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Nervous control of the urinary bladder of the cat
Brain Research, 87 (1975) 201-211 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
201
NERVOUS CONTROL OF THE URINARY BLADDER OF THE CAT
w. c. DE GROAT Department of Pharmacology, School of Medicine, University o]"Pittsburgh, Pittsburgh, Pa. 15261 (u.s.A.)
This paper will focus primarily on recent electrophysiological studies concerning the neural control of the urinary bladder of the cat. More comprehensive reviews of clinical and experimental investigations of micturition in man and animals may be found in the publications by Langworthy et al. 3z, Kuntz 26, Kuru 27, and Bors and Comarr 7. lnnervation of the urinary bladder. The urinary bladder, like most visceral structures receives an innervation from both divisions of the autonomic nervous system29,~°. Parasympathetic fibers, which arise in the sacral segments of the spinal cord and travel in the pelvic nerves, represent the principal excitatory input to the bladder. The integrity of these fibers is essential for the normal performance of micturition. The sympathetic pathways (hypogastric nerves) originate in the lumbar cord and provide an inhibitory input to the detrusor region of the bladder and an excitatory input to the trigone. Afferent activity arising in the bladder is conveyed to the central nervous system over both sets of autonomic nerves. Those afferents which signal bladder distension and which evoke reflex contractions of the detrusor muscle, travel in the pelvic nerves to the sacral cord. Pain impulses from the trigonal region of the bladder pass to the thoracic cord via the hypogastric nerves. Reflex mechanisms underlying micturition. The primary stimulus for micturition is bladder distension which leads to: (1) reflex activation of the parasympathetic excitatory outflow to the bladdedAS, (2) depression of the sympathetic inhibitory outflow12 and (3) depression of the somatic efferent input to the external urethral sphincter. Secondary reflexes elicited by the passage of urine through the urethra '-5 may reinforce these primary reflexes and facilitate the complete emptying of the bladder. Central parasympathetic reflex pathways Cats with an intact neuraxis. Various evidence indicates that the parasympathetic component of the micturition reflex is dependent upon a supraspinal pathway originating in the rostral pons (Fig. 1). Barringtonl, ~ showed that micturition could be elicited in cats decerebrated at the intercollicular level, but that micturition was abolished by transection of the neuraxis at any point below the inferior colliculus. Electrical stimulation in the dorsolateral pons in the region of the locus coeruleus elicited contractions of the urinary bladder3,Zs,45, whereas lesions placed in the same
202
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Fig. 1. Sacral parasympathetic reflex pathways involved in micturition in normal and chronic spinal cats. Records A and B: responses of sacral preganglionic neurons to stimulation of pelvic nerve afferent fibers in a cat with an intact spinal cord (A) and in a chronic spinal cat (B). C: diagram of supraspinal (1) and spinal (2) reflex pathways. D: field potential recorded monophasically in rostral pons (P2,L2,H--4) elicited by a single shock to the pelvic nerve. E: discharge of a postganglionic parasympathetic fiber on the surface of the bladder elicited by a short train of stimuli (7 shocks, 150 c/sec) applied to the rostral pons (P3,L2,H--4) via a metal neurological electrode (0.25 mm, outer diameter). Vertical calibration represents 5 mV in A, 2 mV in B, 50/~V in D and 100 #V in E, negativity upward in this and all subsequent figures. Dots above each record indicate stimulus artifacts. region p r o d u c e d irreversible depression o f b l a d d e r reflexes a. A s n o t e d by o t h e r investigators 1s,lmsl,43,44 areas o f the b r a i n rostral to the pons also have excitatory a n d inhibitory influences on b l a d d e r activity. These s u p r a p o n t i n e centers are believed to have a m o d u l a t i n g a c t i o n on the basic reflex p a t h w a y . M o r e detailed i n f o r m a t i o n a b o u t the o r g a n i z a t i o n o f the sacral p a r a s y m p a t h e t i c outflow to the b l a d d e r has been o b t a i n e d with electrophysiological m e t h o d s z°,11,13-15, 28 Preganglionic p a r a s y m p a t h e t i c neurons, which were identified b y a n t i d r o m i c invasion in response to s t i m u l a t i o n o f the pelvic nerves or sacral ventral roots, were e x a m i n e d with extracellular a n d intracellular recording techniques. N e u r o n s were localized in the i n t e r m e d i o l a t e r a l region o f the spinal gray m a t t e r a n d were differentiated f r o m a l p h a - m o t o n e u r o n s on the basis o f a x o n a l c o n d u c t i o n velocity a n d stimu-
203 lus threshold 13. Two-thirds of the parasympathetic neurons discharged in response to a rapid distension of the bladder or to a maintained increase in bladder pressure 15. The remaining third of the neurons were quiescent at all times and thus most likely provided an innervation to other pelvic viscera (i.e., bowel, sex organs). When the bladder was distended and maintained at constant volume large rhythmic bladder contractions occurred at frequencies ranging from 0.3 to 6/min depending upon the degree of distension. The contractions were preceded or coincided with the firing of parasympathetic neurons 11,15. During periods between bladder contractions or when the bladder was maintained at zero constant pressure, neurons were quiescent. These findings provided direct confirmation of the proposal by Ruch 39 and Plum 37that bladder'tone' is dependent upon the intrinsic properties of the vesical smooth muscle and is unrelated to a tonic parasympathetic efferent discharge. The reflex firing of sacral preganglionic neurons elicited by distension of the bladder or by electrical stimulation of bladder afferents in the pelvic nerve had characteristics of a supraspinal reflex. The reflex discharge occurred after a long latency (80-120 msec) (Fig. 1A), was present in decerebrate animals or in animals with an intact central nervous system, but was absent in acute spinal animals. Intracellular recording revealed that pelvic