95

Brain Research, 549 (1991) 95-105 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116602F BRES 16602

Electrophysiological analysis of the ascending and descending components of the micturition reflex pathway in the rat H. Noto*, J.R. Roppolo, W.D. Steers** and W.C. de Groat Department of Pharmacology Centerfor Neuroscience, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 (U.S.A.) (Accepted 11 December 1990)

Key words: Laterodorsal tegmental nucleus; Periaqueductal gray; Bladder; Pontine micturition center; Electrical stimulation; Evoked potential; Bladder postganglionic nerve

Electrophysioiogical techniques were used to examine the organization of the spinobulbospinal micturition reflex pathway in the rat. Electrical stimulation of afferent axons in the pelvic nerve evoked a long latency (136 + 41 ms) response on bladder postganglionic nerves, whereas stimulation in the dorsal pontine tegmentum elicited shorter latency firing (72 + 25 ms) on these nerves. Transection of the pelvic nerve eliminated these responses. Firing on the bladder postganglionic nerves was evoked by stimulation in a relatively limited area of the pons within and close to the laterodorsal tegmental nucleus (LDT) and adjacent ventral periaqueductal gray. Stimulation at sites ventral to this excitatory area inhibited at lateneies of 107 + 11 ms the asynchronous firing on the bladder postganglionic nerves elicited by bladder distension. Electrical stimulation of afferents in the pelvic nerve evoked short latency (13 + 3 ms) negative field potentials in the dorsal part of the periaqueductal gray as well as long latency (42 + 7 ms) field potentials in and adjacent to the LDT. The responses were not altered by neuromuscular blockade. Similar responses were elicited by stimulation of afferent axons in the bladder nerves. The sum of the lateneies of the ascending and descending pathways between the LDT and the pelvic nerve (i.e. 72 ms plus 42 ms = 114 ms) is comparable although somewhat shorter (22 ms) than the latency of the entire micturition reflex. These results provide further evidence that the micturition reflex in the rat is mediated by a spinobulbospinal pathway which passes through the dorsal pontine tegmentum, and that neurons in the periaqueductal gray as well as the LDT may play as important role in the regulation of the micturition. INTRODUCTION Various studies have indicated that the p a r a s y m p a thetic c o m p o n e n t of the micturition reflex is d e p e n d e n t on a spinobulbospinal p a t h w a y passing through an area of the rostral pons k n o w n as the pontine micturition center (PMC). F o r e x a m p l e , micturition can be elicited after intercollicular d e c e r e b r a t i o n , but is abolished by transection of neuraxis at any point below the inferior coUiculus. Electrical stimulation in the dorsal pontine t e g m e n t u m evokes b l a d d e r contractions 3,8,11-13,15,17,19, 21-23.25,26,29-31,35,37,39, whereas lesioning of the same area results in urinary retention 1,32,36. Single unit recording in this p o n t i n e region identified neurons that r e s p o n d e d to changes in b l a d d e r pressure 2,5,1°,24,25,38. Chemical stim-

reflex have been e x a m i n e d in considerable detail in the cat 3'13. Electrical stimulation of b l a d d e r afferents in the pelvic nerve e v o k e d reflex firing on the b l a d d e r postganglionic nerves at latencies of 80-120 ms. Stimulation in the pons in the region of the locus coeruleus (LC) elicited firing of sacral p a r a s y m p a t h e t i c neurons (latency, 45-60 ms); whereas stimulation of b l a d d e r afferents e v o k e d negative field potential in the same region at latencies of 30-40 ms. The sum of the latencies for the ascending and descending pathways a p p r o x i m a t e s the latency for the entire reflex. R e c e n t electrophysiological studies in our l a b o r a t o r y TM 16,18,22,23,34 using the rat have also indicated the existence

d e m o n s t r a t e d ascending and descending axonal connections between this pontine region and the parasympathetic nucleus in the sacral spinal cord 7,8,12,14,20,28,33.

