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Research report
Role of spinal
a
1-adrenergic mechanisms in the control of lower
urinary tract in the rat
a,b,c ,
*
a,d aMitsuharu Yoshiyama
, Takao Yamamoto
, William C. de Groat
a
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
b
Department of Neurology, Chiba-Higashi National Hospital, 673 Nitona-cho, Chuo-ku, Chiba 260-8712, Japan
c
Department of Neurology, Chiba University School of Medicine, Chiba 260-8670, Japan
d
Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka 532-8514, Japan
Accepted 5 July 2000
Abstract
The role of spinal a1-adrenergic mechanisms in the control of urinary bladder function was examined in urethane (1.2 g / kg s.c.) anesthetized and decerebrate unanesthetized female Sprague–Dawley rats (250–320 g). Bladder activity was recorded via a transurethral catheter during continuous infusion (0.21 ml / min) cystometrograms or under isovolumetric conditions. All drugs were administered intrathecally at the L -S segmental level of spinal cord. During cystometrograms, 3 or 30 nmol of phenylephrine (6 1 a1-adrenergic agonist) did not alter bladder activity; whereas 300 nmol increased the intercontraction interval by 98% and pressure threshold for inducing micturition by 115%, but did not change bladder contraction amplitude. A large dose of phenylephrine (3000 nmol) completely blocked reflex voiding and induced overflow incontinence at a high baseline pressure (mean: 33 cmH O; range: 28–42 cmH O). Under2 2
isovolumetric conditions, 3–30 nmol of phenylephrine abolished bladder activity for 22–45 min; whereas smaller doses (0.003–0.3 nmol) were inactive. Doxazosin (50 nmol), an a1-adrenergic antagonist, decreased intercontraction intervals but did not change bladder contraction amplitude during cystometrograms. Under isovolumetric conditions this dose of doxazosin increased bladder contraction frequency and decreased bladder contraction amplitude. Smaller doses (5 or 25 nmol) of doxazosin did not alter bladder activity. These studies suggest that two types of spinala1-adrenergic mechanisms are involved in reflex bladder activity: (1) inhibitory control of the frequency of voiding reflexes presumably by regulating afferent processing in the spinal cord and (2) facilitatory modulation of the descending limb of the micturition reflex pathway. 2000 Elsevier Science B.V. All rights reserved.
Theme: Endocrine and autonomic regulation
Topic: Urogenital regulation
Keywords: a1-Adrenoceptor; Afferent pathway; Efferent pathway; Locus coeruleus; Spinal cord; Urinary bladder
1. Introduction bulbospinal noradrenergic pathways with
6-hydroxy-dopa-mine did not depress voiding [14,20,21]; however, studies Interest in the role of central noradrenergic pathways in in anesthetized cats, revealed that electrical stimulation of the control of micturition was stimulated by the report of the LC induced bladder contractions that were blocked by
¨
Dahlstrom and Fuxe [1] that sympathetic and parasympa- the intrathecal (i.t.) injection of prazosin, ana1-adrenergic thetic nuclei in the lumbosacral cord receive inputs from antagonist [26,27]. In addition, destruction of noradrener-noradrenergic neurons in the brainstem. A significant gic cells in the LC by microinjection of 6-hydroxy-dopa-proportion of these inputs arise in the locus coeruleus (LC) mine produced a hypoactive bladder, and this effect was [1,14,20,21,23], which has been implicated in the supraspi- partially reversed by the i.t. injection of phenylephrine (an nal control of micturition [3,26,27]. In rats, destruction of a1-adrenergic agonist) suggesting that bulbospinal norad-renergic pathways are involved in the micturition reflex. It is noteworthy, however, that iontophoretic application of *Corresponding author. Tel.: 181-43-261-5171; fax: 1
81-43-268-norepinephrine to bladder preganglionic neurons (PGNs) 2613.