afferent stimulation evoked EPSPs at 65-100 msec latency. Short latency EPSPs and reflex firing were not observed. It was concluded in agreement with Barrington 2 that activation of bladder afferents normally leads to excitation of sacral parasympathetic neurons via a supraspinal pathway. The characteristics of the ascending and descending limbs of the supraspinal pathway have recently been studied by Lalley et al. zs. It was observed that electrical stimulation at various points in the brain stem of cats produced firing of sacral preganglionic neurons and contractions of the bladder. The shortest latency responses (latencies of 45-60 msec) occurred with electrical stimulation in the area of the locus coeruleus (Fig. 1E) and in the same general area designated by Barringtona as the 'pontine micturition center'. Assuming a conduction distance of 40 cm and a pathway with relatively few synapses, it would appear that the descending fibers have peak conduction velocities of 6-9 m/sec. Stimulation of afferents in the pelvic nerve evoked negative field potentials in the rostral pontine areas at latencies of 30-40 msec (Fig. 1D). Making the same assumptions, the ascending fibers could have peak conduction velocities of 10-11 m/sec. Stimulation of areas rostral and medial to the above sites depressed bladder contractions and often produced an initial inhibition and late firing (125-300 msec latency) of sacral preganglionic neurons. It is noteworthy, however, that a strong stimulus applied to a wide variety of sites in the brain elicited the late firing. Thus it might represent a non-specific response which occurs following activation of various central pathways. Chronic spinal preparations. In chronic spinal cats which had developed 'automatic micturition' (7-38 days after transection) long latency excitatory reflexes to the sacral parasympathetic neurons could not be demonstrated; however, short latency reflexes were observed15. Pelvic afferent stimulation evoked EPSPs and reflex firing (Fig. 1B) at latencies of 3-5 msec and 7-25 msec, respectively. Distension of the bladder in chronic spinal animals also evoked reflex firing in sacral parasympathetic path-
204 ways and bladder contractions. These observations contrasted with those of Barrington 1,2 who was unable to evoke excitatory bladder reflexes in chronic spinal preparations. Indeed, Barrington2 proposed that 'automatic micturition' in spinal animals must be mediated by intrinsic properties of the vesical smooth muscle and peripheral nerve plexus or alternatively by spinal reflexes which inhibit the activity of the urethral sphincters. The electrophysiological studies showed, however, that 'automatic micturition' was dependent in part upon the emergence of spinal autonomic reflexes. Whether the appearance of the spinal reflexes was the result of the formation of new pathways34,36 or the unmasking of an existing pathway due to the removal of descending inhibitory control could not be determined. It is interesting in this regard, however, that chronic spinal transection in cats also caused the reappearance of excitatory somatovesical reflexes15which are present in neonatal kittens (Fox'l; De Groat, unpublished observations) but are suppressed during development. It is well known that in many species, including felines, micturition and defecation are elicited in neonates when the mother licks the perineal area. In some species (e.g. guinea pig and rat) this stimulus is essential for the survival of the newborn6, 3s. In kittens the excitatory perineal-bladder reflex disappears at 7-12 weeks of age (De Groat, unpublished) and is supplanted by an inhibitory response to perineal stimulation which is readily demonstrated in adult cats 11,15 and older kittens (De Groat, unpublished). Transection of the spinal cord in adult cats or in kittens which have lost the excitatory perineal-bladder reflex causes the reappearance of the reflex 1-2 weeks after the transection. In these animals electrical stimulation of somatic afferents from the perineal region and the hindlimb elicited short latency (3-5 msec) EPSPs and firing in vesical parasympathetic neurons 15. Stimulation of the same afterents in adult animals with intact spinal cord elicited short latency IPSPs and inhibition followed by EPSPs and firing. Under certain conditions the excitatory perineal-bladder reflexes can be demonstrated in adult animals with an intact cord. For example, after the administration of convulsant doses of strychnine, perineal stimulation commonly produces bladder contractions (De Groat, unpublished). In addition, when the sacral parasympathetic neurons are discharged by electrophoretic administration of an excitant amino acid, perineal stimulation enhances the firing 11. It can be concluded from these observations that the perineal-bladder excitatory reflex in cats is mediated by a spinal pathway which is physiologically important during early neonatal life, but is suppressed by descending supraspinal controls during development. The reappearance of this pathway in chronic spinal animals is probably attributable to removal of supraspinal inhibitory mechanisms. By analogy a similar phenomenon may underlie the appearance of the short latency bladder-bladder reflex in chronic spinal animals. Recurrent inhibition in the central parasympathetic pathway. Another mechanism that might have a role in the regulation of micturition is recurrent inhibition. De Groat and RyalP 4 showed that the activity of the urinary bladder and the firing of parasympathetic neurons could be blocked by antidromic stimulation of sacral ventral roots at frequencies of 10-20 c/sec. This inhibition exhibited a number of important differences from the recurrent inhibition of motoneurons; however, like the latter it
205
