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Effect of parasympathetic denervation on acetylcholine levels in the rat parotid gland. Is there an extraneuronal pool of acetylcholine?
Life Sciences, Vol. 33, pp. 1191-1197 Printed in the U.S.A.
Pergamon Press
EFFECt OF PARASYMPAfHETIC DENERVATION ON ACETYLCHOLINE LEVELS IN THE RAT PAROTID GLAND. IS |HERE AN EXIRANEURONAL POOL OF ACETYLCHOLINE?
M a r v i n R. Markl , Edward F. Domino=,4~ Seong S. Hart=, A u r e l i o Or t i z 4 ~ B e n j a m i n N. Nathews 4, and S a n d r a K. T a i t 4
I) M e d i c a l C o l l e g e of G e o r g i a School of D e n t i s t r y ~ Depar tment o f Oral B i o l o g y - A n a t o m y ~ Augusta~ Georgia 30912. 2) U n i v e r s i t y o f M i c h i g a n School o f Medicine~ D e p a r t m e n t o f P h a r m a c o l o g y , Ann A r b o r ~ M i c h i g a n 48109. 3) University of Michigan School of D e n t i s t r y ~ Cell B i o l o g y L a b o r a t o r y ~ Ann A r b o r ~ M i c h i g a n 48109. 4) L a f ayette Clirlic~ Division of Pharmacology, 951 E. L a f a y e t t e B l v d . , D e t r o i t ~ M i c h i g a n 48207.
(Received in final form July ii, 1983)
~ummary
P a r a s y m p a t h e t i c d e n e r v a t i o n o f t h e r a t p a r o t i d g l a n d by avulsion of the auriculotemporal nerve c a u s e d a marked and l a s t i n g d e c r e a s e i n g l a n d w e i g h t . Parasympathectomy d i d n o t change t h e l e v e l s o f c h o l i n e i n t h e g l a n d b u t d e c r e a s e d b y 60% t h e l e v e l s o f acetylcholine (ACh) t e n d a y s a f t e r s u r g e r y and 65% a t 28 d a y s . It is puzzling that relatively h i g h l e v e l s o f ACh r e m a i n e d a f t e r parasynlpathetic denervation. The p r e s e n c e o f a d d i t i o n al cholinergic n e r v e s t h a t i n n e r v a t e t h e gland~ or pass t h r o u g h i t en r o u t e t o o t h e r s t r u c t u r e s may a c c o u n t f o r some o f t h e r e m a i n i n g ACh. Also, Schwann c e l l s f r o m d e n e r v a t e d n e r v e s m i g h t h a v e c o n t r i b u t e d t o some o f t h e ACh. ] h e e x i s t e n c e o f an e x t r a n e u r o n a l s o u r c e o f ACh is considered.
[he first s t u d y o f t h e t r o p h i c e f f e c t s o f a u t o n o m i c n e r v e s on t h e salivary g l a n d was made b y B e r n a r d i n 1864 ( 1 ) . He d e s c r i b e d a marked atrophy of the submandibular gland after sectioning the chorda tympany n e r v e . More r e c e n t l y ~ S n e l l and Garrett (2) explored t h e e f f e c t o f p o s t g a n g l i o n i c s y m p a t h e t i c d e n e r v a t i o n on the salivary glands of the rat. Their results suggested that the sympathetic input exerts atrophic influence on the glands.
0024,3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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Acetylcholine in Denervated Parotid
Vol. 33, No. 12, 1983
Subsequent studies (3,4,5) demonstrated that salivary gland s t r u c t u r e can be m o d i f i e d b y d i m i n u t i o n as w e l l a s enhancement o f autonomic activity. It is well established that following parasympathetic denervatioi~, pronounced atrophy of the gland occurs w i t h i n a f e w weeks ( 6 ) . It h a s been assumed t h a t most o f t h e e f f e c t s observed following parasympathetic denervatioll are the result of a l o s s of acetylcholine (ACh), w l i i c h o c c u r s when p a r a s y m p a t h e t i c n e r v e t e r m i n a l s d e g e n e r a t e . On t h i s a s s u m p t i o n , i t w o u l d be o f i n t e r e s t t o have a quantitative measuremellt o f ACh i n t h e d e n e r v a t e d g l a n d . This is particularly important in the light of recent studies that have suggested that the par o t i d gland may r e c e i v e part of its autonomic inner vation by s o u r c e s o t h e r than t h o s e classically d e s c r i b e d ( 7 , 8 ) , as w e l l as b y e v i d e n c e s u g g e s t i n g t h e e x i s t e n c e of extraneuronal pools of neuroeffector substances in this gland <9,10,11,12). T h i s r e p o r t documents t h e q u a n t i t a t i v e changes i n ACh and Ch t h a t occur w i t h p a r a s y m p a t h e t i c d e n e r v a t i o n .