E-mail address: [email protected] (M. Yoshiyama). did not excite but rather inhibited synaptic and amino acid
evoked firing [18]. This finding is consistent with the i.t. catheter was inserted according to the technique of electrophysiological data indicating that the descending Yaksh and Rudy [24]. The occipital crest of the skull was noradrenergic excitatory pathway from the LC terminates exposed and the atlanto-occipital membrane was incised at on interneurons in the region of the sacral parasympathetic the midline using the tip of a 16-Gauge needle as a cutting nucleus rather than PGNs and that the interneurons make edge. A catheter (PE-10) was inserted through the slit and excitatory synaptic contacts with the PGNs to complete the passed caudally to the L -level of the spinal cord. Artificial6 descending pathway [28]. Iontophoretic application of cerebrospinal fluid (CSF) (7.5ml) was injected as a flush prazosin blocked the excitatory effect of LC stimulation on following each drug administration (volumes ranging from interneurons but did not block the excitation of PGNs. In 1 to 5 ml). At the end of the experiment a laminectomy contrast to these results in anesthetized cats, experiments was performed to verify the location of the catheter tip. in conscious cats did not reveal an inhibitory effect of i.t. Decerebrations were performed according to published prazosin on voiding [6]. However, 6-hydroxy-dopamine methods [19] which included ligating both carotid arteries treatment did increase bladder capacity. followed by a precollicular decerebration using a blunt Pharmacological studies in rats have also used adren- spatula under halothane anesthesia. Cotton and Avitene ergic drugs to determine the function of bulbospinal (MedChem Products Inc., Woburn, MA, USA) were placed noradrenergic pathways in the regulation of micturition in the intracranial cavity and covered with agar. Experi-[4,25]. Kontani et al. [12] reported that in anesthetized rats, ments were started 2–4 h after the decerebration.
i.t. administration of phenylephrine abolished bladder A transurethral bladder catheter connected to a pressure activity; whereas prazosin did not alter bladder contrac- transducer was used to record the bladder pressure iso-tions during continuous infusion cystometrograms volumetrically with the urethral outlet ligated or to record (CMGs). On the other hand, Ishizuka et al. [10] noted that pressure during a CMG when the bladder was filled with a i.t. administration of doxazosin, an a1-adrenergic antago- constant infusion of physiological saline and allowed to nist, blocked micturition in unanesthetized rats. Thus, there empty around the catheter. Continuous CMGs were per-is evidence for both excitatory and inhibitory adrenergic formed by a constant infusion (0.21 ml / min) of saline into mechanisms in the control of micturition, as well as a the bladder to elicit repetitive voidings, which allowed considerable variation between different studies in the rapid collection of data for a large number of voiding effects of adrenergic agonists and antagonists on reflex cycles [15]. PE-90 and PE-50 cannulae were used for bladder activity. Some of this variation might be due to the isovolumetric recording and constant infusion CMGs, differences between the species, anesthesia and methods. respectively. For isovolumetric recording, the ureters were In the present studies, the involvement of spinal a1- tied distally, cut and the proximal ends cannulated (PE-10) adrenergic pathways in the regulation of micturition was and drained externally. This procedure prevented the examined in anesthetized and decerebrate unanesthetized bladder from filling with urine during the experiment. The rats by studying the effects of drugs administered in- effects of drugs obtained during continuous infusion trathecally on reflex bladder contractions recorded under CMGs could reflect changes in the activity of the urethral isovolumetric conditions or during continuous infusion sphincter (i.e. both internal and external) as well as the CMGs. Our results revealed two types of spinala1-adren- bladder. For example, a decrease in amplitude of bladder ergic mechanisms involved in reflex bladder activity: (1) contractions during voiding could be produced by a inhibitory control of the frequency of reflex bladder reduction of detrusor muscle contraction and / or a decrease contractions, presumably due to modulation of afferent in urethral outlet resistance. Similarly, an increase in the processing in the spinal cord and (2) excitatory modulation intercontraction interval (ICI, interval between two voiding of the amplitude of bladder contractions, presumably due cycles [15]) might be due to an increase in bladder to regulation of the descending limb of the spinobulbospi- capacity and / or improvement of voiding efficiency. Thus, nal bladder reflex pathway. the continuous CMG method is useful for examining drug A preliminary account of this work has been presented effects on voiding, but it can not always distinguish in abstract form [5,30]. between effects on bladder and urethral outlet. On the other hand, any changes in bladder activity recorded under isovolumetric conditions must be attributed to direct
2. Materials and methods effects on the bladder or changes in neural pathways
controlling the bladder. In addition, isovolumetric bladder 2.1. Animal preparation activity appears to be more sensitive to the drug effects than activity recorded during continuous infusion CMGs. Experiments were performed on urethane anesthetized The explanation for this difference in sensitivity is not (1.2 g / kg s.c.) or decerebrate unanesthetized female known [29].