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Fig. 2. Facilitation of the transmission in vesical parasympathetic ganglia by repetitive stimulation of preganglionic fibers in the pelvic nerve. A • records of the postganglionic responses obtained at varying times after the start of continuous preganglionic stimulation (3 c/sec). A1 was the first evoked response and A2, A3, and A4 were obtained, respectively, 1, 2.5 and 4 sec later. B: action potentials recorded on a vesical postganglionic nerve filament in response to submaximal stimulation at a frequency of 3 c/see. First stimulus is marked by a dot. C: time course of the facilitation of transmission in a vesical ganglion. The abscissa indicates the time in seconds after the initiation of the stimulus train and the ordinate is the per cent increase in the amplitude of the postganglionic response. The postganglionic action potentials were elicited by submaximal stimulation of the pelvic nerve. Records are included for frequencies of stimulation at 1 c/see (C)--C)), 2 c/see (O - - O), 5 c/see (El--D), 10 c/see (11--11) and 20 c/see (A--A). was antagonized by the intravenous administration o f strychnine. Strychnine also blocked the depressant action o f glycine on the paraysmpathetic neurons, thus raising the possibility that this amino acid m a y be a transmitter in the inhibitory pathway 1°. Interneurons were encountered in the region of the intermediolateral column, which were activated synaptically by antidromic volleys in the ventral roots. The synaptic reponses of these cells had characteristics which clearly distinguished them f r o m Renshaw cells. Since the firing o f the 'inhibitory' interneurons and the intensity of recurrent inhibition was reduced at high bladder pressures, it is likely that the recurrent pathway is depressed during micturition, thereby facilitating the parasympathetic outflow to the bladder. Facilitation in vesicalparasympathetic ganglia. In their studies of the micturition reflex, De G r o a t and Ryal115 observed that continuous stimulation of vesical afferent fibers at frequencies between 0.5 and 5 c/sec produced a marked recruitment or facilitation o f the evoked discharge in parasympathetic postganglionic pathways to the bladder. It was n o t determined, however, whether the facilitation was related to increased transmission in central pathways or in peripheral ganglia. In recent experiments 42 it was shown that at least part o f this facilitation must be mediated by changes in transmission at ganglionic synapses. As shown in Fig. 2 continuous stimulation o f preganglionic axons in the pelvic nerves elicited responses in vesical postganglionic nerves which gradually increased in amplitude. Depending u p o n the frequency o f stimulation (0.5-20 c/see) the discharge
206 increased 2-20 times over control levels obtained at low frequencies (0.25 c/sec) of stimulation. The facilitation of transmission persisted for several minutes after termination of the stimulus. Synapses in vesical ganglia are effective, therefore, in transmitting only intense parasympathetic activity similar to that which occurs during the micturition reflex. At low frequencies of stimulation the safety factor for transmission is markedly reduced and activation of the entire presynaptic input to a ganglion elicits firing in only a small percentage of the ganglion cells. These observations indicate that vesical parasympathetic ganglia may have more complex functions than previously recognized. The ganglia may act as 'filters' in the micturition pathway: blocking the excitatory input to the bladder when intravesical pressure and parasympathetic firing are low, and facilitating the neural input to the bladder during micturition when preganglionic activity is high. The frequency characteristics of transmission in vesical ganglia are well suited for the maintenance of urinary continence as well as for the production of large, well sustained bladder contractions during micturition. Sympathetic inhibitory pathway to the bladder. Gjone 22 and Edvardsen 18,19 reported that interruption of the sympathetic input to the urinary bladder of the cat enhanced spontaneous and reflexly evoked bladder contractions. They proposed, therefore, that the sympathetic fibers exerted a tonic inhibitory control over bladder activity. Edvardsen18,19 also presented evidence that the sympathetic inhibitory fibers were reflexly activated by distension of the bladder and that the reflex pathway was organized within the lumbosacral cord. Recently, De Groat and Lalleylz obtained direct electrophysiological evidence for such a pathway. They showed that electrical or physiological activation of vesical afferent fibers produced a reflex discharge on the hypogastric nerves and on preganglionic nerves to the inferior mesenteric ganglion. The afferents evoking the reflex had conduction velocities in the range 7-30 m/sec (Ay, 6) and apparently supplied the tension or stretch receptors in the bladder wall. The reflex was present after complete transection of the spinal cord at the lower thoracic level; and, therefore, must have been mediated via a spinal pathway. The estimated central reflex latency was long (mean, 18 msec), indicative of a multisynaptic reflex arc. Lesion experiments demonstrated that the central pathway was partially crossed at the sacral level and ascended in the dorsolateral quadrant of the cord. There was no evidence for crossing above the L7 segment. Study of single or multi-unit activity on preganglionic sympathetic nerves indicated that there were at least 2 distinct populations of lumbar sympathetic fibers which were activated by electrical stimulation of vesical afferents: (1) those which were inhibited (Fig. 3) during micturition and (2) those which were excited during micturition. The former group was also inhibited by artificially raising bladder pressure above the micturition threshold. This inhibitory response to increased intravesical pressure was abolished by transecting the spinal cord at the lower thoracic level, indicating that the inhibition originated from a supraspinal site, possibly the 'pontine micturition center'. The population of sympathetic fibers activated at low bladder pressure and
