nonselective muscarinic acetylcholine receptor (mAChR) agonist), or the vehicle (artificial cerebrospinal fluid (aCSF); 138.6 nM NaCl, 3.35 nM KCl, 1.26 nM CaCl2, 1.16 nM MgCl2, 11.9 nM NaHCO3, pH 7.07.2) on the micturition reflex were investigated in conscious SO or BF rats. Increasing doses of the drug were administered cumulatively at 60-min intervals. The effects of a single dose of pirenzepine (a selective M1 mAChR antagonist) were also examined in conscious SO rats (1, 10, and 100 nmol/rat, i.c.v.) or BF (0.1, 1, and 10 nmol/rat, i.c.v.). To examine the interactions between oxotremorine M and pirenzepine, the former (0.015 pmol/rat, i.c.v.) was administered 5 min after the administration of pirenzepine (1 nmol/rat, i.c.v.) in BF rats. Effects of 4th-ventricle or DPT administration of oxotremorine M or pirenzepine on the micturition reflex were investigated in SO and BF rats under urethane anesthesia. Oxotremorine M or pirenzepine was administered into the 4th ventricle (1 µl/rat) or bilaterally to the DPT (1 µl to each side).
Data are expressed as means±SEM. Statistical comparisons were performed by means of one-way or twoway repeated measures analysis of variance (ANOVA) with subsequent individual comparisons conducted with the aid of Fisher’s PLSD test. A level of P⬍0.05 was considered statistically significant.
3. Results 3.1. Cystometrographically observed influences of ibotenic lesions in the basal forebrain in conscious rats During the 7–10 day post-operative period, BF rats displayed aphagia and ataxia, and needed 5 days to completely recover their preoperative body weight. There was no difference in body weight between BF and SO rats when CMGs were obtained 7–10 days after ibotenic acid/PBS injection. Bilateral lesions of the nuclei basalis caused by ibotenic acid changed bladder capacity as monitored by CMG in conscious rats (Fig. 1). Bladder capacity in BF rats was 0.66±0.05 ml and significantly smaller than that in SO rats (1.51±0.11 ml)[P⬍0.01, Fig. 1(a)]. Residual volumes in BF and SO rats were very small and no significant difference between the groups, so that the micturition volume was nearly equivalent to bladder capacity [Fig. 1(b)]. Bladder contraction pressure in BF rats did not differ from that in SO rats [Fig. 1(c)]. 3.2. Choline acetyltransferase activity Regional brain ChAT activity was assayed at the end of the experiments. A significant decrease in ChAT activity in the frontal cortex was recognized in BF rats, in comparison to that in SO rats or control (untreated) rats (Fig. 2). ChAT activity in BF rats was approximately 74.2% of that in SO rats and 73.4% of that in control rats. 3.3. Effects of oxotremorine M and pirenzepine on micturition reflex in conscious rats
2.7. Drugs Drugs used in this study comprised oxotremorine M, N,N,N-Trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn1-ammonium iodide (Research Biochemical International, Natick, USA) and pirenzepine, 5,11-Dihydro11-[(4-methyl-1-piperazinyl)acetyl]-6H-pyrido[2,3-b][1,4] bonzodiazepin-6-one dihydrochloride (Research Biochemical International). These drugs were dissolved in
To examine the contribution of central muscarinic systems to the micturition reflex, oxotremorine M or pirenzepine was administered into the right lateral ventricle. Increasing doses of oxotremorine M (0.0015-15 pmol/rat, i.c.v.) produced a biphasic change in bladder capacity in BF rats (Fig. 3). Low doses of oxotremorine M (0.015, 0.15 pmol/rat) elicited significant increases in bladder capacity in BF rats compared with vehicle
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Fig. 1. Effects of basal forebrain lesions on (a) bladder capacity, (b) residual volume and (c) bladder contraction pressure. SO: sham operated rats, in which phosphate buffered saline was injected into the basal forebrain. BF: basal forebrain lesion rats, in which ibotenic acid was injected into the basal forebrain. Columns represent means±SEM; n=9 per group. **Indicate value of P⬍0.01 for comparison of bladder capacity between SO and BF rats.
