- Email: [email protected]
Quantitative analysis for innervation of smooth muscle cells in the wall of the urinary bladder
Jow~doftl~e A~,tosomle Nerpo~ 8yMem, 2 (1980) 71--80 0 £ 1 s m 4 w l N o r t h . ~ $1om~lk~l Pesm
71
QUAN'rlTATIVB ANALYS/8 FOR INNKRVATK)N OF SMOOTH MU8CLB ~ IN THKWALL OF ~ URINARY BLADDER ERZSI~BET FEHI~R, J. VAJDA and K. CS./~NYI First Depmrtment of Anatomy, 8emmelwei6 University Mcdicni School. Budap~t
(l~eeived January 4th, 1980) (Accepted March 7th, 1980)
~eyworde: quantitative analysis -- vegetative innervation -- cat -- urinary bladder
ABSTRACT
The distribution of nerve terminals to different parts of the urinary bladder was studied by electron microscopy with the aid of 6-hydroxydopamhw treatment. A quantitative analysis was merle for each region. In the sphincter and trigone areas nerve terminals were found to be much more numerous (3 times more) than in sections from the body and the apex of the bladder. In much of the bladder there were 6--7 smooth muscle cells for each nerve fiber, however in the trigone area 1--2 muscle cells were supplied by terminal nerve fiber. The narrowest gap between nerve terminals and smooth muscle cells was found to be 150--260 nm. It was commonly seen that Desmosome4ike attachments, called nexuses, connected adjacent smooth muscle cells. After 6.hydroxydopsmtne treatment desenerated terminals could be found mostly in the fine periarteris] plexuses but some were also oblerved that were n o t related to blood vessels, especially in the trifone area. Summation of our f l ~ r e s and results for terminal density suggests to us that contractions of the urinary bladder are initiated or controlled in the tr~one region and that other parts of the wall ~ activated by local nerve processes and the numerous nexal contacts.
INTRODUCTION
The relationahip between smooth muscle cells and autonomic ~ onic axons has been examined by many authors. Although recent electron microscopic studies have shown that, predominantly, Unmyelinated nerves
72 are found within the muscle coat [6,14,17], the nature and extent of these nerves which control the activity of the muscle have not yet been satisfactorily established. The precise arrangement of nerve fibers and cells in the wall of the urinary bladder also remains a subject of controversy. We are currently applying quantitative ultrastructural techniques in order to analyze the numbers and distribution of the various types of nerve terminals that have been implicated in the innervation and control of urinary bladder function. MATERIALS A N D M E T H O D S Adult cats of either sex were anesthetized with pentobarbital and perfused with Karnovsky's fixative [16]. Tissue for electron microscopy was obtained from: {1) the trigone area of the bladder; (2) the ventromedian regiov midway between the fundus and apex vesicae; (3) the lateral regions; (4) the m. sphincter vesicae; and (5) the apex of the bladder. The small pieces excised were post~ixed in 1 % osmic acid for 2 h and embedded into Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Tesla BS 500 electron microscope. The identification of adrenergic nerves was further documented with the aid of 6-hydroxydopamine (6-OHDA), 75 mg/kg, recently proved useful as a specific marker for adrenergic nerve terminals in the peripheral parts of the sympathetic nervous system [2,15,20], because 6 - O H D A causes detectable degeneration of noradrenergic nerve terminals. The animals were sacrificed and study of their tissues began 24 or 48 h after treatment with this compound. The density of nerve processes relative to the number of muscle cells was counted bv applying a standard procedure to the micrographs obtained at various lewls through the full thickness of the muscle coat. Profile surfaces of nerve flbers and muscle cells were measured by use of a planimeter and their numbers counted. Areas of 800--900 ~ m 2 were examined in each animal and the number of nerve terminals and smooth muscle cells was calculated for a 100 # m 2 surface thus obtaining the average relationship for each tissue type. Statistical significance was determined by analysis of variance. RESULTS