of a supraspinal reflex p a t h w a y to the b l a d d e r , and that various sites in the dorsal p o n t i n e t e g m e n t u m can m o d u l a t e the activity of the lower urinary tract. Electrical stimulation of the pelvic nerve elicited a long latency (120-155 ms) discharge in the b l a d d e r postganglionic nerves as n o t e d in the cat 3,4,13 and dog 26. T h e discharge

T h e electrophysiological properties of the micturition

was abolished by transection o f the pelvic nerve or

ulation of these neurons m o d u l a t e d b l a d d e r activity 15, 29-31,35,38. R e t r o g r a d e and a n t e r o g r a d e tracing studies

* Present address: Department of Urology, University of Akita, School of Medicine, Akita 010, Japan. ** Present address: Department of Urology, Box 422, University of Virginia, School of Medicine, Charlottesville, VA 22908, U.S.A. Correspondence: J.R. Roppolo, W1356 BMST, Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, U.S.A.

96 thoracic spinal cord transection 16As'34. Electrical stimulation in the dorsal pontine tegmentum elicited excitatory as well as inhibitory effects on bladder activity in the rat 11'22'23. Stimulation in the region of laterodorsal tegmental nucleus (LDT) evoked short latency (0.7-2.0 s) large amplitude, bladder contractions. Stimulation at sites adjacent to the excitatory area inhibited bladder activity22'23. The latency for evoking bladder contractions was similar to that in cat s'35. The similarity in the latencies for evoked bladder contractions among various animals suggests that the properties of ascending and descending pathways of the micturition reflex in rat are similar to those in cats and dogs. This was examined in

t h e p r e s e n t s t u d y u s i n g e l e c t r o p h y s i o l o g i c a l t e c h n i q u e s in u r e t h a n e - a n e s t h e t i z e d rats.

MATERIALS AND METHODS Experiments were performed on 11 female Wistar rats anesthetized with urethane (0.3 g/kg i.p. and 0.9 g/kg s.c.). Following intubation of the trachea, the urinary bladder and its innervation were exposed through a mid-line abdominal incision. The pelvic nerve and postganglionic nerves on the surface of the bladder were isolated and prepared for stimulation or recording. In some experiments the hypogastric nerves and sympathetic chains were transected, and rats were paralyzed with neuromuscular block agents: pancuronium bromide (1.0-1.6 mg/kg, i.v.) or atracurium besylate (0.33 mg/kg, i.v.) and artificially ventilated. Neuromuscular

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Fig. 1. Schematic diagram of the experiment, showing the afferent pathway from bladder to brainstem and the efferent pathway from brainstem back to bladder. Sites of stimulation and recording are indicated and examples of averaged computer evoked responses are shown. A: evoked response in the brainstem following stimulation of pelvic nerve afferents. B: evoked response on a bladder postganglionic nerve (Bladder N.) in response to brainstem stimulation. C: evoked response on a bladder postganglionic nerve following stimulation of the pelvic nerve afferents. D: ratemeter record of asynchronous firing on a bladder postganglionic nerve (upper trace) and a bladder contraction (lower trace) induced by electrical stimulation of the brainstem. The vertical calibrations in A, B, and C correspond to 50 #V, 20/~V, and 10 #V, respectively.

97 blockade was used to eliminate possible afferent input due to contractions of skeletal muscle. The urinary bladder was cannulated by passing a polyethylene tube (5 Fr) through the external urethral orifice. The cannula was secured in place by a ligature around the urethra. The carotid artery and the jugular vein were cannulated for measuring arterial blood pressure and for administration of drugs, respectively. Intravesical pressure and blood pressure were measured by strain gauge transducers (Statham P 23). The rat was placed in a stereotaxic apparatus and a small craniotomy was performed in order to insert an electrode into the dorsal pontine tegmentum for stimulation or recording (Fig. 1). After completion of the surgical procedures in these animals the body was rotated 120°, and abdominal skin flaps were tied to a metal frame to form a pool and the cavity was filled with mineral oil. Isolated bladder postganglionic nerves were transected and the proximal stumps were placed on bipolar silver electrodes for stimulation and recording. The pelvic nerve was also placed on bipolar silver electrodes for stimulation. A monopolar stainless steel electrode (Kopf, tip diameter 250 #m) was used for recording in the bralnstem while a monopolar glass microelectrode filled with Wood's metal (tip diameter 10-20 #m) was used for brainstem stimulation6. After filling the bladder with saline to confirm the presence of reflex bladder contractions, an electrode was introduced stereotaxically into the medial part of the dorsal pontine tegmentum in 0.25or 0.5-mm steps. To examine the electrophysiological properties of the ascending and descending limbs of the spinobulbospinal mictu-