administration (P⬍0.01, P⬍0.05, respectively). However, a larger dose (15 pmol/rat) significantly reduced bladder capacity (P⬍0.01). In SO rats low doses of oxotremorine M did not change bladder capacity, while a larger dose (15 pmol/rat) significantly reduced bladder capacity (P⬍0.01). The amplitude of bladder contraction was not altered in either SO or BF rats after administration of oxotremorine M (data not shown). A single dose of oxotremorine M (0.015, 1.5 pmol/rat, i.c.v.) did not change bladder capacity in SO rats compared with vehicle administration, while a larger dose (15 pmol/rat, i.c.v.) significantly reduced bladder capacity, which reached its minimum value (41.6%
reduction from preadministration capacity) within 15 min [P⬍0.01, Fig. 4(a)]. In contrast in BF rats, a single dose of oxotremorine M (0.015, 1.5, and 15 pmol/rat, i.c.v.) produced a significant increase in bladder capacity, which reached its maximum value (37.9% increase over preadministration capacity for 0.015 pmol/rat) within 15 min after administration [Fig. 4(b)]. No reduction in bladder capacity was recognized even at a dose of 15 pmol of oxotremorine M. None of the oxotremorine M doses produced a significant change in the amplitude of bladder contraction or residual volume in either SO or BF rats (data not shown). A single dose of pirenzepine (100 nmol/rat) elicited a
Fig. 2. Choline acetyltransferase (ChAT) activity in three groups, normal: untreated rats; SO: sham operated rats, in which phosphate buffered saline was injected into the basal forebrain; BF: basal forebrain lesion rats, in which ibotenic acid was injected into the basal forebrain. Columns represent means±SEM; n=9 per group. **Indicates value of P⬍0.01 for comparison of ChAT between normal and BF rats, and SO and BF rats.
Fig. 3. Log dose–response curves showing the effects expressed as percentage changes of increasing doses of oxotremorine M on bladder capacity in sham operated (SO) rats (vehicle, 䊊 with dotted line; oxotremorine M, 䊊 with solid line) and basal forebrain lesion (BF) rats (vehicle, 쐌 with dotted line; oxotremorine M, 쐌 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. BF rats (vehicle); ##P⬍0.01 vs, SO rats (vehicle).
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5(d)] and increased residual volume [Fig. 5(f)]. Lower doses of pirenzepine (0.1, 1 nmol/rat) did not alter bladder capacity, amplitude of bladder contraction, or residual volume. 3.4. Antagonistic effects of pirenzepine on oxotremorine M-induced increase in bladder capacity in conscious rats To study the interactions between oxotremorine M and pirenzepine, we administered a fixed dose of pirenzepine (1 nmol/rat) to BF rat. This dose was selected because it did not cause any significant changes in CMG parameters (Fig. 5). For experiments designed to identify the pharmacological interaction between oxotremorine M and pirenzepine, a single dose of oxotremorine M (0.015 pmol/rat, i.c.v.) was injected 5 min after i.c.v, administration of pirenzepine (1 nmol/rat) (Fig. 6). Intracerebroventricular administration of oxotremorine M after that of aCSF (vehicle–oxotremorine M group) resulted in a statistically significant increase in bladder capacity (P⬍0.01) when compared with aCSF administration (vehicle–vehicle group). In contrast, administration of pirenzepine completely nullified the oxotremorine M-induced increase in bladder capacity (pirenzepine–oxotremorine M group). There was no significant difference in percentage changes in bladder capacity between the pirenzepine–oxotremorine M and pirenzepine–vehicle groups.