Fine varicose nerve terminals, mostly running longitudinally between the bundles of smooth muscle cells, were present throughout the whole bladder. Occasionally fibers were seen to cross the muscle coat transversely, connecting finally with adjacent longitudinally proceeding terminals. The nerve bundles varied in size, containing from 20 to 300 nerve fibers, the majority being unmyelinated. Myelinated fibers occasionally appeared in the most superficial layer of the muscle coat in the trigone area. The smooth muscle cells of the vesical wall were separated from their
73 neighbors by numerous collagen fibers and occasionally by blood vessels; fibrocytes could be observed among them. The fine structure of these muscle cells corresponded closely to that observed in most other smooth muscle tissues. Numerous pinocytotic vesicles were present under the surface and appeared to be continuous with the cell membrane (Fig. 1). The cells were surrounded by thin and incomplete layers of amorphous material and specialized contacts, nexuses, between the muscle cells were common. The narrowest musculo--muscular connection was represented by a tiL~ht junction. At these points the basement membranes of contiguous cells become fused, so that the plasma membranes lie in close apposition (Fig. 2). The narrowest gap between the nerve terminals and the smooth muscle cells was 150--250 nm. In some instances free surfaces of the nerve processes could be distinguished near the muscle cells. The opposing membranes at these points always lacked specialization (Figs. 3, 4). In the adventitia and muscle coat of the trigone area, it was found that clusters of nerve cells are grouped into ganglia of variable size. In sections from the body and apex of the bladder, terminals are relatively few in number. In the region of the sphincter and trigone, however, the number of axons is mu~h greater (Tables I and II). Frequently these axons are encountered within the muscle bundles lying adjacent to individual smooth
Fig. 1. Smooth muscle cells. Arrows pc int to the pinoeytotie vesicles under the cell membranes (X 27,000).
74
•
~
•
as
° ~t..,
'~,~, ~
-~,
•
~,
.
,
\
.'~
~
:
.
,
..
"
',.
"r~,~,,
"4t,
.q~
"
:
,
~'-
.
~ .
~.
"
;'~
-
..:;"
.
.~,
~,,~,~
.
-~
....
Fig. 2. Desmosome-like contacts (arrows) between adjacent muscle cells (× 37,800).
Fig. 3. Nerve terminals among the smooth muscle cells. Arrows show the free surfaces of the nerve processes in the sphincter region (x 21.600).
,
81.48 82.23 83.17 76.26 77.80
F r o n t a l part Side part A p e x o f the bladder M. s p h i n c t e r v ~ i c a e T r i g o n u m vesicae
+ 2.85 -+ 3..57 -+ 4 . 7 2 +- 3.51 + 2.94
Area o f s m o o t h muscle cells
Area/100//m 2
14.32 14.26 14.13 12.37 8.92
-+ 1.05 -+ 2.34 -+ 1.95 -+ 2.05 -+ 1.87
Number of smooth muscle cells 2.05 1.95 1.59 2.40 4.88
-+ 0.04 -+ 0.02 -+ 0.03 -+ 0.02 -+ 0.03
Area o f nerve processes 2.87 2.05 1.90 3.95 4.56
+ 0.03 -+ 0 . 0 5 -+ 0.04 -+ 0.05 -+ 0.06
N u m b e r o f nerve processes
D I S T R I B U T I O N O F T H E D I F F E R E N T T I S S U E E L E M E N T S IN T H E M U S C L E C O A T S O F t l i e U [ t l N A R Y B L A D D E R
TABLE I
16.47 15.81 15.23 21.34 17.31
-+ 2.57 -+ 3.05 -+ 4 . 5 2 -+ 2.75 -+ 2.87
Other tiuue element,
76 TABLE II PERCENTAGE OF NERVE PROCESSES IN THE MAIN PORTION OF THE MUSCLE LAYERS IN TIlE URINARY BLADDER
Corpus vesicae M. sphincter vesicae Trigonum vesicae
Section surface of nerve processes in 100 ~um2
%
Number of nerve profiles//Jm 2
%
1.86 ± 0.03 2.40 -+ 0.02 a •1.88 ± 0.03 b
100 128.82 2.t4.76
2.27 ± 0.03 3.95 "2 0.05 b 4.56 ± 0.06 b
100 173.86 200.75
0.01. b p . : 0.001.