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rition pathway evoked responses from the pelvic nerve to brainstem (Fig. 1A) and from brainstem to bladder postganglionic nerves (Fig. 1B) were recorded under conditions of: (1) a quiescent but partially distended bladder, (2) an empty bladder or (3) a rhythmically contracting bladder. Sites in the pons were identified where pelvic afferent stimulation evoked field potentials and where stimulation elicited bladder contractions and/or responses on the bladder postganglionic nerves (Fig. 1D). In addition, reflex responses elicited on the bladder postganglionic nerves by pelvic nerve afferent stimulation were recorded to estimate the latency of the entire micturition reflex (Fig. 1C). Electrical stimulation in the rostral pons consisted of trains (20-30 ms) of pulses 1-30 V, 0.2 ms in duration at 100-150 Hz. Pelvic nerve stimulation was 1-15 V, 0.05 ms in duration, 100-200 Hz, 10-40 ms trains. These stimulus parameters were based on previous stimulation experiments on the pelvic nerve and brainstem 17'2t'33. Neural activity was displayed on an oscilloscope, averaged with digital computer, and plotted on a Cal Comp plotter. Asynchronous firing was quantitated with an amplitude discriminator/ratemeter and displayed on a rectilinear paper recorder with bladder and blood pressure (Fig. 1D). The latency measurements were made from both oscilloscope display and from computer averaged evoked potentials. The latencies were measured from the onset of stimulation to a point where the amplitude of the evoked response was above baseline activity. Train durations were adjusted to minimize interference of the stimulus artifact from our measurements. Stimulus parameters were varied to give an evoked response of maximal amplitude and shortest measurable latency.

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200Ms Fig. 2. Effect of neuromuscular blockade and nerve transections on the evoked reflex response on bladder postganglionic nerves elicited by electrical stimulation of afferents in the pelvic nerve. A: electrical stimulation of the pelvic nerve (0.05 ms, 100 Hz, 25 ms train duration, 3 V) evoked a long latency (128 ms) supraspinal reflex (SSR) on the bladder postganglionic nerves. In this animal (Rat #4) the lumbar sympathetic chain was bilaterally transected. B: transection of the hypogastric nerve and neuromuscular blockade (pancuronium bromide 1.6 mg/kg, i.v.) did not affect this evoked response. C: electrical stimulation of the pelvic nerve (0.05 ms, 100 Hz, 40 ms train duration, 9 V) in a different animal (Rat #5) elicited a long latency response (188 ms) on the bladder postganglionic nerves. D: transection of the pelvic nerve centrally eliminated the long latency response. Each record shows an average of 3 successive responses. The vertical calibrations correspond to 20AuV.

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lOOMs 200MS Fig. 3. Effect of neuromuscular blockade and nerve transections on the response in bladder postgangliomc nerves evoked by electrical stimulation of the dorsal pontine tegmentum. A: electrical stimulation (0.2 ms, 100 Hz, 30 ms train duration, 20 V) of the dorsal pontine tegmentum (Bregma -8.3 mm, L 1.0 mm, H 3.7 mm) evoked a response at a latency of 69 ms on a bladder postganglionic nerve (Rat #2). B: neuromuscular blockade (pancuronium bromide 1.6 mg/kg, i.v.) did not abolish this response. C: electrical stimulation (0.2 ms, 100 Hz, 22 ms train duration, 30 V) of the dorsal pontine tegmentum (Bregma -8.8 mm, L 0.8 mm, H 3.5 mm) in a different animal (Rat #3) elicited an evoked response at a latency of 95 ms on a bladder postganglionic nerve. In this animal the ipsilateral hypogastric nerve and lumbar sympathetic chain were transected. D: transection of the ipsilateral pelvic nerve abolished the evoked response. Each record shows an average of 2 (A and B) or 3 (C and D) successive responses. The vertical calibrations correspond to 20/~V.