Fig. 4. Time course of changes in bladder capacity after intracerebroventricular administration of oxotremorine M in (a) sham operated rats and (b) basal forebrain (BF) rats. Three doses of oxotremorine M were administered to SO rats (vehicle, 䊊 with dotted line; 0.015 pmol, 䊊 with solid line; 1.5 pmol, 왕 with solid line; 15 pmol, 䊐 with solid line) and BF rats (vehicle, 쐌 with dotted line; 0.015 pmol, 쐌 with solid line; 1.5 pmol, 왖 with solid line; 15 pmol, 䊏 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. SO rats (vehicle) or BF rats (vehicle).
small but significant increase in bladder capacity in SO rats compared with vehicle administration [43.7% increase over preadministration capacity, Fig. 5(a)]. This dose of pirenzepine also reduced the amplitude of bladder contraction [Fig. 5(c)] and increased residual volume [Fig. 5(e)]. Lower doses of pirenzepine (1, 10 nmol/rat) did not alter bladder capacity, amplitude of bladder contraction, or residual volume, In BF rats 10 nmol of pirenzepine, 10 times less than that used for SO rats, produced a significant increase in bladder capacity compared with vehicle administration [189.4% increase over preadministration capacity, Fig. 5(b)]. This dose produced a reduction in the amplitude of bladder contraction [Fig.
3.5. Effects of 4th ventricle or DPT administration of oxotremorine M or pirenzepine in anesthetized rats To examine the contribution of muscarinic systems in the pons to the micturition reflex, oxotremorine M or pirenzepine was administered into the 4th ventricle or DPT. Oxotremorine M (15 pmol), when injected into the 4th ventricle in urethane anesthetized rats, produced a transient enhancement of the micturition reflex in SO and BF rats within 1 minute after the administration [Fig. 7(b)]. This enhancing effect, which lasted a few min, was followed by inhibition of the micturition reflex in BF rats. No increase in residual volume was seen. Administration of the vehicle (aCSF) into the 4th ventricle had no effect on the micturition reflex. Bilateral administration of oxotremorine M (1.5 pmol) into the DPT, enhanced the micturition reflex without causing any change in residual volume [Fig. 7(a)]. Pirenzepine (100 nmol, injected into the 4th ventricle), produced an inhibitory effect on the micturition reflex accompanied by an increase in residual volume [Fig. 8(b)], but when it was administered (10 nmol) into the DPT, it did not change the micturition reflex [Fig. 8(a)].
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Fig. 5. Time course of changes in (a and b) bladder capacity, (c and d) bladder contraction pressure, and (e and f) residual volume after intracerebroventricular administration of pirenzepine in (a, c, and e) sham operated (SO) rats and (b, d, and f) basal forebrain (BF) rats. Three doses of pirenzepine were administered to SO rats (vehicle, 䊊 with dotted line; 1 nmol, 䊊 with solid line; 10 nmol, 왕 with solid line; 100 nmol, 䊐 with solid line) and BF rats (vehicle, 쐌 with dotted line; 0.1 nmol, 䉬 with solid line; 1 nmol, 쐌 with solid line; 10 nmol, 왖 with solid line). Values represent means±SEM; n=6 per group. **P⬍0.01, *P⬍0.05 vs. SO rats (vehicle) or BF rats (vehicle).
4. Discussion Anatomical and electrophysiological studies have revealed that activation of bladder parasympathetic nerves during micturition is mediated by a spinobulbospinal pathway passing through a micturition center (i.e., the potine micturition center, PMC) in the DPT (Morrison, 1987). The on–off switch of the micturition reflex is believed to be located in the PMC, which receives inhibitory or excitatory input from the forebrain (de Groat et al., 1993). Anatomical tracing studies in
animals using the transneuronal transport of a pseudorabies virus have revealed multiple sites in the cerebral cortex, diencephalon and the brainstem that are likely to be the source of the modulatory mechanisms in micturition reflex (Nadelhaft et al., 1992; Vizzard et al., 1995). Lesions of the cerebral cortex resulting from tumors, aneurysms, or vascular disease appear to enhance the micturition reflex at least in part by removing the cortical inhibitory control of the micturition center in the brain stem. Various neurotransmitters have been implicated in the supraspinal control of micturition in animals (de
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Fig. 6. Time course of changes in bladder capacity after intracerebroventricular administration of oxotremorine M (0.015 pmol/rat) in basal forebrain lesion (BF) rats pretreated with pirenzepine (1 nmol/ rat, i.c.v.). Data were compared among 4 groups (vehicle–vehicle, 쐌 with dotted line; pirenzepine–vehicle, 쐌 with solid line; vehicle– oxotremorine M, 왖 with solid line; pirenzepine–oxotremorine M, 䊏 with solid line). Values represent means±SEM; n=7 per group. **P⬍0.01, *P⬍0.05 vs. BF rats (pirenzepine–oxotremorine M, 䊏 with solid line).