a p <
m u s c l e cells. T h e p a t t e r n s o f nerve d i s t r i b u t i o n a n d t e r m i n a t i o n s a p p e a r e d t o be t h e s a m e in t h e b o d y , a p e x a n d lateral parts o f t h e b l a d d e r . F o u r t y p e s o f n e r v e t e r m i n a l s o r s y n a p s e s c o u l d be d i s t i n g u i s h e d b y t h e i r vesicular c o n t e n t , as has b e e n d e s c r i b e d p r e v i o u s l y b y F e h ~ r e t al. [ 1 1 ] . O n e t y p e o f t e r m i n a l possesses small ( 4 0 - - 6 0 n m in d i a m e t e r ) clear vesicles
Fig. 4. Nerve terminal containing 150--250 nm granulated vesicles (arrows) near the muscle cells in the body part of the bladder (x ~.~7,800).
77
Fig. 5. Degenerated nerve terminals (arrows) between the muscle cells in the trigone area after 6-hydroxydopamine treatment (x 16.200).
(Fig. 3); the second type contains mainly small vesicles with a central dense core; the third type of terminal contains large granulated vesicles of 80--150 nm in diameter; the fourth type possesses neurosecretory vesicles of 150-250 nm (Fig. 4). In a few instances, single smooth muscle cells are associated with all 4 types of terminals, each of which occupies a position that conforms with the recognized limits for effective neuromuscular transmission. However, nerve terminals do not innervate each muscle cell, but neurotransmitters may be released from "en passage" varicosities of the nerves and thus one fiber may influence several adjacent muscle cells. After 6-OHDA treatment degenerating terminal varicosi?~es could be seen in the nerves of the fine perivascular plexus in all parts of tl, e body. This also has been observed previously by Hoyes et al. [ 14 ]. In the trigone region and in the m. sphincter urethrae, (~egenerated nerve terminals could be found in muscle layers unassociated with blood vessels (Fig. 5); however, in the rest of the bladder the muscle layers were essentially devoid of such degenerating nerve fiber terminals. DISCUSSION Review of the literature r~veals conflicting opinions as to the role of the autonomic nervous system in the control of urinary bladder function. Some
"/8 authors were unable to distinguish neuromorphologically the triBone area from the rest of the bladder [6], however, accol~llng to Talra [19] the bladder consists of two functionally different units, the detrusor and the trigone, and the two units respond differently to sympathetic nerve stimulation and to exogenous catecholamines. The density of nerve terminals was proportionately greater in the trigone area than in other parts of the bladder; the difference in average was statisticaJly significant (Fig. 6, ref. 21). This differer.~e is supposed to be due to the fact that most of the ganglionic cells are lucated in this area and most of the extrinsic nerves synapse on their soma [ 10]. The majority of postganglionic efferents emanate from cell bodies located in the intramural ganglia, their numbers are not reduced significantly by extirpation of both hypogastric ganglia [9,11]. Elbadawi and Schenk [6] observed that the parasympathetic innervation is uniformly rich throughout the bladder with a 1 : 1 nerve fiber to muscle ratio; and the vesicourethral junction has an extremely rich innervation with a 1 : 1 or higher nerve to muscle ratio ['/]. Our results showed t h a t the trigone area has 3 times more nerve terminals than do other regions of the bladder. According to our precise counting, in most areas of the bladder wall 6--7 smooth muscle cells share one nerve fiber, however, in the trigone area 1--2 muscle tells have one nerve fiber, while in the sphincter area 3 - 4 smoc~th muscle cells are supplied by one fiber. The function of sympathetic noradrenergic nerves within the detrusor of the ~:at bladder is believed to be one of inhibition (5,12]. According to Dixon and Gosling [8] the sympathetic effect is apparently mediated by a direct action upon smooth muscle cells. Because nerve fibers are scarce in the body and apex region in proportion to the amount of muscle tissue, it is evident that every smooth muscle cell does not receive a neural process or terminal synapse. The nature of the transmitter materials in the local nerve
//••-
1,593 pm21 100 p m z
,.~)046 HrN2/ 100j.lrz"l2 ,m',,00
INI ...... 2 01 iam'/100 pro' Fig. 6. Distribution of nerve fibers in the different parts of the bladder.