At the end of the experiment, a small lesion was made at the tip of the electrode by electrocoagulation (1 mA for I0 s with Kopf electrodes and 50/~A for 50 s with Wood's metal electrodes) at the tip of the electrode to identify the stimulating or recording site. The brainstem was fixed with 10% formalin, sectioned through the area of rostral pons (56/tm) and stained with Neutral red. The location of the lesions was noted and marked on representative sections from a stereotaxic atlas.

RESULTS

Bladder contractions elicited by electrical stimulation in the pons Electrical stimulation (0.2 ms pulses at 50 Hz intratrain frequency, 3 s train duration, 1-15 V) using a microelectrode positioned in the medial part of the dorsal p o n t i n e t e g m e n t u m evoked not only a short latency bladder contraction but also asynchronous firing on bladder postganglionic nerves (3 rats; Fig. 1D). The latency for the bladder contraction was 1-2 s. The

o p t i m u m sites for evoking a bladder contraction were centered in the region of laterodorsal tegmental nucleus (LDT) and ventral periaqueductal gray. This area extended 0.5-1.2 m m in the rostrocaudal direction 22.

Reflex responses on bladder postganglionic nerves following stimulation of afferents in the pelvic nerve Electrical stimulation of the pelvic nerve elicited long latency reflex responses on the bladder postganglionic nerves in 9 rats (Fig. 2 and Table I). The shortest latency response obtained in each animal ranged from 86 to 203 ms (136 + 41 ms, m e a n + S.D.). Stimulus parameters consisted of 0.05 ms pulses at 100-150 Hz intratrain frequency and 15 to 40 ms train durations. Threshold intensities ranged from 2 - 6 V. The responses evoked during bladder contractions were larger than those elicited during the intervals b e t w e e n contractions. The long latency responses were unaffected by neuromuscular blockade (Fig. 2B). Transection of the hypogastric nerve

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on the bladder postganglionic nerves in each rat (Rats #1, 2, 3, 4 and 7). Lesions were located in the rostral to middle part of the LDT and adjacent periaqueductal gray. Abbreviations as in Fig. 4.

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Fig. 4. Correlation between stimulation sites in the dorsal pontine tegmentum and £esponses in the bladder postganglionic nerves evoked by electrical stimulation in Rat #2. A shows the sites (a,b,c,d and e) at which electrical stimulation (0.2 ms, 100 Hz, 30 ms train duration, 20 V) was applied. Arrows show the electrode tract (Bregma -8.3 mm, L 1.0 mm). B shows averaged records of 3 successive evoked responses. Records marked a to e correspond to sites a to e indicated in A respectively. The shortest latency (72 ms) and largest amplitude responses were recorded in proximity to the LDT (Bregma -8.3 mm, L 1.0 mm, H 3.7 mm). Vertical calibration corresponds to 20/zV. Abbreviations in Figs. 4,5,8, and 9: Aq, aqueduct; CG, central gray; Cnf, cuneiform nucleus; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; IC, inferior colliculus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; PnO, pontine reticular nucleus, oral; scp, superior cerebellar peduncle: VTg, ventral tegmental nucleus.

and lumbar sympathetic chains (Fig. 2C) did not affect the evoked responses. However, transection of the

ipsilateral pelvic nerve central to the site of stimulation eliminated the long latency responses (Fig. 2D).