Methods
Twelve, 2 5 - d a y o l d S p r a g u e - D a w l e y r a t s were o b t a i n e d from S p a r t a n Laboratories ( H a s l e t t , Michigan 48840). A t t h i s age t h e p a r o t i d gland reaches a b i o c h e m i c a l l y a d u l t s t a t e <13). The r a t s were housed in a temperature-controlled room w i t h a 12-hour light cycle. The a n i m a l s w e r e g i v e n f o o d ( P u r i n a Rat Chow) and water ad libitum uw~til 17 h o u r s p r i o r to removal of the glands when f o o d was s t o p p e d . A n e s t h e s i a was a c h i e v e d b y a c o m b i n a t i o n o f 25 mg/kg chloral hydrate and 75 mg/kg k e t a m i ne hydrochl oride (Ketalar , Parke-Davis Co., Detroit, Michigan 48207), both g i v e n i n t r a p e r i t o n e a l I y. P o s t g a n g l i or~i c par asympathectomy was performed under anesthesia by a v u l s i o n o f t h e left auriculot e m p o r a l n e r v e a c c o r d i n g t o t h e p r o c e d u r e used b y Mark (14) a f t e r Burgen <15). The t i s s u e s w e r e r e p l a c e d to their anatomical p o s i t i o n s and t h e wou~d c l o s e d w i t h f o u r number 5 - 0 s i l k sutures. The i,tact right p a r o t i d g l a n d s e r v e d as t h e control in all experiments. A t 10 and 28 d a y s a f t e r d e n e r v a t i o n the animals were killed by c e r v i c a l d i s l o c a t i o n . Both p a r o t i d g l a n d s were r a p i d l y removed and t r i m m e d u n d e r a s u r g i c a l m i c r o s c o p e f o l l o w i n g the technique used b y Mark ( 1 4 ) . the g l a n d s were immediately weighed and then homogenized i n i c e c o l d 0 . 4 N p e r c h l o r i c acid using glass homogenizers, rlle samples were t r a n s f e r r e d to a refrigerated c e n t r i f u g e and t r e a t e d f o r 20 m i n u t e s a t 167000 x g . The s u p e r n a t a n t was t r a n s f e r r e d t o 12 ml t u b e s ; t h e precipitate was discarded. 130 p l o f 7 . 5 N p o t a s s i u m a c e t a t e w e r e a d d e d t o each tube to precipitate e>:cess p e r c h l o r a t e i o n and t o b r i n g t h e pH o f each s a m p l e t o pH 4 . 2 - 4 . 4 . A t t h i s p o i n t t h e s a m p l e s were frozen and s t o r e d f o r l a t e r a n a l y s i s . Glands were a l s o removed from unoperated cof]trol a n i m a l s and w e i g h e d t o determine the possibility of genetic right to left weight differences. Acetylcholine (ACh) arid c h o l i n e (Ch) were d e t e r m i n e d u s i n g a modification of the procedure of Zahniser et al. (16). On the d a y o f a s s a y t h e s a m p l e s w e r e thawed~ t h e s u p e r n a t a n t t r a n s f e r r e d
Vol.
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Acetylcholine
in D e n e r v a t e d
Parotid
1193
to clean test tubes, and the pH adjusted to 4.2-4.4. Internal standards for ACh (proprionylcholine) and for Ch (3-hydroxypropyltrimethyl ammonium iodide, also known as methylated preanol) were added (17). lhe quaternary compounds were isolated or, Dowex 50W-8X al~d e l u t e d with 50% 2M NaCI in methanol. The eluate was then evaporated, ai~d t h e r e s i d u e extracted with acetonitrile and esterified with butyryl chloride. The esters were demetbylated wiLh sodium benzenethiolate under rigorous anhydrous co!~ditiolts (18). [lle p r o d u c t s were assayed in a Hewlett-Packard 5730A gas chr o m a t o g r aplk with a niLr ogen detector. Standard curves fo~ A C h alld C h w e r e o b t a i J ~ e d . A C h a n d C h in t h e intact and dener vated glands was calculated from the ratios of ACh to p r o p r i o I i y l c h o l i i ~ e and Ch t o m e t h y l a t e d p r e a n o l . The data were analysed orl a n A p p l e computer with statistical s o l twar e from Unicomp Associates inc. The mean, standard deviation and standard error were calculated for each of the experimer~tal groul~s, rl-le p a i r e d l-statistic was made between control and the contralateral denervated gland for both the tenday and the 28-day groups of animals (31). The P--value was also calculated for these same groups.
Results
GLAND W E I G H T . P a r a s y m p a t h e c t o m y by a v u l s i o n o f the auriculot e m p o r a l n e r v e was f o u n d t o h a v e a m a r k e d e f f e c t on g l a n d w e i g h t q c a u s i n g a 46% ( P < . O 0 0 4 ) d e c r e a s e 10 d a y s a f t e r denervation. This deficit persisted i n g l a n d s a s s a y e d a t 28 d a y s ( T a b l e I ) . The weight of both the control and t h e d e n e r v a t e d g l a n d s increased during Lhis period. Wllile t h e i n c r e a s e in absolute terms was greater ir~ t h e c o n t r o l glands, the rate of g r o w t h was nearly the same in b o t h the normal and the de,,ervated glands. CHOLINE LEVELS. P o s t g a n g I i on i c par-asympathectomy did significantly change t h e c o n t e n t o f Ch i n t h e r a t p a r o t i d c~mpared w i t h t h e c o n t r o l glands (see Discussion).
not when
ACETYLCHOLINE LEVELS. P a r a s y m p a t h e c t o m i z e d g l a n d s showed a l a r g e reduction i n ACh a t b o t h 10 and 28 d a y s a f t e r surgery (Table I). The a v e r a g e ACh l e v e l o f t h e c o n t r o l g l a n d s a t 10 d a y s was 7.02 nmol/g (S.E.+0.39). ACh i n d e n e r v a t e d g l a n d s was 2.50 nmol/g (S.E.+0.65). This represents a decrease of 65%. When ACh was measured 28 days after avulsion of the nerve, the control glands aver aged 4.88 nmol/g (S.E.+0.47) and the parasympathectomized glands 1.58 nmol/g (+0.38). In this case the ACh in the denervated glands was about 69% less than that in the contralateral controls. The d i f f e r e n c e i n ACh levels between the normal and t h e d e n e r v a t e d g l a l l d s a t b o t h t e n and 28 d a y s after surgery were highly significallt with a P-value of less than 0.0002 in both cases.