79 ~rminals has not been fully determined. Ambach and Zar [1] observed that most, if not all, of the po6tganglionic nerves are non-cholinergic in nature; ~ccording to Burnstock [4] somp of them may be. purinergic. Go,cling ant* Dixon [13], and Schulman [18] speak of two types of autonomic nerves; Hoyes et al. [14] claim that 4 types of terminals can be recognized within the muscle cells of the ureter. Our observations are similar to the latter. Tight junctions appear between adjacent cells when examined by electron microscopy [3,17] and it is generally agreed that a nexus represents a pathway of lower electric resistance to the transmission of impulse from one muscle cell to another. However, there can be little doubt that the nerves are primarily responsible for controlling the contraction of smooth muscle cells. Summation of our results for terminal densities of fibers suggests that contraction of the urinary bladder smooth muscle cells is generated in the trigone area and that the presence of the local nerve terminals and numerous nexal contacts aid propagation of the impulse. REFERENCES I Ambach, N. and Zar, M., Non
80 16 Karnowdxy, M.J., A formaldehyde-11utanddehyde fixative of hith ommolarity for use in electronmicr0ecopy, J. Cell. Biol., 27 (1968) 137--138. 17 Schulman, C.C., Electron mlctoecopy of the hmmm umterlc I n n m a t l o n , Brit. J. UroI., 46 (1974) 609"-623. 18 Sehulman, C.C., Ultrutruetural evidence for sdreneqllc and choliz;eqliC Innervatlon of the human ureter, J. Urol. (Baltlmore), 113 (1975) 788--771. 19 Talra, N., The autonomic pharmacology of the bladder, Ann. Rev. Phanuaeol., 12 (1972) 197--208. 20 Tranaer, J.P. und Thoenen, H., Ultramorpholollleche Veri/nderunilen der sympathi~hen NervenendiEungen der Katze naeh Vorbehandlung mit 6- und 6-hydroxydopamlne, Naunyn-Sehmiedeberg's Arch. exp. Path Pharmak., 287 (1967) 343--344. 21 Uemura, E., Fletcher, T.F., Dirks, V.A. and Bradley, W.E., Diltribut/on of sacral afferent u o n s in cat urinary bladder, Amer. J. Armt, 136 (1973) 309--314.
71
QUAN'rlTATIVB ANALYS/8 FOR INNKRVATK)N OF SMOOTH MU8CLB ~ IN THKWALL OF ~ URINARY BLADDER ERZSI~BET FEHI~R, J. VAJDA and K. CS./~NYI First Depmrtment of Anatomy, 8emmelwei6 University Mcdicni School. Budap~t
(l~eeived January 4th, 1980) (Accepted March 7th, 1980)
~eyworde: quantitative analysis -- vegetative innervation -- cat -- urinary bladder
ABSTRACT
The distribution of nerve terminals to different parts of the urinary bladder was studied by electron microscopy with the aid of 6-hydroxydopamhw treatment. A quantitative analysis was merle for each region. In the sphincter and trigone areas nerve terminals were found to be much more numerous (3 times more) than in sections from the body and the apex of the bladder. In much of the bladder there were 6--7 smooth muscle cells for each nerve fiber, however in the trigone area 1--2 muscle cells were supplied by terminal nerve fiber. The narrowest gap between nerve terminals and smooth muscle cells was found to be 150--260 nm. It was commonly seen that Desmosome4ike attachments, called nexuses, connected adjacent smooth muscle cells. After 6.hydroxydopsmtne treatment desenerated terminals could be found mostly in the fine periarteris] plexuses but some were also oblerved that were n o t related to blood vessels, especially in the trifone area. Summation of our f l ~ r e s and results for terminal density suggests to us that contractions of the urinary bladder are initiated or controlled in the tr~one region and that other parts of the wall ~ activated by local nerve processes and the numerous nexal contacts.