Excitatory responses on the bladder postganglionic nerves elicited by stimulation of the brainstem Electrical stimulation using a microelectrode (0.2 ms pulses at 100-150 Hz intratrain frequency, 20-30 ms train duration) in the medial part of the dorsal pontine tegmentum evoked firing on the bladder postganglionic nerves in 7 of 8 rats at latencies of 49 to 117 ms (72 + 25 ms, mean + S.D.) (Fig. 3 and Table I). Threshold intensities ranged from 3 to 10 V. The responses were not changed by the administration of a neuromuscular blocking agent (Fig. 3B) or transection of the hypogastric nerves and lumbar sympathetic chains (Fig. 3C); whereas, transection of the ipsilateral pelvic nerve abolished the evoked responses (Fig. 3D). Brainstem stimu-

TABLE I La~n~ofevokedrespons~ Stimulus~evoked response Pelvic N. /Bladder postganglionic n.

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42+ 7 33 - 47 5

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Fig. 6. Responses on bladder postganglionic nerves evoked by electrical stimulation of the dorsal pontine tegmentum between and during bladder contractions. A: electrical stimulation (0.2 ms, 100 I-Iz, 25 ms train duration, 15 V) (Bregrna -8.8 rnm, L 0.8 mm, H 4.0 mm) during the interval between bladder contractions evoked a response (latency, 90 ms) on the postganglionic nerve (Rat #3). B: stimulation during a bladder contraction at the same site enhanced the response. C: electrical stimulation (0.2 ms, 200 I-Iz, 30 ms train duration, 30 V) (Bregma -8.8 ram, L 0.4 mm and H 2.5 mm) between bladder contractions did not elicit any response on a bladder postganglionic nerve (Rat #4), whereas D: stimulation at the same site during a bladder contraction inhibited the asynchronous firing on the bladder postganglionic nerve at a latency of 110 ms. Location of stimulation site was 1.5 mm ventral to the site which elicited an evoked response on the bladder postganglionic nerves. Each record shows an average of 5 successive responses. Vertical calibrations represent 40 gV in A,B and 100 #V in C,D.

lation induced firing on the contralateral as well as ipsilateral b l a d d e r postganglionic nerves. The sites that e v o k e d firing on the postganglionic nerves were located in a relatively limited area in the dorsal pontine t e g m e n t u m (Fig. 4). The dorsoventral distribution of these sites was 0.5-1.5 m m in each animal. Figure 5 shows the location of electrolytic lesions marking the sites which e v o k e d the shortest latency and largest amplitude neural activity in 5 animals. The lesions were located in and close to the L D T and adjacent ventral periaqueductal gray. The responses e v o k e d during b l a d d e r contractions (Fig. 6B) were larger than those elicited in the interval between b l a d d e r contractions (Fig. 6A). The responses were minimal when the b l a d d e r was empty.

Inhibitory responses elicited by stimulation of ventral pontine sites Electrical stimulation during the intervals between b l a d d e r contractions at sites 1-3 mm ventral to the area

that elicited reflex firing did not induce firing on the b l a d d e r postganglionic nerves (Fig. 6C). H o w e v e r , stimulation at the same sites during b l a d d e r contractions inhibited the asynchronous firing on postganglionic nerves which a c c o m p a n i e d each b l a d d e r contraction (Fig. 6D). The latencies for the inhibition r a n g e d from 97 to 122 ms (107 + 12 ms). D u r a t i o n of the inhibition was 118-158 ms (142 + 19 ms). T h e inhibitory responses were still o b s e r v e d after n e u r o m u s c u l a r b l o c k a d e o r hypogastric nerve transection.

Evoked responses in the medial part of the rostral pons following stimulation of afferent pathways in the pelvic nerve Electrical stimulation with single shocks o r trains (0.05 ms pulses at 100-200 H z intratrain frequency, 15-20 ms train duration) of afferent axons in the pelvic nerve e v o k e d short latency and short duration negative field potentials in the dorsal part of the rostral pons in 5 of 6 rats (Fig. 7 A , C ) . The shortest latencies and durations for