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Acetylcholine
in Denervated Parotid
Vol. 33, No. 12, 1983
IABLE I
IDAYSN
CONIROL
DENERVATED
G l a n d Wet W e i g h t
I
10
28
5
7
5 7
121(+ii)
53.5
332(+18)
207(+17)
62.3
Levels
266(+19) 209(+17)
(nmol/g) 304(+42) 229<+23)
Acetylcl~oline Levels i0 28
5 7
(mg)
226(+12)
Choline 10 28
D/Cxl00 I
7.02(+0.4) 4.85(+0.5)
117.8 113.6
(nmol/g)
2.50(+0.6) 1.49(+0.3)
35.6 30.7
C o n t r o l and d e n e r v a t e d v a l u e s : Means + S t a n d a r d Error (S.E.>. Significance of data determined by C o r r e l a t e d T - s t a t i s t i c : P0.1 f o r Ch; P
Discussion
of
It i s now w e l l d o c u m e n t e d t h a t p a r a s y m p a t h e t i c d e n e r v a t i o n a salivary gland results in pronounced a t r o p h y w i t h i n a few weeks (3,6,14,19). Our r e s u l t s c o n f i r m t h e i d e a t h a t parasympathetic nerves exert atrophic effect on t h i s g l a n d and quantitatively relates the c h a n g e t o c h o l i n e and a c e t y l c h o l i n e levels in the tissue. MEASUREMENT OF CHOLINE. Because t h e a n i m a l s w e r e s a c r i f i c e d by cervical dislocation r a t h e r t h a n by m i c r o w a v e i r r a d i a t i o n , the l e v e l s o f c h o l i n e we measured p r o b a b l y a r e a r t i f i c i a l l y high. In the rat brain i t has been shown t h a t c h o l i n e levels increase rapidly following death (20). Guidotti et. al. < 2 0 ) showed t h a t microwave irradiation of tissue produces a rapid precise in situ inactivation of cholinergic enzymes (21). In the present experiments microwave fi×ation was n o t used because of the difficulty in achieving even i r r a d i a t i o n of the two glands. Also, once i r r a d i a t e d , t h e g l a n d s were d i f f i c u l t to distinguish from tissue.
the surrounding
POSSIBLE SOURCES OF ACh IN DENERVATED GLANDS. this report represents the first quantitative
To o u r knowledge documentation of
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33, No.
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A c e t y l c h o l i n e in D e n e r v a t e d P a r o t i d
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the effect of auriculotemporal n e u r o t o m y on t h e c o n c e n t r a t i o n of ACII i n tlJe r a t par o t i d glal~d. Sectioning of t h e a u r i c u l o t e m p o r a l nerve caused ACh corlcelltr a t i o n Lo decrease to 55% of the contralateral gland after I0 days, and remained essentially unchanged at 28 days. lhe large amount of residual ACh indicates that tlme ~Jeur o n s of tlme aur i c u l o t e m p o r a l nerve are not the exclusive source o~ ACb in the par otid gland. Several possible sour ces o f t h e "~ e s i d u a l " A C h m a y b e c o n s i d e r e d in the light of c e r t a i ~ e::per i m e n t s . FOSSIBILI[Y OF A CHEMICAL ARTIFACT. Perclllorlc acid is a known catalyst for esterification. Hence t h e p r e s e n c e o f a c e t a t e i o n , perclhloric a c i d al,d e n d o g e n o u s c h o l i n e from the tissue a t pH 4 . 2 could result in a non-enzymatic svnthesis o f ACh. This possibility was t e s t e d by an i n v i t r o e,xperiment using similar amounts of each o f t h e r e a g e l l t s i n c l u d i n g added c h o l i n e (200 n m o l ) . ACh was observed as a result of ~his mixture in which the time sequences were similar to those for a tissue assay. However, the maximal amount was i_~.283% (r).56 i,nol) o f A C h f o r 2 0 0 nmol of choline. Although this is a clearly detectable amourlt of ACh, the mean l e v e l o f ACh i l l t h e d e J ~ e r v a t e d g l a n d s v a r i e d f r o m 3.47 to 2.14 nmol/g of tissue. HeiDce, uI1der t h e w o r s t c i r c u m s t a n c e s , a total o f 2 . 1 4 r a i l , u s JI . 5 6 o r 1 . 5 8 n m o l / g m u s t h a v e come f r o m t h e glands, l h e d a t a i n T a b l e i has been c o r r e c t e d for this none n z y m a t i c s o u r c e o f ACh. P O S S I B I L I [ Y OF OTHER NEURONAL SOURCES. S e v e r a l l i n e s of evidence argue that at least part of the residual ACh i n t h e denervated glands is d u e t o tl~e p r e s e n c e o f p a r a s y m p a t h e t i c nerves other tharl the auriculotemporal. This contention was a d d r e s s e d d u r i n g several careful d i s s e c t i o J ~ s o f t h e g l a n d (14>. The f a c i a l nerve was seeal t o come i n t o c l o s e a p p o s i t i o n t o t h e g l a n d as i t emerged from behind the ear. Several small branches were traced from the main t r u n k and p e n e t r a t e d the gland capsule. Their exact terminations~ however~ w e r e n o t o b s e r v e d . Thus, w h e t h e r t h i s nerve makes any functioilal connections with the gland is not known. However, a c c o r d i n g t o A l m and E k s t r o m ( 7 ) i t p r o b a b l y does not. [hey failed to s e e a n y c l l a n g e i n t h e number of AChE-positive nerves in the parotid g l a n d when t h e y a v u l s e d t h i s nerve along with the auriculotemporal. In our dissection some cervical nerves w e r e a l s o seelJ t o p a s s t h r o u g h t h e s u p e r f i c i a l stroma of the gland. But, again, fibers c o u l d n o t be f o l l o w e d to their termination. To o u r k n o w l e d g e , n o b o d y h a s t e s t e d t h e p o s s i b i l i t y that braiiches from these nerves might innervate the gland. Since it is impossible to dissect out nerves lying within the capsule of the gland at tile time the gland is excised, it is probable that a portion o f t h e ACh m e a s u r e d i n tlme a s s a y i s t h a t present within axons of the facial . e r v e and c e r v i c a l nerves-whether or not they are destined for the gland in a functional sense. The r e s u l t s of certain o t h e r e x p e r i m e n t s h a v e been i n t e r p r e t e d to indicate that nerves other than those classically described innervate the rat parotid gland. E k s t r o m (8) c o n c l u d e d t h a t persistence o f ChAT (3%) i n t h e p a r o t i d gland following avulsion of the auriculotemporal nerve indicated the presence of intact parasympathetic nerves. Alto and E k s t r o m ( 7 ) w e r e l e d t o the same c o n c l u s i o n when r e t r o g r a d e injection of eserine into the salivary duct led to salivary secretion and t h e h i s t o c h e m i c a l presence of AChE activity. Time s e c r e t i o , was l i k e l y : h o w e v e r , t o be highly
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Acetylcholine in Denervated Parotid
exagerated due t o t h e e x i s t e n c e in the secretory ceils.