INTRODUCTION
The relationahip between smooth muscle cells and autonomic ~ onic axons has been examined by many authors. Although recent electron microscopic studies have shown that, predominantly, Unmyelinated nerves
72 are found within the muscle coat [6,14,17], the nature and extent of these nerves which control the activity of the muscle have not yet been satisfactorily established. The precise arrangement of nerve fibers and cells in the wall of the urinary bladder also remains a subject of controversy. We are currently applying quantitative ultrastructural techniques in order to analyze the numbers and distribution of the various types of nerve terminals that have been implicated in the innervation and control of urinary bladder function. MATERIALS A N D M E T H O D S Adult cats of either sex were anesthetized with pentobarbital and perfused with Karnovsky's fixative [16]. Tissue for electron microscopy was obtained from: {1) the trigone area of the bladder; (2) the ventromedian regiov midway between the fundus and apex vesicae; (3) the lateral regions; (4) the m. sphincter vesicae; and (5) the apex of the bladder. The small pieces excised were post~ixed in 1 % osmic acid for 2 h and embedded into Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Tesla BS 500 electron microscope. The identification of adrenergic nerves was further documented with the aid of 6-hydroxydopamine (6-OHDA), 75 mg/kg, recently proved useful as a specific marker for adrenergic nerve terminals in the peripheral parts of the sympathetic nervous system [2,15,20], because 6 - O H D A causes detectable degeneration of noradrenergic nerve terminals. The animals were sacrificed and study of their tissues began 24 or 48 h after treatment with this compound. The density of nerve processes relative to the number of muscle cells was counted bv applying a standard procedure to the micrographs obtained at various lewls through the full thickness of the muscle coat. Profile surfaces of nerve flbers and muscle cells were measured by use of a planimeter and their numbers counted. Areas of 800--900 ~ m 2 were examined in each animal and the number of nerve terminals and smooth muscle cells was calculated for a 100 # m 2 surface thus obtaining the average relationship for each tissue type. Statistical significance was determined by analysis of variance. RESULTS
Fine varicose nerve terminals, mostly running longitudinally between the bundles of smooth muscle cells, were present throughout the whole bladder. Occasionally fibers were seen to cross the muscle coat transversely, connecting finally with adjacent longitudinally proceeding terminals. The nerve bundles varied in size, containing from 20 to 300 nerve fibers, the majority being unmyelinated. Myelinated fibers occasionally appeared in the most superficial layer of the muscle coat in the trigone area. The smooth muscle cells of the vesical wall were separated from their
73 neighbors by numerous collagen fibers and occasionally by blood vessels; fibrocytes could be observed among them. The fine structure of these muscle cells corresponded closely to that observed in most other smooth muscle tissues. Numerous pinocytotic vesicles were present under the surface and appeared to be continuous with the cell membrane (Fig. 1). The cells were surrounded by thin and incomplete layers of amorphous material and specialized contacts, nexuses, between the muscle cells were common. The narrowest musculo--muscular connection was represented by a tiL~ht junction. At these points the basement membranes of contiguous cells become fused, so that the plasma membranes lie in close apposition (Fig. 2). The narrowest gap between the nerve terminals and the smooth muscle cells was 150--250 nm. In some instances free surfaces of the nerve processes could be distinguished near the muscle cells. The opposing membranes at these points always lacked specialization (Figs. 3, 4). In the adventitia and muscle coat of the trigone area, it was found that clusters of nerve cells are grouped into ganglia of variable size. In sections from the body and apex of the bladder, terminals are relatively few in number. In the region of the sphincter and trigone, however, the number of axons is mu~h greater (Tables I and II). Frequently these axons are encountered within the muscle bundles lying adjacent to individual smooth
Fig. 1. Smooth muscle cells. Arrows pc int to the pinoeytotie vesicles under the cell membranes (X 27,000).
74
•
~
•
as
° ~t..,
'~,~, ~
-~,
•
~,
.
,
\
.'~
~
:
.
,
..
"
',.
"r~,~,,
"4t,
.q~
"
:
,
~'-
.
~ .
~.
"
;'~
-
..:;"
.
.~,
~,,~,~
.
-~
....
Fig. 2. Desmosome-like contacts (arrows) between adjacent muscle cells (× 37,800).
Fig. 3. Nerve terminals among the smooth muscle cells. Arrows show the free surfaces of the nerve processes in the sphincter region (x 21.600).