the electrical activity of the striated muscle. A 30-Gauge tion (PT, the intravesical pressure required to trigger a needle with a hooked EMG electrode positioned at the tip reflex voiding contraction) and ICI during continuous was inserted into sphincter approximately 5–10 mm lateral CMGs. All values are expressed as mean6S.E.M. Analysis to the urethra and then withdrawn leaving the EMG wires of variance (ANOVA), the paired t-test and Dunnett’s embedded in the muscle [13]. The EMG activity was multiple comparison test were used when appropriate for passed through a discriminator / ratemeter and recorded on statistical data analysis. For all statistical tests, P,0.05 chart recorder. The peak firing rate during each micturition was considered significant.
contraction was measured.
The protocols in these studies were approved by the
Animal Care and Use Committee of the University of 3. Results
Pittsburgh. All efforts were made to minimize animal
suffering, to reduce the number of animals used, and to 3.1. Effects of i.t. phenylephrine on the lower urinary utilize alternatives to in vivo techniques, if available. tract during continuous CMGs
2.2. Drugs As shown in Figs. 1 and 2, high doses (300 and 3000
nmol) of phenylephrine increased the ICI and PT during Drugs used in these studies included urethane (ethyl continuous CMGs; whereas small doses (3 and 30 nmol) carbamate, Sigma, St. Louis, MO), phenylephrine (Sigma) occasionally increased the ICI but in most experiments and doxazosin (a gift from Roche Bioscience). Phenylep- were inactive. The 300 nmol of the drug prolonged the hrine was dissolved in artificial CSF [9,16] to prepare intervals between the contractions, and the 3000 nmol 0.3–3000mM solutions. Doxazosin was dissolved in 100% induced long-lasting elevation of the intravesical pressure dimethyl sulfoxide to prepare a 20-mM solution, which (Fig. 1). The increase in intravesical pressure is presumab-was diluted to 10 mM with artificial CSF. Drug doses were ly due to overdistension of the bladder by continuous calculated for the base of each compound. infusion of saline after drug-induced suppression of void-ing. A high dose (300 nmol, n55) consistently increased 2.3. Evaluation and statistical analysis the PTs and the ICIs (Fig. 2). In one rat, this dose produced an initial decrease in the ICIs accompanied by a The effects of phenylephrine and doxazosin were evalu- slightly higher baseline pressure (3 cmH O higher). This2 ated on various parameters of bladder activity including: effect persisted for 20 min after which the ICIs increased the amplitude and frequency of reflex bladder contractions as in other experiments. The dose–response curves in Fig. under isovolumetric conditions and the bladder contraction 2 were constructed from data obtained at least 30 min after amplitude (BCA), pressure threshold for inducing micturi- drug administration.
Fig. 2. These graphs show the effects of phenylephrine (3–3000 nmol i.t.) on bladder contraction amplitude (A), pressure threshold for inducing micturition (B), and intercontraction intervals (C), during continuous CMGs. ** P,0.01 (Dunnett’s multiple comparison test following repeated measures ANOVA, n55). C5control (artificial CSF) injection. O.I.5overflow incontinence.
The largest dose (3000 nmol) of phenylephrine com- 30 nmol were also administered while recording bladder pletely blocked reflex voiding and induced the overflow activity under isovolumetric conditions. As shown in Figs. incontinence in all animals (n55). After this dose, the 3 and 4, low doses of phenylephrine (0.03 and 0.3 nmol) intravesical pressure gradually increased (Fig. 1) and had little effect on the frequency and amplitude of bladder reached peak pressures ranging from 28 to 57 cmH O2 contractions; whereas higher doses (3 and 30 nmol) (mean: 45 cmH O) within 5–8 min (mean: 6 min).2 completely eliminated bladder contractions for 19–35 min. Overflow incontinence then persisted for 15–40 min In this paper, the period of complete inhibition will be (mean: 25 min, n54) at intravesical pressures ranging termed ‘disappearance time’ (Fig. 4). Recovery from from 28 to 42 cmH O (mean: 33 cmH O). In four of five2 2 inhibition was usually characterized by the abrupt appear-rats, partial recovery of bladder activity occurred within 50 ance of large amplitude bladder contractions that were very min. In these animals, the activity consisted of small similar to those occurring before the drug (Fig. 3). In two amplitude contractions (10–22 cmH O) with higher2 animals, a second administration of phenylephrine (3 baseline pressures and shorter ICIs, implying inefficient nmol) produced a similar depressant effect.
voiding and increased residual volumes. In one rat, partial recovery of bladder activity occurred within 10 min.