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Vol. 33, No, 12, 1983
supersensitivity
IS THERE AN EXTRANEURONAL POOL OF ACh? Our f i n d i n g o f a large residual p o o l o f ACh i n c h r o n i c a l l y parasympathectomized glands suggests the possibility o f an e x t r a n e u r o n a l p o o l o f ACh i n t h i s gland. Support of this contention, however, is indirect. In t h e frog sartorius m u s c l e , f o r e x a m p l e , ACh l e v e l s f a l l t o a minimum in e i g h t d a y s , b u t t h e m u s c l e ACh l e v e l n e v e r f a l l s b e l o w 25% o f t h e c o n t r o l v a l u e . S i m i l a r f i n d i n g s h a v e been r e p o r t e d i n c e r t a i n mammalian muscle preparations (10,22). While it is generally believed t h a t AChE o r i g i n a t e s primarily within the neuronal cell body (23), it is likely t h a t t h e r e i s some contribution from other cell types within the gland. At the present time, there is little direct evidence to indicate the location of this presumptive extraneuronal pool. Several l i n e s o f e v i d e n c e s u g g e s t t h a t t h e Schwann c e l l c o u l d be one such site (9,10~24,25,26). Even i f t h i s w e r e t h e c a s e , i t w o u l d s t i l l need to b e d e t e r m i n e d w h e t h e r t h e h i g h c o n c e n t r a t i o n o f ACh in the d e n e r v a t e d g l a n d i s c o m p a r a b l e t o t h a t seen i n the intact gland or is an adaptive response to denervation. Further investigations must be p e r f o r m e d b e f o r e t h e s e q u e s t i o n s can be answered. An e x t r a n e u r o n a l p o o l o f nor e p i n e p h r i n e h a s been o b s e r v e d i n t h e salivary g l a n d (27,28,29,30). The p r e s e n c e o f a s i m i l a r e×traneuronal p o o l o f ACh, e s p e c i a l l y in the presence of the significant l e v e l s o f c h o l i n e i n t h e g l a n d , i s an a t t r a c t i v e hypothesis to pursue.
References
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13. 14.
C. BARNARD, 3 . A n a t . ( P a r i s ) 1 5 0 7 - 5 1 3 ( 1 8 6 4 ) . R.S. SNELL and J . R . GARRETT, Z e i t . f u r Z e l l f o r s c h 48 6 3 9 - 6 5 2 (1958). L . H . SCHNEYER and C. SCHNEYER, Ann. N . Y . A c a d . S c i . 85 189200 ( 1 9 6 0 ) . C . A . SCHNEYER, Am. 3 . P h y s i o l . 203 2 3 2 - 2 3 6 ( 1 9 6 2 ) . C . A . SCHNEYER and H.D. HALL, Am. 3 . P h y s i o l . 211 943 ( 1 9 6 6 ) . C.A. SCHNEYER and H.D. HALL, Am. 3 . P h y s i o l . 207 308 ( 1 9 6 4 ) . P. ALM and 3 . EKSTROM, A r c h . O r a l B i o l . 21 417-421 ( 1 9 7 6 ) . J . EKSTROM, Q. J . E x p . P h y s i o l . 59 1 9 1 - 1 9 9 ( 1 9 7 4 ) . M.J. D E N N I S and R. MELEDI, J . P h y s i o l . ( L o n d . ) 237 431-452 (1974). C . O . HEBB, J . P h y s i o l . (Lond.) 163 294-306 (1962). S. TUCEK, E × p l . N e u r o l . 40 2 3 - 3 5 (1973) 0. ALMGREN and J . JONASON, The Mechanism o f Neuronal and E×traneuronal Transport o f C a t e c h o l a m i n e s , D.M. Paton ed. p p . 2 9 9 - 3 1 2 , Raven P r e s s , New Y o r k ( 1 9 7 6 ) . R.S. REDMAN and L.M. SREEBNY, D e v e l o p . Biol. 25 248-279 (1971). M.MARK, R o l e o f t h e A u t o n o m i c N e u r o n s System i n R e g u l a t i o n o f the Structure and Function of the Rat Parotid Gland~
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15.
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24. 25. 26. 27. 28. 29. 30. 31.