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Fig. 7. Evoked responses in the dorsal part of rostral pons following electrical stimulation of the pelvic nerve, a bladder nerve and the ureterovesical junction. A: electrical stimulation of the pelvic nerve (0.05 ms, 200 Hz, 15 ms train duration, 6 V) evoked a short latency (19 ms) response in the dorsal part of periaqueductal gray (PAG) (Bregma -8.3 mm, L 0.5 ram, H 5.3 mm in Rat #5). B: High intensity stimulation (50 V) of the ureterovesicai junction in the same animal elicited a similar response (latency, 13 ms) at the same site. C: electrical stimulation of the pelvic nerve (0.05 ms, 150 Hz, 20 ms train duration, 5 V) in a different animal (Rat #9) evoked a similar response (latency, 13 ms) in the dorsal part of the rostral pons (Bregma -8.8 mm, L 0.9 mm, H 6.0 mm). D: stimulation of the proximal stump of a bladder nerve with the same intensity in this animal induced an evoked response at a latency of 27 ms. These evoked responses were still observed after neuromuscular blockade (pancuronium bromide 1.6 mg/kg, i.v.). Records in A, B, C and D are averaged records of 10, 10, 20 and 20 successive evoked responses, respectively. Vertical calibrations correspond to 50 gV. these evoked potentials ranged from 9 to 17 ms (13 + 3 ms) and from 16 to 35 ms (25 + 6 ms), respectively. The intensities of stimulation (2-10 V) were similar to those for evoking reflex firing on bladder nerves. Electrical stimulation of the ureterovesical junction or bladder nerve afferents elicited similar responses at these sites in the brainstem (Fig. 7B,D). The latency for bladder nerve to brainstem responses was slightly longer (12-20 ms) than that for pelvic nerve to brainstem responses. These evoked responses were still observed after neuromuscular blockade with pancuronium. The sites at which pelvic nerve stimulation elicited short latency responses were located in a relatively limited area in the dorsal part of the periaqueductal gray (Figs. 8 and 9). Longer latency (33-47 ms, mean 42 + 7 ms) and long duration (100-160 ms; mean 130 + 15 ms) responses were elicited by pelvic nerve stimulation at sites 1.5-2.5 mm ventral to the area where short latency responses were obtained (Fig. 8). These sites were located in and adjacent to the laterodorsal tegmental nucleus (LDT) (Fig. 8 and 9). The short and long latency responses were elicited on both sides of the brainstem by unilateral pelvic nerve stimulation. The short latency responses were more prominent than the long latency responses, and could be evoked by single stimuli, whereas the latter were elicited more readily with short trains of stimuli. The amplitude

of both types of evoked potentials was markedly reduced by increasing the frequency of stimulation from 1 Hz to 5 - 8 Hz. DISCUSSION The present study revealed afferent and efferent connections between the urinary bladder and nuclei in the rostral pontine tegmentum. These connections, which are conveyed through autonomic relay centers in the lower lumbar and sacral segments of the spinal cord (L6-S1) very likely represent the ascending and descending limbs of a spinobulbospinal ('long-loop') micturition reflex pathway. Two components of the ascending limb were detected with stimulation of afferent pathways in bladder nerves or the pelvic nerves: (1) a short latency (13 + 3 ms) component identified by evoked field potentials in the dorsal part of the periaqueductal gray ( P A G ) and (2) a longer latency (42 + 7 ms) component demonstrated by evoked potentials in the region of the laterodorsal tegmental nucleus (LDT). It is not known whether these two evoked potentials represent different ascending spinopontine pathways or one pathway consisting of a primary receiving area in the P A G which then projects to the LDT. Descending projections from the L D T to the

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controlling micturition in rat. Bilateral lesions occupying the caudal part of the L D T produced urinary retention 32, whereas electrical stimulation within and in the proximity to the L D T evoked a bladder contraction 22'23. Microinjection of an excitatory amino acid into the same area elicited bladder contractions 38. Single unit recordings identified neurons in this region with firing patterns related to changes in bladder pressure 38. Axonal tracing studies revealed that neurons in the L D T have direct projections to the lumbosacral intermediate cell column 7' 14,33, and also demonstrated that the L D T receives projections from spinal tract cells in the lumbosacral spinal cord 7. Electrical stimulation of the pelvic nerve evoked reflex firing on bladder postganglionic nerves at latencies of 86-203 ms (136 _+ 41 ms) as reported by other investigators in cat (latencies, 80-120 ms) 3"4, dog (150-200 ms) 26 and rat (80-170 ms) 16'18'a4. Although neuromuscular blockade and transection of the hypogastric nerves and lumbar sympathetic chain did not affect this reflex, lesioning of the pelvic nerve centrally abolished this reflex firing. The long latency reflex was present in the precollicular decerebrate rat 16']8 and absent in the spinal rat (T 8 transection) 16A8'34. These observations are consistent with the findings in cat 4 and dog 26, and indicate that the long latency reflex in rat is also relayed through the brainstem and is mediated by parasympathetic component through the pelvic nerve.