,
81.48 82.23 83.17 76.26 77.80
F r o n t a l part Side part A p e x o f the bladder M. s p h i n c t e r v ~ i c a e T r i g o n u m vesicae
+ 2.85 -+ 3..57 -+ 4 . 7 2 +- 3.51 + 2.94
Area o f s m o o t h muscle cells
Area/100//m 2
14.32 14.26 14.13 12.37 8.92
-+ 1.05 -+ 2.34 -+ 1.95 -+ 2.05 -+ 1.87
Number of smooth muscle cells 2.05 1.95 1.59 2.40 4.88
-+ 0.04 -+ 0.02 -+ 0.03 -+ 0.02 -+ 0.03
Area o f nerve processes 2.87 2.05 1.90 3.95 4.56
+ 0.03 -+ 0 . 0 5 -+ 0.04 -+ 0.05 -+ 0.06
N u m b e r o f nerve processes
D I S T R I B U T I O N O F T H E D I F F E R E N T T I S S U E E L E M E N T S IN T H E M U S C L E C O A T S O F t l i e U [ t l N A R Y B L A D D E R
TABLE I
16.47 15.81 15.23 21.34 17.31
-+ 2.57 -+ 3.05 -+ 4 . 5 2 -+ 2.75 -+ 2.87
Other tiuue element,
76 TABLE II PERCENTAGE OF NERVE PROCESSES IN THE MAIN PORTION OF THE MUSCLE LAYERS IN TIlE URINARY BLADDER
Corpus vesicae M. sphincter vesicae Trigonum vesicae
Section surface of nerve processes in 100 ~um2
%
Number of nerve profiles//Jm 2
%
1.86 ± 0.03 2.40 -+ 0.02 a •1.88 ± 0.03 b
100 128.82 2.t4.76
2.27 ± 0.03 3.95 "2 0.05 b 4.56 ± 0.06 b
100 173.86 200.75
0.01. b p . : 0.001.
a p <
m u s c l e cells. T h e p a t t e r n s o f nerve d i s t r i b u t i o n a n d t e r m i n a t i o n s a p p e a r e d t o be t h e s a m e in t h e b o d y , a p e x a n d lateral parts o f t h e b l a d d e r . F o u r t y p e s o f n e r v e t e r m i n a l s o r s y n a p s e s c o u l d be d i s t i n g u i s h e d b y t h e i r vesicular c o n t e n t , as has b e e n d e s c r i b e d p r e v i o u s l y b y F e h ~ r e t al. [ 1 1 ] . O n e t y p e o f t e r m i n a l possesses small ( 4 0 - - 6 0 n m in d i a m e t e r ) clear vesicles
Fig. 4. Nerve terminal containing 150--250 nm granulated vesicles (arrows) near the muscle cells in the body part of the bladder (x ~.~7,800).
77
Fig. 5. Degenerated nerve terminals (arrows) between the muscle cells in the trigone area after 6-hydroxydopamine treatment (x 16.200).
(Fig. 3); the second type contains mainly small vesicles with a central dense core; the third type of terminal contains large granulated vesicles of 80--150 nm in diameter; the fourth type possesses neurosecretory vesicles of 150-250 nm (Fig. 4). In a few instances, single smooth muscle cells are associated with all 4 types of terminals, each of which occupies a position that conforms with the recognized limits for effective neuromuscular transmission. However, nerve terminals do not innervate each muscle cell, but neurotransmitters may be released from "en passage" varicosities of the nerves and thus one fiber may influence several adjacent muscle cells. After 6-OHDA treatment degenerating terminal varicosi?~es could be seen in the nerves of the fine perivascular plexus in all parts of tl, e body. This also has been observed previously by Hoyes et al. [ 14 ]. In the trigone region and in the m. sphincter urethrae, (~egenerated nerve terminals could be found in muscle layers unassociated with blood vessels (Fig. 5); however, in the rest of the bladder the muscle layers were essentially devoid of such degenerating nerve fiber terminals. DISCUSSION Review of the literature r~veals conflicting opinions as to the role of the autonomic nervous system in the control of urinary bladder function. Some