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U n i v e r s i t y M i c r o f i l m s , Ann A r b o r ( 1 9 8 0 ) . A.S.V. BURGEN, S a l i v a r y G l a n d s and T h e i r Secretions~ L.J. G r e e b n y and J . Meyer e d s . p p . 3 0 3 - 3 0 7 , Pergamon P r e s s , New York (1964). N.R. ZAHNISER, D. CHOU and I . HAHIN, J . P h a r m a c o l . Exp. T h e r . 200 5 4 5 - 5 5 9 ( 1 9 7 7 ) . D.N. BUCHANAN, F . R . FUCEK and E . F . DOMINO, J. C h r o m a t o g . 181 329-335 (1979). D . J . JENDEN and I . HANIN, Handbook o f C h e m i c a l Assay M e t h o d s , I . H a n i n e d . , p p . 1 3 5 - 1 5 0 , Raven P r e s s , New Y o r k ( 1 9 7 4 ) . C.R. ARNETT and J . N . DAVIS, J . P h a r m a c o l . Exp. T h e r . 211 3 9 4 400 ( 1 9 7 0 ) . A. GUIDOTTI, D . L . CHENEY, M. TRABUCCHI, M. DOTEUTCHI, C. WANG and R.A. HAWKINS, N e u r o p h a r m a c o l o g y 13 1115-1122 ( 1 9 7 4 ) . W.B. STAVINOHA, 3. FRAZER and A.T.MODAK, Cholinergic Mechanisms and P s y c h o p h a r m a c o l o g y , D . J . Jenden e d . , p p . 169179, Plenum P r e s s , New Y o r k ( 1 9 7 7 ) . J.EKSTROM, Q. 3 . E x p . P h y s i o l . 59 1 9 1 - 1 9 9 ( 1 9 7 4 ) . S.P. BHATNAGAR and F . C . MACINTOSH, P r o c . Canad. Fed. Biol. Soc. 3 1 2 - 1 3 ( 1 9 & 0 ) . J . R . COOPER, F . E . BLOOM and R.H. ROTH, The B i o c h e m i c a l B a s i s o~ Neuropharmacology (Fourth Edition) Oxford ( N e w York) (1982). B . I . BOGART, Am. J . A n a t . 132 2 5 9 - 2 6 6 ( 1 9 7 1 ) . S.TUCEK, J. ZELENA, I . GE and F. VYSKOCIL, Neuroscience 3 707-704 (1978). R. BIRKS and F . C . MACINTOSH, Canad. J . B i o c h e m . P h y s i o l . 39 787-827 (1961). B . C . R . STROMBLAD, E x p e r i e n t i a ( B a s e l ) 16 4 1 7 - 4 1 8 ( 1 9 6 0 ) . B.C.R. STROMBLAD, J . P h a r m a c o l . Exp. Ther. 134 154-159 (1961). U.S. yon EULER and G. RYD, A c t a P h y s i o l . S t a n d . 59 62-66 (1963). U.S. von EULER and A. PURKHOLD, A c t a P h y s i o l . S t a n d . 24 212217 ( 1 9 5 1 ) . R.C. DUNCAN, R.G. KNAPP AND M. C. MILLER I I I , Introductory Biostatistics f o r t h e H e a l t h S c i e n c e s , p p . 7 1 - 8 3 , John Wiley and Sons, New Y o r k ( 1 9 7 7 ) .
Pergamon Press
EFFECt OF PARASYMPAfHETIC DENERVATION ON ACETYLCHOLINE LEVELS IN THE RAT PAROTID GLAND. IS |HERE AN EXIRANEURONAL POOL OF ACETYLCHOLINE?
M a r v i n R. Markl , Edward F. Domino=,4~ Seong S. Hart=, A u r e l i o Or t i z 4 ~ B e n j a m i n N. Nathews 4, and S a n d r a K. T a i t 4
I) M e d i c a l C o l l e g e of G e o r g i a School of D e n t i s t r y ~ Depar tment o f Oral B i o l o g y - A n a t o m y ~ Augusta~ Georgia 30912. 2) U n i v e r s i t y o f M i c h i g a n School o f Medicine~ D e p a r t m e n t o f P h a r m a c o l o g y , Ann A r b o r ~ M i c h i g a n 48109. 3) University of Michigan School of D e n t i s t r y ~ Cell B i o l o g y L a b o r a t o r y ~ Ann A r b o r ~ M i c h i g a n 48109. 4) L a f ayette Clirlic~ Division of Pharmacology, 951 E. L a f a y e t t e B l v d . , D e t r o i t ~ M i c h i g a n 48207.
(Received in final form July ii, 1983)
~ummary
P a r a s y m p a t h e t i c d e n e r v a t i o n o f t h e r a t p a r o t i d g l a n d by avulsion of the auriculotemporal nerve c a u s e d a marked and l a s t i n g d e c r e a s e i n g l a n d w e i g h t . Parasympathectomy d i d n o t change t h e l e v e l s o f c h o l i n e i n t h e g l a n d b u t d e c r e a s e d b y 60% t h e l e v e l s o f acetylcholine (ACh) t e n d a y s a f t e r s u r g e r y and 65% a t 28 d a y s . It is puzzling that relatively h i g h l e v e l s o f ACh r e m a i n e d a f t e r parasynlpathetic denervation. The p r e s e n c e o f a d d i t i o n al cholinergic n e r v e s t h a t i n n e r v a t e t h e gland~ or pass t h r o u g h i t en r o u t e t o o t h e r s t r u c t u r e s may a c c o u n t f o r some o f t h e r e m a i n i n g ACh. Also, Schwann c e l l s f r o m d e n e r v a t e d n e r v e s m i g h t h a v e c o n t r i b u t e d t o some o f t h e ACh. ] h e e x i s t e n c e o f an e x t r a n e u r o n a l s o u r c e o f ACh is considered.
[he first s t u d y o f t h e t r o p h i c e f f e c t s o f a u t o n o m i c n e r v e s on t h e salivary g l a n d was made b y B e r n a r d i n 1864 ( 1 ) . He d e s c r i b e d a marked atrophy of the submandibular gland after sectioning the chorda tympany n e r v e . More r e c e n t l y ~ S n e l l and Garrett (2) explored t h e e f f e c t o f p o s t g a n g l i o n i c s y m p a t h e t i c d e n e r v a t i o n on the salivary glands of the rat. Their results suggested that the sympathetic input exerts atrophic influence on the glands.