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Fig. 3. Relationships between bladder activity and reflexes evoked in pelvic (A,C) and hypogastric (B,D) nerves by electrical stimulation of afferents in the pelvic nerve. Records A-D are computer averaged responses (10 responses) recorded between (A and B) and during (C and D) isovolumetric bladder contractions. Vertical calibration represents 30/~V in A and B and 60/~V in C and D. E: diagram depicting the effects of bladder pressure on the autonomic outflow to the bladder. See text for further details. inhibited at high bladder pressures during micturition fits nicely into the inhibitory schema proposed by Edvardsen 18. These fibers would be activated during bladder filling to produce a feedback inhibition of vesical ganglia and smooth muscle 16,17, thereby allowing the bladder to accommodate to larger volumes. With the onset of micturition, however, they would be depressed allowing the micturition reflex to proceed uninhibited and the bladder to empty completely (Fig. 3). That population of fibers which was activated during micturition might have several actions. The fibers might have a vasomotor function, since an increase in blood pressure is often recorded during the micturition reflex, and they might also contract the ureters and ureterovesical 'sphincters' to prevent the reflux of urine into the ureters during micturition. Mechanisms underlying sympathetic inhibition in the bladder. It has been reported that catecholamines administered by close intraarterial injection or released endogenously during stimulation o f the hypogastric nerves elicit two distinct inhibitory responses in the urinary bladder16,17,40, 41. One type of inhibition, which was apparent
208
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Fig. 4. Effects of electrical stimulation of the hypogastric nerve (HGN-STIM) and injected norepinephrine (NEPI) on transmissionin pelvic ganglia before (I) and after (II) dihydroergotamine (200 #g). Recordings were made on a postganglionic nerve filament on the surface of the urinary bladder. Action potentials were elicited by submaximal preganglionic pelvic nerve stimulation at a frequency of 0.5 c/sec. Records A and B correspond respectively to HGN-STIM (20 V, 20 c/sec) and the intraarterial injection of norepinephrine (NEPI, 0.5 pg). The bars indicate the duration of HGN-STIM. Drug injections are indicated by a dot above each record. Vertical calibration represents 400 #V, negativity upwards. as a depression of s p o n t a n e o u s or evoked bladder contractions was antagonized by beta-adrenergic blocking agents. Since this i n h i b i t i o n could be d e m o n s t r a t e d in n o r m a l l y innervated as well as decentralized u n s t i m u l a t e d bladder preparations, it must have been mediated by a direct action o n the vesical s m o o t h muscle cells. The second type of i n h i b i t i o n occurred in the parasympathetic ganglia o n the surface of the u r i n a r y bladder. This i n h i b i t i o n was unaffected by beta-blocking agents, b u t was ADRENERGIC INHIBITION OF THE BLADDER
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Fig. 5. Diagrammatic representation of receptors mediating adrenergic inhibition in the urinary bladder of the cat. The dashed line from the adrenergic inhibitory neuron indicates that the postganglionic axon to the bladder and the pelvic ganglia do not necessarily originate from the same adrenergic inhibitory neuron.
209 completely antagonized by alpha-adrenergic blocking agents (Fig. 4). Thus, adrenergic modulation of bladder activity seems to involve depressant actions at different sites in the vesical neuromuscular apparatus and also actions on differe~ adrenergic receptors: beta-receptors in vesical smooth muscle and alpha-receptors in parasympathetic ganglia (Fig. 5). The inhibition observed in parasympathetic ganglia was of particular interest, since it represented the first demonstration of ganglionic blockade by an adrenergic synaptic mechanism. The existence of such an inhibitory mechanism had been a subject of speculation for many years s,35 and is now supported by a considerable body of experinaental evidence (for reviews, see refs. 9, 23 and 33). An anatomical substrate for adrenergic inhibition in vesical ganglia was described by Hamberger and Norberg 24,25 and E1-Badawi and Schenk z0. Using histochemical techniques these investigators showed that adrenergic terminals occurred in close apposition to the vesical cholinergic ganglion cells. Since the terminals did not degenerate following section of the hypogastric nerves or the lumbar sympathetic chain, it was concluded that they arose from adrenergic neurons located in the pelvic plexus. Electrophysiological evidence indicates that these neurons receive synaptic connections from preganglionic fibers in the hypogastric nerve 17,4°. It has also been shown that the administration of various cholinomimetic agents, including acetylcholine, excited the inhibitory neurons and in turn produced adrenergic inhibition in vesical ganglia 40. It has not been determined, however, whether acetylcholine is the transmitter mediating synaptic activation of the inhibitory neurons. This research was supported in part by G r a n t 07923 from the National Institute of Neurological Diseases and Stroke, G r a n t Q-66 from the Health Research and Services Fund of Pittsburgh, Pa., and a Research Career Development Award, NS 13854. I am grateful to Dr. P. M. Lalley and Dr. W. R. Saum for their contributions to this work and to Mr. J. Douglas, Mr. J. von Hedemann and Mr. T. T o k a r for expert technical assistance.
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211 38 ROSENBLATr,J. S., AND LEHRMAN, D. S., Maternal behavior of the laboratory rat. In H. L. RHEINGOLD (Ed.), Maternal Behavior in Mammals, Wiley, New York, 1963, pp. 8-57. 39 RUCH, T. C., Central control of the bladder. In H. W. MAGOUN(Ed.), Handbook of Physiology, Section 1. Neurophysiology, Vol. I1, Williams and Wilkins, Baltimore, Md., 1960, pp. 1207-1223. 40 SAUM,W. R., AND DE GROAT, W. C., Parasympathetic ganglia: activation of an adrenergic inhibitory mechanism by cholinomimetic agents, Science, 175 (1972) 659-661. 41 SAUM, W. R., AND DE GROAT, W. C., Antagonism by bulbocapnine of adrenergic inhibition in parasympathetic ganglia in the urinary bladder, Brain Research, 37 (1972) 340-344. 42 SAUM, W. R., AND DE GROAT, W. C., Nicotinic and muscarinic mechanisms in pelvic parasympathetic ganglia in the urinary bladder of the cat, Fed. Proc., 32 (1973) 800. 43 TANG~P. C.~Levels ~f brain stem and diencepha~n c~ntr~lling micturiti~n re~ex~J. Neur~physi~L~ 18 (1955) 583-595. 44 TANG, P. C., AND RUCH, T. C., Localization of brain stem and diencephalic areas controlling the micturition reflex, J. comp. Neurol., 106 (1956) 213-245. 45 WANG, S. C., AND RANSON, S. W., Autonomic responses to electrical stimulation of the lower brain stem, J. comp. Neurol., 71 (1939) 437-455.