Groat et al., 1993), but no reports have dealt with cortical diencephalic neurotransmitter systems exerting an inhibitory control on the PMC except for this and one other study (Yoshimura et al., 1993). Dopaminergic pathways from the substantia nigra to the basal ganglia have an inhibitory effect on the micturition reflex via activation of D1 receptors. The inhibitory action of dopamine is indicated by the changes in voiding function in patients with Parkinson’s disease and in a monkey
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with Parkinsonism induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine. The present pharmacological studies have implicated central muscarinic mechanisms as playing important roles in both the inhibitory and excitatory control of the micturition reflex. Ibotenic lesions in the basal forebrain, inducing severe neuronal damage to the nucleus basalis magnocellularis, were found to reduce bladder capacity and ChAT activity in the frontal cortices. These results indicate that the cholinergic pathway between the frontal cortex and the basal nucleus contributes to the inhibitory control of the micturition reflex. Impairment of this regulatory system is likely to cause neurogenic bladder dysfunction in patients with Alzheimer type senile dementia (Resnick and Baumann, 1988). No increase in residual volume or reduction in bladder contraction pressure was observed in our rat model, which means that the forebrain cholinergic pathway appears to raise the bladder threshold of the micturition switching circuit. Furthermore, impairment of this regulatory pathway is largely responsible for the development of the overactive bladder induced by cerebral infarction (Nakada et al., 2000). The quantitative analysis of mAChR and subtype mRNA in the acute phase of a middle cerebral artery occlusion model demonstrated a tendency toward a reduction in mRNA levels in the ischemic cortex (Kuji et al., 1997). Even a transient cerebral ischemia causes a reduction in ChAT activity, ACh contents, and release in chronic phase (Iwasaki et al., 1996; Kakihara et al., 1984). Intracerebroventricular administration of oxotremorine M, a nonselective mAChR agonist, produced primarily an inhibitory effect on the micturition reflex in BF rats, although some enhancement effects were also noted. Oxotremorine M did not necessarily make up for the total reduction in bladder capacity resulting from BF lesions, which indicates that dysfunction of the central
Fig. 7. Effects of administration of oxotremorine M into (a) the dorsal pontine tegmentum (DPT, 1.5 pmol/rat) or (b) the 4th ventricle (15 pmol/rat) on cystometrogram and electromyographic (EMG) finding of the external urethral sphincter in urethane anesthetized rats. ECP, bladder contraction pressure; RV, residual volume.
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Fig. 8. Effects of administration of pirenzepine into (a) the dorsal pontine tegmentum (DPT, 10 nmol/rat) or (b) the 4th ventricle (100 nmol/rat) on cystometrogram and electromyographic (EMG) finding of the external urethral sphincter in urethane anesthetized rats. BCP, bladder contraction pressure; RV, residual volume.