0024,3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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Subsequent studies (3,4,5) demonstrated that salivary gland s t r u c t u r e can be m o d i f i e d b y d i m i n u t i o n as w e l l a s enhancement o f autonomic activity. It is well established that following parasympathetic denervatioi~, pronounced atrophy of the gland occurs w i t h i n a f e w weeks ( 6 ) . It h a s been assumed t h a t most o f t h e e f f e c t s observed following parasympathetic denervatioll are the result of a l o s s of acetylcholine (ACh), w l i i c h o c c u r s when p a r a s y m p a t h e t i c n e r v e t e r m i n a l s d e g e n e r a t e . On t h i s a s s u m p t i o n , i t w o u l d be o f i n t e r e s t t o have a quantitative measuremellt o f ACh i n t h e d e n e r v a t e d g l a n d . This is particularly important in the light of recent studies that have suggested that the par o t i d gland may r e c e i v e part of its autonomic inner vation by s o u r c e s o t h e r than t h o s e classically d e s c r i b e d ( 7 , 8 ) , as w e l l as b y e v i d e n c e s u g g e s t i n g t h e e x i s t e n c e of extraneuronal pools of neuroeffector substances in this gland <9,10,11,12). T h i s r e p o r t documents t h e q u a n t i t a t i v e changes i n ACh and Ch t h a t occur w i t h p a r a s y m p a t h e t i c d e n e r v a t i o n .
Methods
Twelve, 2 5 - d a y o l d S p r a g u e - D a w l e y r a t s were o b t a i n e d from S p a r t a n Laboratories ( H a s l e t t , Michigan 48840). A t t h i s age t h e p a r o t i d gland reaches a b i o c h e m i c a l l y a d u l t s t a t e <13). The r a t s were housed in a temperature-controlled room w i t h a 12-hour light cycle. The a n i m a l s w e r e g i v e n f o o d ( P u r i n a Rat Chow) and water ad libitum uw~til 17 h o u r s p r i o r to removal of the glands when f o o d was s t o p p e d . A n e s t h e s i a was a c h i e v e d b y a c o m b i n a t i o n o f 25 mg/kg chloral hydrate and 75 mg/kg k e t a m i ne hydrochl oride (Ketalar , Parke-Davis Co., Detroit, Michigan 48207), both g i v e n i n t r a p e r i t o n e a l I y. P o s t g a n g l i or~i c par asympathectomy was performed under anesthesia by a v u l s i o n o f t h e left auriculot e m p o r a l n e r v e a c c o r d i n g t o t h e p r o c e d u r e used b y Mark (14) a f t e r Burgen <15). The t i s s u e s w e r e r e p l a c e d to their anatomical p o s i t i o n s and t h e wou~d c l o s e d w i t h f o u r number 5 - 0 s i l k sutures. The i,tact right p a r o t i d g l a n d s e r v e d as t h e control in all experiments. A t 10 and 28 d a y s a f t e r d e n e r v a t i o n the animals were killed by c e r v i c a l d i s l o c a t i o n . Both p a r o t i d g l a n d s were r a p i d l y removed and t r i m m e d u n d e r a s u r g i c a l m i c r o s c o p e f o l l o w i n g the technique used b y Mark ( 1 4 ) . the g l a n d s were immediately weighed and then homogenized i n i c e c o l d 0 . 4 N p e r c h l o r i c acid using glass homogenizers, rlle samples were t r a n s f e r r e d to a refrigerated c e n t r i f u g e and t r e a t e d f o r 20 m i n u t e s a t 167000 x g . The s u p e r n a t a n t was t r a n s f e r r e d t o 12 ml t u b e s ; t h e precipitate was discarded. 130 p l o f 7 . 5 N p o t a s s i u m a c e t a t e w e r e a d d e d t o each tube to precipitate e>:cess p e r c h l o r a t e i o n and t o b r i n g t h e pH o f each s a m p l e t o pH 4 . 2 - 4 . 4 . A t t h i s p o i n t t h e s a m p l e s were frozen and s t o r e d f o r l a t e r a n a l y s i s . Glands were a l s o removed from unoperated cof]trol a n i m a l s and w e i g h e d t o determine the possibility of genetic right to left weight differences. Acetylcholine (ACh) arid c h o l i n e (Ch) were d e t e r m i n e d u s i n g a modification of the procedure of Zahniser et al. (16). On the d a y o f a s s a y t h e s a m p l e s w e r e thawed~ t h e s u p e r n a t a n t t r a n s f e r r e d
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Acetylcholine
in D e n e r v a t e d
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to clean test tubes, and the pH adjusted to 4.2-4.4. Internal standards for ACh (proprionylcholine) and for Ch (3-hydroxypropyltrimethyl ammonium iodide, also known as methylated preanol) were added (17). lhe quaternary compounds were isolated or, Dowex 50W-8X al~d e l u t e d with 50% 2M NaCI in methanol. The eluate was then evaporated, ai~d t h e r e s i d u e extracted with acetonitrile and esterified with butyryl chloride. The esters were demetbylated wiLh sodium benzenethiolate under rigorous anhydrous co!~ditiolts (18). [lle p r o d u c t s were assayed in a Hewlett-Packard 5730A gas chr o m a t o g r aplk with a niLr ogen detector. Standard curves fo~ A C h alld C h w e r e o b t a i J ~ e d . A C h a n d C h in t h e intact and dener vated glands was calculated from the ratios of ACh to p r o p r i o I i y l c h o l i i ~ e and Ch t o m e t h y l a t e d p r e a n o l . The data were analysed orl a n A p p l e computer with statistical s o l twar e from Unicomp Associates inc. The mean, standard deviation and standard error were calculated for each of the experimer~tal groul~s, rl-le p a i r e d l-statistic was made between control and the contralateral denervated gland for both the tenday and the 28-day groups of animals (31). The P--value was also calculated for these same groups.