201
NERVOUS CONTROL OF THE URINARY BLADDER OF THE CAT
w. c. DE GROAT Department of Pharmacology, School of Medicine, University o]"Pittsburgh, Pittsburgh, Pa. 15261 (u.s.A.)
This paper will focus primarily on recent electrophysiological studies concerning the neural control of the urinary bladder of the cat. More comprehensive reviews of clinical and experimental investigations of micturition in man and animals may be found in the publications by Langworthy et al. 3z, Kuntz 26, Kuru 27, and Bors and Comarr 7. lnnervation of the urinary bladder. The urinary bladder, like most visceral structures receives an innervation from both divisions of the autonomic nervous system29,~°. Parasympathetic fibers, which arise in the sacral segments of the spinal cord and travel in the pelvic nerves, represent the principal excitatory input to the bladder. The integrity of these fibers is essential for the normal performance of micturition. The sympathetic pathways (hypogastric nerves) originate in the lumbar cord and provide an inhibitory input to the detrusor region of the bladder and an excitatory input to the trigone. Afferent activity arising in the bladder is conveyed to the central nervous system over both sets of autonomic nerves. Those afferents which signal bladder distension and which evoke reflex contractions of the detrusor muscle, travel in the pelvic nerves to the sacral cord. Pain impulses from the trigonal region of the bladder pass to the thoracic cord via the hypogastric nerves. Reflex mechanisms underlying micturition. The primary stimulus for micturition is bladder distension which leads to: (1) reflex activation of the parasympathetic excitatory outflow to the bladdedAS, (2) depression of the sympathetic inhibitory outflow12 and (3) depression of the somatic efferent input to the external urethral sphincter. Secondary reflexes elicited by the passage of urine through the urethra '-5 may reinforce these primary reflexes and facilitate the complete emptying of the bladder. Central parasympathetic reflex pathways Cats with an intact neuraxis. Various evidence indicates that the parasympathetic component of the micturition reflex is dependent upon a supraspinal pathway originating in the rostral pons (Fig. 1). Barringtonl, ~ showed that micturition could be elicited in cats decerebrated at the intercollicular level, but that micturition was abolished by transection of the neuraxis at any point below the inferior colliculus. Electrical stimulation in the dorsolateral pons in the region of the locus coeruleus elicited contractions of the urinary bladder3,Zs,45, whereas lesions placed in the same
202
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Fig. 1. Sacral parasympathetic reflex pathways involved in micturition in normal and chronic spinal cats. Records A and B: responses of sacral preganglionic neurons to stimulation of pelvic nerve afferent fibers in a cat with an intact spinal cord (A) and in a chronic spinal cat (B). C: diagram of supraspinal (1) and spinal (2) reflex pathways. D: field potential recorded monophasically in rostral pons (P2,L2,H--4) elicited by a single shock to the pelvic nerve. E: discharge of a postganglionic parasympathetic fiber on the surface of the bladder elicited by a short train of stimuli (7 shocks, 150 c/sec) applied to the rostral pons (P3,L2,H--4) via a metal neurological electrode (0.25 mm, outer diameter). Vertical calibration represents 5 mV in A, 2 mV in B, 50/~V in D and 100 #V in E, negativity upward in this and all subsequent figures. Dots above each record indicate stimulus artifacts. region p r o d u c e d irreversible depression o f b l a d d e r reflexes a. A s n o t e d by o t h e r investigators 1s,lmsl,43,44 areas o f the b r a i n rostral to the pons also have excitatory a n d inhibitory influences on b l a d d e r activity. These s u p r a p o n t i n e centers are believed to have a m o d u l a t i n g a c t i o n on the basic reflex p a t h w a y . M o r e detailed i n f o r m a t i o n a b o u t the o r g a n i z a t i o n o f the sacral p a r a s y m p a t h e t i c outflow to the b l a d d e r has been o b t a i n e d with electrophysiological m e t h o d s z°,11,13-15, 28 Preganglionic p a r a s y m p a t h e t i c neurons, which were identified b y a n t i d r o m i c invasion in response to s t i m u l a t i o n o f the pelvic nerves or sacral ventral roots, were e x a m i n e d with extracellular a n d intracellular recording techniques. N e u r o n s were localized in the i n t e r m e d i o l a t e r a l region o f the spinal gray m a t t e r a n d were differentiated f r o m a l p h a - m o t o n e u r o n s on the basis o f a x o n a l c o n d u c t i o n velocity a n d stimu-
203 lus threshold 13. Two-thirds of the parasympathetic neurons discharged in response to a rapid distension of the bladder or to a maintained increase in bladder pressure 15. The remaining third of the neurons were quiescent at all times and thus most likely provided an innervation to other pelvic viscera (i.e., bowel, sex organs). When the bladder was distended and maintained at constant volume large rhythmic bladder contractions occurred at frequencies ranging from 0.3 to 6/min depending upon the degree of distension. The contractions were preceded or coincided with the firing of parasympathetic neurons 11,15. During periods between bladder contractions or when the bladder was maintained at zero constant pressure, neurons were quiescent. These findings provided direct confirmation of the proposal by Ruch 39 and Plum 37that bladder'tone' is dependent upon the intrinsic properties of the vesical smooth muscle and is unrelated to a tonic parasympathetic efferent discharge. The reflex firing of sacral preganglionic neurons elicited by distension of the bladder or by electrical stimulation of bladder afferents in the pelvic nerve had characteristics of a supraspinal reflex. The reflex discharge occurred after a long latency (80-120 msec) (Fig. 1A), was present in decerebrate animals or in animals with an intact central nervous system, but was absent in acute spinal animals. Intracellular recording revealed that pelvic afferent stimulation evoked EPSPs at 65-100 msec latency. Short