3.3. Effects of i.t. doxazosin on the lower urinary tract Phenylephrine (3000 nmol) administered to decerebrate
during continuous CMGs
unanesthetized rats (n52) also produced overflow inconti-nence.
Doxazosin was administered in 25 nmol doses at 30-min The effects of increasing doses of phenylephrine on
intervals to construct a cumulative dose–response curve. A EUS EMG activity were examined in two animals. In one
small dose (25 nmol) was inactive, whereas 50 nmol rat, doses of 3–300 nmol of phenylephrine decreased the
significantly decreased the ICI (58% reduction) without EMG activity by 26–62%; whereas in the other rat these
altering BCA or PT (Fig. 5 and Table 1). The 100 nmol doses had no effect. During the overflow incontinence
dose of doxazosin slightly suppressed the BCA (from induced by a higher dose (3000 nmol), EUS EMG activity
3461 to 2662 cmH O, 24% reduction, P2 50.081, n54) was totally suppressed in both rats. In another group of
and reduced the ICI (from 150643 to 34614 s, 77% animals (n54), a single dose (300 nmol) of phenylephrine
reduction, P50.062, n54). Injections of vehicle (50% was given. In three rats, EUS EMG activity was
sup-dimethyl sulfoxide in saline) did not affect bladder activity. pressed 41–62%; while ICIs increased 42–111% (from
169626 to 306670 s) and PTs increased 141–419% (from
661 to 2567 cmH O). However, the BCA was not2 3.4. Effects of i.t. doxazosin on urinary bladder activity changed. In one rat, the EUS EMG was not affected by under isovolumetric conditions
this dose of phenylephrine.
Small doses of doxazosin (5–25 nmol) did not alter 3.2. Effects of i.t. phenylephrine on urinary bladder isovolumetric bladder contractions however, 50 nmol
Fig. 3. The effects of phenylephrine (0.03 and 3 nmol i.t.) on bladder activity under isovolumetric conditions. Multiple doses of phenylephrine could be injected in this animal. (The effect of 0.3 nmol is not presented in this figure.) Note that the 0.03 nmol of the drug produced decrease of bladder contraction ‘frequency’ whereas artificial CSF had little effect on the parameter, and that the 3 nmol eliminated bladder activity for 15 min and bladder activity recovered with the intravesical pressure almost as high as control values.
Table 1
Effects of doxazosin (i.t.) on bladder activity during continuous infusion CMGs
Dose of Bladder contraction Intercontraction interval doxazosin amplitude (cmH O)2 (s)
25 nmol (n56)
Before 3462 152642
(30–39) (42–366)
After 3163 104654
(25–38) (10–352)
50 nmol (n57)
Before 3463 124628
(25–50) (39–225)
After 3062 52614**
(18–38) (2–117)
**P,0.01 (paired t-test, in comparison with before). Values in parenthe-ses are the ranges measured in the present studies.
activity completely recovered to control values within 30 min.
3.5. Interactions between phenylephrine and doxazosin
on the micturition reflex
Fig. 4. The graph shows the time of disappearance induced by
phenylep-hrine (0.03–30 nmol i.t.) of bladder activity under isovolumetric con- When phenylephrine and doxazosin were administered ditions. DT5disappearance time, the interval until the 90% of bladder in the same experiments (n510) in an attempt to demon-contraction amplitude recovered after the inhibition by phenylephrine.
strate antagonistic interactions, the results were variable. In Values at each dose were obtained from five to seven experiments.