Results
GLAND W E I G H T . P a r a s y m p a t h e c t o m y by a v u l s i o n o f the auriculot e m p o r a l n e r v e was f o u n d t o h a v e a m a r k e d e f f e c t on g l a n d w e i g h t q c a u s i n g a 46% ( P < . O 0 0 4 ) d e c r e a s e 10 d a y s a f t e r denervation. This deficit persisted i n g l a n d s a s s a y e d a t 28 d a y s ( T a b l e I ) . The weight of both the control and t h e d e n e r v a t e d g l a n d s increased during Lhis period. Wllile t h e i n c r e a s e in absolute terms was greater ir~ t h e c o n t r o l glands, the rate of g r o w t h was nearly the same in b o t h the normal and the de,,ervated glands. CHOLINE LEVELS. P o s t g a n g I i on i c par-asympathectomy did significantly change t h e c o n t e n t o f Ch i n t h e r a t p a r o t i d c~mpared w i t h t h e c o n t r o l glands (see Discussion).
not when
ACETYLCHOLINE LEVELS. P a r a s y m p a t h e c t o m i z e d g l a n d s showed a l a r g e reduction i n ACh a t b o t h 10 and 28 d a y s a f t e r surgery (Table I). The a v e r a g e ACh l e v e l o f t h e c o n t r o l g l a n d s a t 10 d a y s was 7.02 nmol/g (S.E.+0.39). ACh i n d e n e r v a t e d g l a n d s was 2.50 nmol/g (S.E.+0.65). This represents a decrease of 65%. When ACh was measured 28 days after avulsion of the nerve, the control glands aver aged 4.88 nmol/g (S.E.+0.47) and the parasympathectomized glands 1.58 nmol/g (+0.38). In this case the ACh in the denervated glands was about 69% less than that in the contralateral controls. The d i f f e r e n c e i n ACh levels between the normal and t h e d e n e r v a t e d g l a l l d s a t b o t h t e n and 28 d a y s after surgery were highly significallt with a P-value of less than 0.0002 in both cases.
1194
Acetylcholine
in Denervated Parotid
Vol. 33, No. 12, 1983
IABLE I
IDAYSN
CONIROL
DENERVATED
G l a n d Wet W e i g h t
I
10
28
5
7
5 7
121(+ii)
53.5
332(+18)
207(+17)
62.3
Levels
266(+19) 209(+17)
(nmol/g) 304(+42) 229<+23)
Acetylcl~oline Levels i0 28
5 7
(mg)
226(+12)
Choline 10 28
D/Cxl00 I
7.02(+0.4) 4.85(+0.5)
117.8 113.6
(nmol/g)
2.50(+0.6) 1.49(+0.3)
35.6 30.7
C o n t r o l and d e n e r v a t e d v a l u e s : Means + S t a n d a r d Error (S.E.>. Significance of data determined by C o r r e l a t e d T - s t a t i s t i c : P
Discussion
of
It i s now w e l l d o c u m e n t e d t h a t p a r a s y m p a t h e t i c d e n e r v a t i o n a salivary gland results in pronounced a t r o p h y w i t h i n a few weeks (3,6,14,19). Our r e s u l t s c o n f i r m t h e i d e a t h a t parasympathetic nerves exert atrophic effect on t h i s g l a n d and quantitatively relates the c h a n g e t o c h o l i n e and a c e t y l c h o l i n e levels in the tissue. MEASUREMENT OF CHOLINE. Because t h e a n i m a l s w e r e s a c r i f i c e d by cervical dislocation r a t h e r t h a n by m i c r o w a v e i r r a d i a t i o n , the l e v e l s o f c h o l i n e we measured p r o b a b l y a r e a r t i f i c i a l l y high. In the rat brain i t has been shown t h a t c h o l i n e levels increase rapidly following death (20). Guidotti et. al. < 2 0 ) showed t h a t microwave irradiation of tissue produces a rapid precise in situ inactivation of cholinergic enzymes (21). In the present experiments microwave fi×ation was n o t used because of the difficulty in achieving even i r r a d i a t i o n of the two glands. Also, once i r r a d i a t e d , t h e g l a n d s were d i f f i c u l t to distinguish from tissue.
the surrounding
POSSIBLE SOURCES OF ACh IN DENERVATED GLANDS. this report represents the first quantitative
To o u r knowledge documentation of
Vol.
33, No.