latency EPSPs and reflex firing were not observed. It was concluded in agreement with Barrington 2 that activation of bladder afferents normally leads to excitation of sacral parasympathetic neurons via a supraspinal pathway. The characteristics of the ascending and descending limbs of the supraspinal pathway have recently been studied by Lalley et al. zs. It was observed that electrical stimulation at various points in the brain stem of cats produced firing of sacral preganglionic neurons and contractions of the bladder. The shortest latency responses (latencies of 45-60 msec) occurred with electrical stimulation in the area of the locus coeruleus (Fig. 1E) and in the same general area designated by Barringtona as the 'pontine micturition center'. Assuming a conduction distance of 40 cm and a pathway with relatively few synapses, it would appear that the descending fibers have peak conduction velocities of 6-9 m/sec. Stimulation of afferents in the pelvic nerve evoked negative field potentials in the rostral pontine areas at latencies of 30-40 msec (Fig. 1D). Making the same assumptions, the ascending fibers could have peak conduction velocities of 10-11 m/sec. Stimulation of areas rostral and medial to the above sites depressed bladder contractions and often produced an initial inhibition and late firing (125-300 msec latency) of sacral preganglionic neurons. It is noteworthy, however, that a strong stimulus applied to a wide variety of sites in the brain elicited the late firing. Thus it might represent a non-specific response which occurs following activation of various central pathways. Chronic spinal preparations. In chronic spinal cats which had developed 'automatic micturition' (7-38 days after transection) long latency excitatory reflexes to the sacral parasympathetic neurons could not be demonstrated; however, short latency reflexes were observed15. Pelvic afferent stimulation evoked EPSPs and reflex firing (Fig. 1B) at latencies of 3-5 msec and 7-25 msec, respectively. Distension of the bladder in chronic spinal animals also evoked reflex firing in sacral parasympathetic path-
204 ways and bladder contractions. These observations contrasted with those of Barrington 1,2 who was unable to evoke excitatory bladder reflexes in chronic spinal preparations. Indeed, Barrington2 proposed that 'automatic micturition' in spinal animals must be mediated by intrinsic properties of the vesical smooth muscle and peripheral nerve plexus or alternatively by spinal reflexes which inhibit the activity of the urethral sphincters. The electrophysiological studies showed, however, that 'automatic micturition' was dependent in part upon the emergence of spinal autonomic reflexes. Whether the appearance of the spinal reflexes was the result of the formation of new pathways34,36 or the unmasking of an existing pathway due to the removal of descending inhibitory control could not be determined. It is interesting in this regard, however, that chronic spinal transection in cats also caused the reappearance of excitatory somatovesical reflexes15which are present in neonatal kittens (Fox'l; De Groat, unpublished observations) but are suppressed during development. It is well known that in many species, including felines, micturition and defecation are elicited in neonates when the mother licks the perineal area. In some species (e.g. guinea pig and rat) this stimulus is essential for the survival of the newborn6, 3s. In kittens the excitatory perineal-bladder reflex disappears at 7-12 weeks of age (De Groat, unpublished) and is supplanted by an inhibitory response to perineal stimulation which is readily demonstrated in adult cats 11,15 and older kittens (De Groat, unpublished). Transection of the spinal cord in adult cats or in kittens which have lost the excitatory perineal-bladder reflex causes the reappearance of the reflex 1-2 weeks after the transection. In these animals electrical stimulation of somatic afferents from the perineal region and the hindlimb elicited short latency (3-5 msec) EPSPs and firing in vesical parasympathetic neurons 15. Stimulation of the same afterents in adult animals with intact spinal cord elicited short latency IPSPs and inhibition followed by EPSPs and firing. Under certain conditions the excitatory perineal-bladder reflexes can be demonstrated in adult animals with an intact cord. For example, after the administration of convulsant doses of strychnine, perineal stimulation commonly produces bladder contractions (De Groat, unpublished). In addition, when the sacral parasympathetic neurons are discharged by electrophoretic administration of an excitant amino acid, perineal stimulation enhances the firing 11. It can be concluded from these observations that the perineal-bladder excitatory reflex in cats is mediated by a spinal pathway which is physiologically important during early neonatal life, but is suppressed by descending supraspinal controls during development. The reappearance of this pathway in chronic spinal animals is probably attributable to removal of supraspinal inhibitory mechanisms. By analogy a similar phenomenon may underlie the appearance of the short latency bladder-bladder reflex in chronic spinal animals. Recurrent inhibition in the central parasympathetic pathway. Another mechanism that might have a role in the regulation of micturition is recurrent inhibition. De Groat and RyalP 4 showed that the activity of the urinary bladder and the firing of parasympathetic neurons could be blocked by antidromic stimulation of sacral ventral roots at frequencies of 10-20 c/sec. This inhibition exhibited a number of important differences from the recurrent inhibition of motoneurons; however, like the latter it
205