Fig. 6. The effects of doxazosin (50 nmol i.t.) on bladder activity under isovolumetric conditions. Note that doxazosin decreased bladder contraction amplitude and increased frequency of bladder contractions.
two anesthetized rats during continuous CMGs, 300 nmol decreased the frequency and increased the intravesical of phenylephrine elicited a significant increase in ICI and pressure threshold for inducing reflex voiding; whereas PT. Thirty min later while the effects of phenylephrine still doxazosin, an a1-adrenergic antagonist, increased the persisted, administration of doxazosin (50 nmol) reversed frequency but decreased the amplitude of reflex bladder the effects of phenylephrine. A second injection of contractions. These findings raise the possibility that phenylephrine 30 min after doxazosin, did not alter bladder activation of a1-adrenergic receptors can inhibit spinal activity. In another anesthetized rat, doxazosin (50–100 sensory mechanisms that trigger voiding, but also facilitate nmol) did not reverse the overflow incontinence induced the parasympathetic efferent pathways that induce bladder by a large dose of phenylephrine (3000 nmol, n51). In emptying. Thus, brainstem noradrenergic neurons which unanesthetized decerebrate rats (n52), doxazosin (50 project to the lumbosacral spinal cord may exert two nmol) reversed the overflow incontinence induced by opposing influences on the lower urinary tract: (1) en-phenylephrine (3000 nmol) in one rat but not the other rat. hancement of urine storage by delaying the initiation of the Under isovolumetric conditions, the effect of phenylep- micturition reflex and (2) facilitation of voiding by increas-hrine (3 nmol) to reduce the frequency or completely ing the excitatory neural input to the urinary bladder. inhibit bladder contractions was blocked or markedly These effects on the parasympathetic outflow to the reduced by doxazosin pretreatment (50 and 10 nmol, bladder are likely to work in concert with other spinal respectively) in two experiments after the administration of adrenergic mechanisms that regulate the sympathetic and doxazosin in doses (50 nmol) that depressed the BCA. In somatic neural pathways to the urethra and EUS, respec-two other experiments the inhibitory effects of phenylep- tively [2,6,17].
hrine (3–30 nmol) were not altered. The function of central noradrenergic pathways in the control of micturition has been a subject of controversy for many years. Early studies in anesthetized cats showed that norepinephrine applied iontophoretically to bladder
para-4. Discussion sympathetic PGNs suppressed firing induced synaptically
or by a glutamatergic receptor agonist [18]. Other experi-The present studies revealed thata1-adrenergic receptor ments in anesthetized cats revealed that descending norad-mechanisms in the spinal cord may have a complex role in renergic projections from the LC provided an excitatory the neural control of voiding function in the rat. Intrathecal input to bladder PGN via activation of interneurons in the administration of phenylephrine, ana1-adrenergic agonist, spinal cord [28]. However, subsequent studies in unanes-thetized cats were unable to demonstrate this excitatory pathway [6,8]. Experiments in rats have also yielded Table 2
variable results. In anesthetized rats, i.t. administration of Effects of doxazosin (i.t.) on bladder activity under isovolumetric
phenylephrine depressed reflex micturition, whereas conditions
prazosin, an a1-adrenergic antagonist, was inactive [12]. Dose of Bladder contraction Contraction frequency
On the other hand, in unanesthetized rats, some studies doxazosin amplitude (cmH O)2 (contractions / min)
were unable to demonstrate an involvement of spinal 5 nmol (n55)
a1-adrenergic receptors in micturition [7], but other
in-Before 4664 0.4560.06
vestigations revealed that doxazosin, an a -adrenergic
(37–64) (0.21–0.65) 1
After 4565 0.4760.07 antagonist, depressed the intravesical pressure during (32–62) (0.20–0.63) voiding [10]. Based on the latter finding, it was suggested 50 nmol (n57)
that a1-adrenergic receptors have a facilitatory role in
Before 5362 0.5560.05
micturition. Our present data support such a conclusion.