12, 1983
A c e t y l c h o l i n e in D e n e r v a t e d P a r o t i d
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the effect of auriculotemporal n e u r o t o m y on t h e c o n c e n t r a t i o n of ACII i n tlJe r a t par o t i d glal~d. Sectioning of t h e a u r i c u l o t e m p o r a l nerve caused ACh corlcelltr a t i o n Lo decrease to 55% of the contralateral gland after I0 days, and remained essentially unchanged at 28 days. lhe large amount of residual ACh indicates that tlme ~Jeur o n s of tlme aur i c u l o t e m p o r a l nerve are not the exclusive source o~ ACb in the par otid gland. Several possible sour ces o f t h e "~ e s i d u a l " A C h m a y b e c o n s i d e r e d in the light of c e r t a i ~ e::per i m e n t s . FOSSIBILI[Y OF A CHEMICAL ARTIFACT. Perclllorlc acid is a known catalyst for esterification. Hence t h e p r e s e n c e o f a c e t a t e i o n , perclhloric a c i d al,d e n d o g e n o u s c h o l i n e from the tissue a t pH 4 . 2 could result in a non-enzymatic svnthesis o f ACh. This possibility was t e s t e d by an i n v i t r o e,xperiment using similar amounts of each o f t h e r e a g e l l t s i n c l u d i n g added c h o l i n e (200 n m o l ) . ACh was observed as a result of ~his mixture in which the time sequences were similar to those for a tissue assay. However, the maximal amount was i_~.283% (r).56 i,nol) o f A C h f o r 2 0 0 nmol of choline. Although this is a clearly detectable amourlt of ACh, the mean l e v e l o f ACh i l l t h e d e J ~ e r v a t e d g l a n d s v a r i e d f r o m 3.47 to 2.14 nmol/g of tissue. HeiDce, uI1der t h e w o r s t c i r c u m s t a n c e s , a total o f 2 . 1 4 r a i l , u s JI . 5 6 o r 1 . 5 8 n m o l / g m u s t h a v e come f r o m t h e glands, l h e d a t a i n T a b l e i has been c o r r e c t e d for this none n z y m a t i c s o u r c e o f ACh. P O S S I B I L I [ Y OF OTHER NEURONAL SOURCES. S e v e r a l l i n e s of evidence argue that at least part of the residual ACh i n t h e denervated glands is d u e t o tl~e p r e s e n c e o f p a r a s y m p a t h e t i c nerves other tharl the auriculotemporal. This contention was a d d r e s s e d d u r i n g several careful d i s s e c t i o J ~ s o f t h e g l a n d (14>. The f a c i a l nerve was seeal t o come i n t o c l o s e a p p o s i t i o n t o t h e g l a n d as i t emerged from behind the ear. Several small branches were traced from the main t r u n k and p e n e t r a t e d the gland capsule. Their exact terminations~ however~ w e r e n o t o b s e r v e d . Thus, w h e t h e r t h i s nerve makes any functioilal connections with the gland is not known. However, a c c o r d i n g t o A l m and E k s t r o m ( 7 ) i t p r o b a b l y does not. [hey failed to s e e a n y c l l a n g e i n t h e number of AChE-positive nerves in the parotid g l a n d when t h e y a v u l s e d t h i s nerve along with the auriculotemporal. In our dissection some cervical nerves w e r e a l s o seelJ t o p a s s t h r o u g h t h e s u p e r f i c i a l stroma of the gland. But, again, fibers c o u l d n o t be f o l l o w e d to their termination. To o u r k n o w l e d g e , n o b o d y h a s t e s t e d t h e p o s s i b i l i t y that braiiches from these nerves might innervate the gland. Since it is impossible to dissect out nerves lying within the capsule of the gland at tile time the gland is excised, it is probable that a portion o f t h e ACh m e a s u r e d i n tlme a s s a y i s t h a t present within axons of the facial . e r v e and c e r v i c a l nerves-whether or not they are destined for the gland in a functional sense. The r e s u l t s of certain o t h e r e x p e r i m e n t s h a v e been i n t e r p r e t e d to indicate that nerves other than those classically described innervate the rat parotid gland. E k s t r o m (8) c o n c l u d e d t h a t persistence o f ChAT (3%) i n t h e p a r o t i d gland following avulsion of the auriculotemporal nerve indicated the presence of intact parasympathetic nerves. Alto and E k s t r o m ( 7 ) w e r e l e d t o the same c o n c l u s i o n when r e t r o g r a d e injection of eserine into the salivary duct led to salivary secretion and t h e h i s t o c h e m i c a l presence of AChE activity. Time s e c r e t i o , was l i k e l y : h o w e v e r , t o be highly
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Acetylcholine in Denervated Parotid
exagerated due t o t h e e x i s t e n c e in the secretory ceils.
of
denervation
Vol. 33, No, 12, 1983
supersensitivity
IS THERE AN EXTRANEURONAL POOL OF ACh? Our f i n d i n g o f a large residual p o o l o f ACh i n c h r o n i c a l l y parasympathectomized glands suggests the possibility o f an e x t r a n e u r o n a l p o o l o f ACh i n t h i s gland. Support of this contention, however, is indirect. In t h e frog sartorius m u s c l e , f o r e x a m p l e , ACh l e v e l s f a l l t o a minimum in e i g h t d a y s , b u t t h e m u s c l e ACh l e v e l n e v e r f a l l s b e l o w 25% o f t h e c o n t r o l v a l u e . S i m i l a r f i n d i n g s h a v e been r e p o r t e d i n c e r t a i n mammalian muscle preparations (10,22). While it is generally believed t h a t AChE o r i g i n a t e s primarily within the neuronal cell body (23), it is likely t h a t t h e r e i s some contribution from other cell types within the gland. At the present time, there is little direct evidence to indicate the location of this presumptive extraneuronal pool. Several l i n e s o f e v i d e n c e s u g g e s t t h a t t h e Schwann c e l l c o u l d be one such site (9,10~24,25,26). Even i f t h i s w e r e t h e c a s e , i t w o u l d s t i l l need to b e d e t e r m i n e d w h e t h e r t h e h i g h c o n c e n t r a t i o n o f ACh in the d e n e r v a t e d g l a n d i s c o m p a r a b l e t o t h a t seen i n the intact gland or is an adaptive response to denervation. Further investigations must be p e r f o r m e d b e f o r e t h e s e q u e s t i o n s can be answered. An e x t r a n e u r o n a l p o o l o f nor e p i n e p h r i n e h a s been o b s e r v e d i n t h e salivary g l a n d (27,28,29,30). The p r e s e n c e o f a s i m i l a r e×traneuronal p o o l o f ACh, e s p e c i a l l y in the presence of the significant l e v e l s o f c h o l i n e i n t h e g l a n d , i s an a t t r a c t i v e hypothesis to pursue.
References
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