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Fig. 2. Facilitation of the transmission in vesical parasympathetic ganglia by repetitive stimulation of preganglionic fibers in the pelvic nerve. A • records of the postganglionic responses obtained at varying times after the start of continuous preganglionic stimulation (3 c/sec). A1 was the first evoked response and A2, A3, and A4 were obtained, respectively, 1, 2.5 and 4 sec later. B: action potentials recorded on a vesical postganglionic nerve filament in response to submaximal stimulation at a frequency of 3 c/see. First stimulus is marked by a dot. C: time course of the facilitation of transmission in a vesical ganglion. The abscissa indicates the time in seconds after the initiation of the stimulus train and the ordinate is the per cent increase in the amplitude of the postganglionic response. The postganglionic action potentials were elicited by submaximal stimulation of the pelvic nerve. Records are included for frequencies of stimulation at 1 c/see (C)--C)), 2 c/see (O - - O), 5 c/see (El--D), 10 c/see (11--11) and 20 c/see (A--A). was antagonized by the intravenous administration o f strychnine. Strychnine also blocked the depressant action o f glycine on the paraysmpathetic neurons, thus raising the possibility that this amino acid m a y be a transmitter in the inhibitory pathway 1°. Interneurons were encountered in the region of the intermediolateral column, which were activated synaptically by antidromic volleys in the ventral roots. The synaptic reponses of these cells had characteristics which clearly distinguished them f r o m Renshaw cells. Since the firing o f the 'inhibitory' interneurons and the intensity of recurrent inhibition was reduced at high bladder pressures, it is likely that the recurrent pathway is depressed during micturition, thereby facilitating the parasympathetic outflow to the bladder. Facilitation in vesicalparasympathetic ganglia. In their studies of the micturition reflex, De G r o a t and Ryal115 observed that continuous stimulation of vesical afferent fibers at frequencies between 0.5 and 5 c/sec produced a marked recruitment or facilitation o f the evoked discharge in parasympathetic postganglionic pathways to the bladder. It was n o t determined, however, whether the facilitation was related to increased transmission in central pathways or in peripheral ganglia. In recent experiments 42 it was shown that at least part o f this facilitation must be mediated by changes in transmission at ganglionic synapses. As shown in Fig. 2 continuous stimulation o f preganglionic axons in the pelvic nerves elicited responses in vesical postganglionic nerves which gradually increased in amplitude. Depending u p o n the frequency o f stimulation (0.5-20 c/see) the discharge
206 increased 2-20 times over control levels obtained at low frequencies (0.25 c/sec) of stimulation. The facilitation of transmission persisted for several minutes after termination of the stimulus. Synapses in vesical ganglia are effective, therefore, in transmitting only intense parasympathetic activity similar to that which occurs during the micturition reflex. At low frequencies of stimulation the safety factor for transmission is markedly reduced and activation of the entire presynaptic input to a ganglion elicits firing in only a small percentage of the ganglion cells. These observations indicate that vesical parasympathetic ganglia may have more complex functions than previously recognized. The ganglia may act as 'filters' in the micturition pathway: blocking the excitatory input to the bladder when intravesical pressure and parasympathetic firing are low, and facilitating the neural input to the bladder during micturition when preganglionic activity is high. The frequency characteristics of transmission in vesical ganglia are well suited for the maintenance of urinary continence as well as for the production of large, well sustained bladder contractions during micturition. Sympathetic inhibitory pathway to the bladder. Gjone 22 and Edvardsen 18,19 reported that interruption of the sympathetic input to the urinary bladder of the cat enhanced spontaneous and reflexly evoked bladder contractions. They proposed, therefore, that the sympathetic fibers exerted a tonic inhibitory control over bladder activity. Edvardsen18,19 also presented evidence that the sympathetic inhibitory fibers were reflexly activated by distension of the bladder and that the reflex pathway was organized within the lumbosacral cord. Recently, De Groat and Lalleylz obtained direct electrophysiological evidence for such a pathway. They showed that electrical or physiological activation of vesical afferent fibers produced a reflex discharge on the hypogastric nerves and on preganglionic nerves to the inferior mesenteric ganglion. The afferents evoking the reflex had conduction velocities in the range 7-30 m/sec (Ay, 6) and apparently supplied the tension or stretch receptors in the bladder wall. The reflex was present after complete transection of the spinal cord at the lower thoracic level; and, therefore, must have been mediated via a spinal pathway. The estimated central reflex latency was long (mean, 18 msec), indicative of a multisynaptic reflex arc. Lesion experiments demonstrated that the central pathway was partially crossed at the sacral level and ascended in the dorsolateral quadrant of the cord. There was no evidence for crossing above the L7 segment. Study of single or multi-unit activity on preganglionic sympathetic nerves indicated that there were at least 2 distinct populations of lumbar sympathetic fibers which were activated by electrical stimulation of vesical afferents: (1) those which were inhibited (Fig. 3) during micturition and (2) those which were excited during micturition. The former group was also inhibited by artificially raising bladder pressure above the micturition threshold. This inhibitory response to increased intravesical pressure was abolished by transecting the spinal cord at the lower thoracic level, indicating that the inhibition originated from a supraspinal site, possibly the 'pontine micturition center'. The population of sympathetic fibers activated at low bladder pressure and