(44–56) (0.40–0.79)
The variability in the results of different studies are very
After 2464*** 0.9360.17*
(11–38) (0.55–1.88) likely due to multiple factors including: the presence in the spinal cord of both inhibitory and facilitatory adrenergic * P,0.05, *** P,0.001 (paired t-test, in comparison with before). Values
outlet, as well as the different experimental conditions for through spinal pathways to the switching circuitry in the each of the studies. For example, during continuous PMC [4]. If it can be assumed that i.t. administration of infusion CMGs, intravesical voiding pressures are depen- phenylephrine does not act in the periphery to alter afferent dent not only on the amplitude of bladder contractions but activity, then it seems reasonable to conclude that it acts also on urethral outlet resistance. Thus, adrenergic block- centrally to reduce the transmission of afferent activity to ing agents that can alter urethral as well as bladder activity the PMC. The effect of doxazosin to increase the fre-might lower voiding pressure by several actions [2]. In the quency of reflex bladder contractions under isovolumetric present study, the contribution of the urethra was elimi- conditions is consistent with this concept. On the other nated in some experiments by recording under isovolumet- hand, doxazosin did not alter the PT during continuous ric conditions. In these experiments, doxazosin suppressed CMGs. This might be attributable to the fact that the bladder contraction amplitudes, indicating that the drug control PTs were very low (1.5–3 cmH O) and therefore it2 acted by depressing the parasympathetic excitatory input to is technically difficult to detect a further reduction after the bladder. During continuous infusion CMGs, this effect doxazosin.
was not detected, suggesting that a simultaneous action on In some but not all experiments, doxazosin antagonized the neural control of the urethra or rapid distension of the the inhibitory effect of phenylephrine, suggesting that the bladder negated the inhibitory effect on the parasympa- latter effect is due to activation ofa1-adrenergic receptors. thetic reflex pathway. Alternatively, it is possible that The reason for the variable effects of doxazosin is un-during rapid filling of the bladder, the spinala1-adrenergic known. However, it should be noted that the doxazosin– excitatory mechanisms do not make an important contribu- phenylephrine interaction studies are difficult to conduct tion to micturition. because both agents have inhibitory actions. For example, However, during both isovolumetric recording and the inhibitory effect of doxazosin on bladder contraction constant infusion CMGs, doxazosin did increase the fre- amplitude limited the dose that could be tested. In addition, quency of voiding reflexes, indicating that an a1-adren- this inhibitory effect could have interfered with the ability ergic inhibitory pathway was tonically controlling the of doxazosin to reverse the phenylephrine inhibition of triggering mechanism for voiding. The micturition reflex is afferent mechanisms; because a doxazosin-induced de-initiated by afferent receptors in the bladder wall, which crease in bladder contraction amplitude would reduce respond to distension. These afferents travel in the pelvic afferent input from the bladder and in turn indirectly nerve to the sacral spinal cord, where they activate a long enhance the inhibitory effects of phenylephrine.
ascending supraspinal pathway that projects to an area in In summary, the present results indicate that two types the dorsolateral pons known as the ‘pontine micturition of spinal a1-adrenergic mechanisms are involved in the center’ (PMC). The PMC provides the on–off switching control of reflex bladder activity in rats: (1) inhibitory function, which generates the rhythmicity of reflex bladder control of afferent processing in the spinal cord and (2) contractions. A descending pathway then projects from the excitatory modulation of the descending limb of bladder PMC back to the parasympathetic PGNs in the lumbosacr- reflex pathway (Fig. 7). These two mechanisms acting in al cord and axons which in turn provide excitatory input to concert would facilitate urine storage by increasing bladder the bladder [4]. Because voiding is activated by afferent capacity and also enhance voiding efficiency by increasing input from the bladder, it is reasonable to speculate that the parasympathetic nerve activity and the amplitude of blad-adrenergic inhibitory pathway regulates afferent processing der contractions. Although it might seem paradoxical that in spinal cord. The finding that phenylephrine increased bulbospinal noradrenergic pathways would promote oppos-the ICIs during continuous CMGs, supports this specula- ing activities of the bladder (i.e. storage and voiding), it tion. The action of phenylephrine to completely inhibit should be noted that activation of central and peripheral voiding under isovolumetric conditions indicates that this a1-adrenergic receptors also facilitates other urine storage adrenergic inhibitory mechanism has the potential to exert (e.g. reflex control of the urethra and external urethral a powerful influence on micturition. However, as evi- sphincter [2,6,17]) and voiding mechanisms (e.g. choliner-denced by the modest effect of doxazosin on voiding gic transmission in bladder ganglia [11] and acetylcholine frequency (i.e. approximately two-fold increase) this inhib- release from parasympathetic nerves in the bladder [22]). itory mechanism is only submaximally active under normal Thus, it might be appropriate to consider that bulbospinal
conditions. noradrenergic projections to the sacral parasympathetic
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Acknowledgements
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294 (1995) 645–650. (Center for Biological Research, Roche Bioscience, Palo
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