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Fig. 8. Correlation between pelvic nerve afferent evoked responses and locations in the medial part of the rostral pons in Rat #5. A shows recording sites (a through i) in the rostral pons. Arrows indicate the electrode tract. B shows averaged records of 20 successive evoked responses elicited by electrical stimulation of the pelvic nerve (0.05 ms, 200 Hz, 15 ms train duration, 8 V). Records a to i in B correspond to brainstem sites a to i in A, respectively. Short latency (17 ms) responses were obtained in the dorsal part of the periaqueductal gray, and 40 to 50 ms latency, long duration responses were recorded adjacent to the LDT. Background neuronal activity was high at site i. Vertical calibration indicates 50 #V. Abbreviations as in Fig. 4.

bladder postganglionic neurons in the pelvic plexus occurred with a mean latency of 72 + 25 ms. The sum of the latencies (114 ms) for the ascending and descending pathways was slightly less than the latency (136 ms) of the entire reflex (i.e. pelvic nerve afferent to bladder postganglionic nerve). This small but consistent difference in latency may represent, in part, synaptic delay at the brainstem level, which may be excluded in the latency measurements when the descending pathway is activated by direct stimulation of the LDT. These observations complement previous studies 7A4' 22,23,32,33,38 indicating that the L D T is an important integration center for a spinobulbospinal reflex pathway

103 Electrophysiological studies of the descending limbs of the supraspinal micturition reflex pathway in cats revealed that the shortest latency responses (latencies of 45-60 ms) in the sacral preganglionic neurons occurred with electrical stimulation in the locus coeruleus 3. Similar responses on the pelvic nerves or on the bladder postganglionic nerves were reported in cats (60-110 ms) 19 and dogs (50-150 ms) 26. The present study showed that the shortest latencies in the rat ranged from 49 to 117 ms (mean, 72 + 25 ms). Thus, the latencies of the descending limbs of the supraspinal micturition reflex were quite similar across a range of animals (cat, dog and rat). Since there is a considerable difference in the length of this pathway in these animals, the conduction velocity of the fibers must be slower or the synaptic organization different in the rat as compared to the cat and dog. Latencies of the ascending limb of the supraspinal micturition reflex have been reported in cat 3'9'24. Electrical stimulation of afferents in the pelvic nerve evoked negative field potentials in the PMC at latencies of 30-40 ms 3, or excited some PMC neurons at latencies of 30-60 ms 24. These responses were obtained in the same region where electrical stimulation elicited an excitatory effect on the bladder activity. On the other hand, Ikeda 9 demonstrated in the cat that pelvic nerve stimulation evoked responses bilaterally in the dorsal pontine tegmentum at latencies of 9-23 ms or 38-40 ms. The present study showed that pelvic nerve stimulation in rat also elicited two different latency responses bilaterally in different areas in the dorsal pontine tegmentum. Recently, peripheral nerve conduction times were calculated for bladder afferent and efferent pathways in the rat TM. Mallory et al. TM reported that with an average conduction distance of 70 mm, the efferent conduction time ranged from 58 to 173 ms while the afferent conduction time ranged from 7 to 86 ms. Subtracting the peripheral conduction times for the fastest conducting myelinated afferents and unmyelinated efferents TM from the total ascending (42 + 7 ms) and descending times (72 + 25 ms) yields, an ascending spinopontine conduction time of 35 ms and descending conduction time of 14 ms. The sum of these two times yields an estimated central delay of 49 ms, which is somewhat shorter than the 60 to 76 ms central delay of the spinobulbospinal micturition reflex in the cat 3. Pelvic afferent evoked potentials in the bladder excitatory area occurred at longer latencies than responses evoked in the region 1.5-2.5 mm dorsal in the periaqueductal gray (PAG). Electrical stimulation of afferents in bladder nerves as well as pelvic nerve evoked short latency responses. The magnitude of the short latency response was influenced by changes of the bladder volume. However, stimulation in this area did not evoke