cholinergic system can not be counteracted only by supplementation with ACh. Other neurotransmitter systems relaying ACh neurons may also undergo changes in BF rats. In previous reports we suggested that NMDA (N-methyl-d-aspartate) glutamatergic or D1 dopaminergic mechanisms, which originate in the forebrain, contributed to inhibitory control of the micturition reflex (Yokoyama et al. 1999, 2000). Moreover, a recent study has shown that muscarinic activation inhibits the release of glutamate in the corticostriatal pathway (Niittykoski et al., 1999). Cholinergic neurons may thus influence the glutamatergic pathways that regulate the micturition reflex in the brain. Selective M1 mAChR antagonist pirenzepine, when administered at one-tenth of the concentration which had effect on the micturition reflex, counteracted the oxotremorine M-induced increase in bladder capacity in BF rats. This indicates that the M1 muscarinic receptors in the cerebral cortex have an inhibitory effect on the micturition reflex pathway. An autoradiographic study has provided the evidence of M1 mAChR over many forebrain structures including the cerebral cortex, while a very small fraction of the total number of receptors sites in the thalamus and brainstem (Mash and Potter, 1986). No critical studies have been published which focus on the functional role of the forebrain M1 muscarinic system in the micturition reflex, but it is impaired in BF models, thus resulting in enhancement of the micturition reflex. The excitatory effect of a large dose of oxotremorine M on the micturition reflex requires an explanation. A single dose (15 pmol/rat) of intracerebroventricular oxotremorine M reduced bladder capacity in SO rats, and cumulative doses (up to 15 pmol/rat) had the same effect in both SO and BF rats. Decerebration experiments have revealed that activation of cholinergic recep-
tors in the pontine-mesencephalic brain region with mAChR agonist oxotremorine induces bladder hyperactivity (Sille´ n et al., 1982), while focally injected carbachol into the DPT, which activates cholinoceptive neuronal cell, elicited the micturition reflex in an acute decerebrate cat (Sugaya et al., 1987). Activation of cholinoceptive neurons in the DPT presumably becomes a trigger for the recruitment of any one of a number of neuronal circuits involved in micturition. Our study demonstrated that chemical stimulation with oxotremorine M injected into the DPT facilitated the micturition reflex. Since M1 mAChR does not exist in the pons (Mash and Potter, 1986) and pirenzepine injected into the DPT had no effect on the micturition reflex, M2 mAChR might mediate this response. Injection into the 4th ventricle close to the DPT elicited primarily an excitatory response, which was always followed by an inhibitory response. These results suggest that muscarinic mechanisms in the DPT contribute to enhancement of the micturition reflex, and that the prolonged inhibitory effect of oxotremorine M injected into the 4th ventricle seems to be caused by its inhibitory influence on the PMC via M1 muscarinic receptors in the cerebral cortex. Therefore, the excitatory effect of a large dose of oxotremorine M injected into the lateral ventricle is likely to be the results of its effect on cholinoceptive neurons in the DPT. In BF rats, however, the difference in response between the cumulatively applied oxotremorine M and single dose of oxotremorine M is unexplainable. Another question that needs answering is what the mechanism underlies the action of selective M1 mAChR antagonist pirenzepine? In our study pirenzepine had an inhibitory effect on the micturition reflex when injected into the lateral ventricle, but none when injected into the DPT. As mentioned earlier M1 mAChR does not exist in the pons, so that the inhibitory influence of pirenzep-
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ine might be due to its effect on the forebrain via M1 mAChR. The release of GABA from the cerebral cortical GABA neurons is reportedly modulated by presynaptic M1 mAChR (Hashimoto et al., 1994). Suppression of GABA release by carbachol can be counteracted by pirenzepine in the cerebral cortex, meaning that pirenzepine induces GABA release through M1 mAChR. Intracerebroventricular administration of GABAergic agents (muscimol and baclofen) produces dose-dependent inhibition of the micturition reflex at the supraspinal level (Kanie et al., 2000). Therefore, pirenzepine possibly suppresses the micturition reflex by means of activating GABAergic mechanisms in the cerebral cortex. The effects of pirenzepine on bladder capacity, bladder contraction pressure and residual volume were clearly different in SO and BF rats with BF rats being more sensitive and responding to one-tenth of the dose which had an effect on SO rats. This difference in sensitivity between SO and BF rats may reflect changes in GABAergic mechanisms in the cerebral cortex following BF lesions. Another possibility is that pirenzepine can act as a M2 mAChR antagonist at high doses. Pirenzepine preferentially binds to M1 mAChR, and also exhibit low affinity to M2 and M3 mAChR (Wess et al., 1991). An inhibitory effect of pirenzepine on the micturition reflex when applied to the lateral ventricle might be due to its affinity for M2 mAChR in the pons. Using an M2 mAChR antagonist, this problem will be solved in more detail in future experiments. In conclusion, M1 mAChR appears to be involved in forebrain inhibitory mechanisms in the micturition reflex. Our findings suggest that injury to the basal forebrain causes a change in M1 muscarinic inhibitory sytems, which results in enhancement of the micturition reflex. Therefore, M1 mAChR agonists can be expected to be beneficial for the treatment of the overactive bladder caused by Alzheimer type dementia.
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