"/8 authors were unable to distinguish neuromorphologically the triBone area from the rest of the bladder [6], however, accol~llng to Talra [19] the bladder consists of two functionally different units, the detrusor and the trigone, and the two units respond differently to sympathetic nerve stimulation and to exogenous catecholamines. The density of nerve terminals was proportionately greater in the trigone area than in other parts of the bladder; the difference in average was statisticaJly significant (Fig. 6, ref. 21). This differer.~e is supposed to be due to the fact that most of the ganglionic cells are lucated in this area and most of the extrinsic nerves synapse on their soma [ 10]. The majority of postganglionic efferents emanate from cell bodies located in the intramural ganglia, their numbers are not reduced significantly by extirpation of both hypogastric ganglia [9,11]. Elbadawi and Schenk [6] observed that the parasympathetic innervation is uniformly rich throughout the bladder with a 1 : 1 nerve fiber to muscle ratio; and the vesicourethral junction has an extremely rich innervation with a 1 : 1 or higher nerve to muscle ratio ['/]. Our results showed t h a t the trigone area has 3 times more nerve terminals than do other regions of the bladder. According to our precise counting, in most areas of the bladder wall 6--7 smooth muscle cells share one nerve fiber, however, in the trigone area 1--2 muscle tells have one nerve fiber, while in the sphincter area 3 - 4 smoc~th muscle cells are supplied by one fiber. The function of sympathetic noradrenergic nerves within the detrusor of the ~:at bladder is believed to be one of inhibition (5,12]. According to Dixon and Gosling [8] the sympathetic effect is apparently mediated by a direct action upon smooth muscle cells. Because nerve fibers are scarce in the body and apex region in proportion to the amount of muscle tissue, it is evident that every smooth muscle cell does not receive a neural process or terminal synapse. The nature of the transmitter materials in the local nerve
//••-
1,593 pm21 100 p m z
,.~)046 HrN2/ 100j.lrz"l2 ,m',,00
INI ...... 2 01 iam'/100 pro' Fig. 6. Distribution of nerve fibers in the different parts of the bladder.
79 ~rminals has not been fully determined. Ambach and Zar [1] observed that most, if not all, of the po6tganglionic nerves are non-cholinergic in nature; ~ccording to Burnstock [4] somp of them may be. purinergic. Go,cling ant* Dixon [13], and Schulman [18] speak of two types of autonomic nerves; Hoyes et al. [14] claim that 4 types of terminals can be recognized within the muscle cells of the ureter. Our observations are similar to the latter. Tight junctions appear between adjacent cells when examined by electron microscopy [3,17] and it is generally agreed that a nexus represents a pathway of lower electric resistance to the transmission of impulse from one muscle cell to another. However, there can be little doubt that the nerves are primarily responsible for controlling the contraction of smooth muscle cells. Summation of our results for terminal densities of fibers suggests that contraction of the urinary bladder smooth muscle cells is generated in the trigone area and that the presence of the local nerve terminals and numerous nexal contacts aid propagation of the impulse. REFERENCES I Ambach, N. and Zar, M., Non
80 16 Karnowdxy, M.J., A formaldehyde-11utanddehyde fixative of hith ommolarity for use in electronmicr0ecopy, J. Cell. Biol., 27 (1968) 137--138. 17 Schulman, C.C., Electron mlctoecopy of the hmmm umterlc I n n m a t l o n , Brit. J. UroI., 46 (1974) 609"-623. 18 Sehulman, C.C., Ultrutruetural evidence for sdreneqllc and choliz;eqliC Innervatlon of the human ureter, J. Urol. (Baltlmore), 113 (1975) 788--771. 19 Talra, N., The autonomic pharmacology of the bladder, Ann. Rev. Phanuaeol., 12 (1972) 197--208. 20 Tranaer, J.P. und Thoenen, H., Ultramorpholollleche Veri/nderunilen der sympathi~hen NervenendiEungen der Katze naeh Vorbehandlung mit 6- und 6-hydroxydopamlne, Naunyn-Sehmiedeberg's Arch. exp. Path Pharmak., 287 (1967) 343--344. 21 Uemura, E., Fletcher, T.F., Dirks, V.A. and Bradley, W.E., Diltribut/on of sacral afferent u o n s in cat urinary bladder, Amer. J. Armt, 136 (1973) 309--314.