a bladder contraction or firing on bladder nerves. The results raise the possibility that the dorsal part of the P A G is one of the receiving areas of afferent input from the bladder, and may send information to the PMC or other sites in the central nervous system. It is unlikely that the P A G is a relay station in the micturition reflex pathway, but it may have a modulatory influence on micturition. The evoked responses on bladder postganglionic nerves elicited by L D T stimulation were not influenced following neuromuscular blockade or transection of lumbar sympathetic pathways, but were abolished after lesioning of the pelvic nerve. In addition, the evoked responses during bladder contractions were larger than those elicited between bladder contractions, and were minimal when the bladder was empty. These results indicate that the evoked response from this pontine region to the bladder postganglionic nerves was mediated by the sacral parasympathetic excitatory pathway through the pelvic nerve, and that the response was modulated by the level of afferent input from the bladder. In cats, firing on bladder nerves in response to electrical stimulation of the brainstem was also dependent on the level of the intravesical pressure and afferent input from the bladder. However, recent experiments in the cat revealed that interruption of bladder afferent pathways by bilateral section of the L7-S 3 dorsal roots abolished spontaneous bladder contractions, but abolished neither the bladder contraction nor external urethral sphincter relaxation elicited by electrical stimulation of the PMC 17. These observations suggest that the coordination of detrusorsphincter responses in cat can be activated by stimulation of the PMC, independent of sacral afferent input. In the rat, afferent input appears to have a more prominent modulatory influence on the descending limb of the micturition reflex pathway. Electrical stimulation of the dorsal pontine tegmentum evoked firing in contralateral as well as ipsilateral bladder postganglionic nerves. This observation is consistent with the results of neuroanatomical studies in which anterograde tracing revealed that the descending fibers from the L D T terminated bilaterally in the sacral intermediolateral column 14. Retrograde tracing studies demonstrated that following unilateral injection of a neurotracer into the sacral intermediolateral column, PMC neurons were labeled bilaterally in approximately equal numbers 2°. Electrical stimulation in the dorsal pontine tegmentum during bladder contractions also inhibited reflex firing on bladder postganglionic nerves. The sites that inhibited nerve activity were located in the region 1-3 mm ventral to the bladder excitatory area. This observation is consistent with former studies 22,23 where electrical stimulation at sites ventral to the bladder excitatory area

104 aborted or terminated an ongoing bladder contraction. This inhibition was observed in paralyzed animals and after transection of the

lumbar sympathetic nerves

Afferent stimulation elicited longer latency responses (42 + 7 ms) within and in the proximity to the LDT. These observations provide further evidence that the micturi-

ranged from 97 to 122 ms (107 + 12 ms) which is approximately 35 ms longer than excitatory response. These results suggest that the inhibitory pathway may be

tion reflex in the rat is mediated by a supraspinal pathway which passes through the dorsal p o n t i n e t e g m e n t u m , and that neurons in the periaqueductal gray as well as the L D T have a role in regulation of micturition. Although the localization of the bladder excitatory area is not

mediated by a more complicated polysynaptic pathway or by more slowly conducting descending fibers than those

identical with that in cat and dog, the area is located in the same general area of the rostral pons and the

of the excitatory pathway. In summary, electrical stimulation in an area encom-

latencies of the ascending and descending limbs of the supraspinal micturition reflex are consistent across a

passing the L D T and adjacent periaqueductal gray elicited bladder contractions and firing on bladder post-

range of animals.

indicating that inhibition occurred at some point on the parasympathetic pathway. The latencies for inhibition

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