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Fig. 3. Relationships between bladder activity and reflexes evoked in pelvic (A,C) and hypogastric (B,D) nerves by electrical stimulation of afferents in the pelvic nerve. Records A-D are computer averaged responses (10 responses) recorded between (A and B) and during (C and D) isovolumetric bladder contractions. Vertical calibration represents 30/~V in A and B and 60/~V in C and D. E: diagram depicting the effects of bladder pressure on the autonomic outflow to the bladder. See text for further details. inhibited at high bladder pressures during micturition fits nicely into the inhibitory schema proposed by Edvardsen 18. These fibers would be activated during bladder filling to produce a feedback inhibition of vesical ganglia and smooth muscle 16,17, thereby allowing the bladder to accommodate to larger volumes. With the onset of micturition, however, they would be depressed allowing the micturition reflex to proceed uninhibited and the bladder to empty completely (Fig. 3). That population of fibers which was activated during micturition might have several actions. The fibers might have a vasomotor function, since an increase in blood pressure is often recorded during the micturition reflex, and they might also contract the ureters and ureterovesical 'sphincters' to prevent the reflux of urine into the ureters during micturition. Mechanisms underlying sympathetic inhibition in the bladder. It has been reported that catecholamines administered by close intraarterial injection or released endogenously during stimulation o f the hypogastric nerves elicit two distinct inhibitory responses in the urinary bladder16,17,40, 41. One type of inhibition, which was apparent
208
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Fig. 4. Effects of electrical stimulation of the hypogastric nerve (HGN-STIM) and injected norepinephrine (NEPI) on transmissionin pelvic ganglia before (I) and after (II) dihydroergotamine (200 #g). Recordings were made on a postganglionic nerve filament on the surface of the urinary bladder. Action potentials were elicited by submaximal preganglionic pelvic nerve stimulation at a frequency of 0.5 c/sec. Records A and B correspond respectively to HGN-STIM (20 V, 20 c/sec) and the intraarterial injection of norepinephrine (NEPI, 0.5 pg). The bars indicate the duration of HGN-STIM. Drug injections are indicated by a dot above each record. Vertical calibration represents 400 #V, negativity upwards. as a depression of s p o n t a n e o u s or evoked bladder contractions was antagonized by beta-adrenergic blocking agents. Since this i n h i b i t i o n could be d e m o n s t r a t e d in n o r m a l l y innervated as well as decentralized u n s t i m u l a t e d bladder preparations, it must have been mediated by a direct action o n the vesical s m o o t h muscle cells. The second type of i n h i b i t i o n occurred in the parasympathetic ganglia o n the surface of the u r i n a r y bladder. This i n h i b i t i o n was unaffected by beta-blocking agents, b u t was ADRENERGIC INHIBITION OF THE BLADDER
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209 completely antagonized by alpha-adrenergic blocking agents (Fig. 4). Thus, adrenergic modulation of bladder activity seems to involve depressant actions at different sites in the vesical neuromuscular apparatus and also actions on differe~ adrenergic receptors: beta-receptors in vesical smooth muscle and alpha-receptors in parasympathetic ganglia (Fig. 5). The inhibition observed in parasympathetic ganglia was of particular interest, since it represented the first demonstration of ganglionic blockade by an adrenergic synaptic mechanism. The existence of such an inhibitory mechanism had been a subject of speculation for many years s,35 and is now supported by a considerable body of experinaental evidence (for reviews, see refs. 9, 23 and 33). An anatomical substrate for adrenergic inhibition in vesical ganglia was described by Hamberger and Norberg 24,25 and E1-Badawi and Schenk z0. Using histochemical techniques these investigators showed that adrenergic terminals occurred in close apposition to the vesical cholinergic ganglion cells. Since the terminals did not degenerate following section of the hypogastric nerves or the lumbar sympathetic chain, it was concluded that they arose from adrenergic neurons located in the pelvic plexus. Electrophysiological evidence indicates that these neurons receive synaptic connections from preganglionic fibers in the hypogastric nerve 17,4°. It has also been shown that the administration of various cholinomimetic agents, including acetylcholine, excited the inhibitory neurons and in turn produced adrenergic inhibition in vesical ganglia 40. It has not been determined, however, whether acetylcholine is the transmitter mediating synaptic activation of the inhibitory neurons. This research was supported in part by G r a n t 07923 from the National Institute of Neurological Diseases and Stroke, G r a n t Q-66 from the Health Research and Services Fund of Pittsburgh, Pa., and a Research Career Development Award, NS 13854. I am grateful to Dr. P. M. Lalley and Dr. W. R. Saum for their contributions to this work and to Mr. J. Douglas, Mr. J. von Hedemann and Mr. T. T o k a r for expert technical assistance.
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211 38 ROSENBLATr,J. S., AND LEHRMAN, D. S., Maternal behavior of the laboratory rat. In H. L. RHEINGOLD (Ed.), Maternal Behavior in Mammals, Wiley, New York, 1963, pp. 8-57. 39 RUCH, T. C., Central control of the bladder. In H. W. MAGOUN(Ed.), Handbook of Physiology, Section 1. Neurophysiology, Vol. I1, Williams and Wilkins, Baltimore, Md., 1960, pp. 1207-1223. 40 SAUM,W. R., AND DE GROAT, W. C., Parasympathetic ganglia: activation of an adrenergic inhibitory mechanism by cholinomimetic agents, Science, 175 (1972) 659-661. 41 SAUM, W. R., AND DE GROAT, W. C., Antagonism by bulbocapnine of adrenergic inhibition in parasympathetic ganglia in the urinary bladder, Brain Research, 37 (1972) 340-344. 42 SAUM, W. R., AND DE GROAT, W. C., Nicotinic and muscarinic mechanisms in pelvic parasympathetic ganglia in the urinary bladder of the cat, Fed. Proc., 32 (1973) 800. 43 TANG~P. C.~Levels ~f brain stem and diencepha~n c~ntr~lling micturiti~n re~ex~J. Neur~physi~L~ 18 (1955) 583-595. 44 TANG, P. C., AND RUCH, T. C., Localization of brain stem and diencephalic areas controlling the micturition reflex, J. comp. Neurol., 106 (1956) 213-245. 45 WANG, S. C., AND RANSON, S. W., Autonomic responses to electrical stimulation of the lower brain stem, J. comp. Neurol., 71 (1939) 437-455.