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The relationship between the antidiuretic activity of rat eye plexus blood and passive avoidance behaviour
Physiology and Behavior, Vol. 11, pp. 377-380. Brain Research Publications Inc., 1973. Printed in the U.S.A.
The Relationship Between the Antidiuretic Activity of Rat Eye Plexus Blood and Passive Avoidance Behaviour E L I Z A B E T H A. T H O M P S O N 1
The Medical Research Council Unit for Metabolic" Studies in Psychiatry, University Department of Psychiatry, Middlewood Hospital, Sheffield, $6 1 TP, England AND D A V I D DE W I E D
The Rudolf Magnus Institute o f Pharmacology, University of Utrecht, Medical Faculty, Utrecht, The Netherlands
(Received 5 F e b r u a r y 1 9 7 3 ) THOMPSON, E. A. AND D. DE WlED. The relationship between the antidiuretic activity of rat eye plexus blood and passive avoidance behaviour. PHYSIOL. BEHAV. 11(3) 3 7 7 - 3 8 0 , 1973.-Antidiuretic activity (AD-activity) of eye plexus blood in rats increases during a 24 hr postshock retention trial of a passive step-through avoidance response. The rate of increase in AD-activity was related to the intensity of the shock to which the animals had been exposed previously. If avoidance latency was increased by the administration of ACTH4_ ~o or desglycinamide lysine vasopressin (DG-LVP) 1 hr prior to the 24 hr postshock retention trial, AD-activity rose concomitantly. The influence of A C T H , _ 1o on avoidance latency which generally has disappeared 24 hr after a single injection in contrast to a single injection of DG-LVP which affects avoidance latency for several days, was reflected in a time dependent rise in AD-activity of eye plexus blood. Passive avoidance behaviour Antidiuretic activity Desglycinamide lysine vasopressin
Eye plexus blood
T H E P O S T E R I O R p i t u i t a r y and vasopressin p r o f o u n d l y a f f e c t a c q u i s i t i o n a n d m a i n t e n a n c e o f aversively s t i m u l a t e d active a n d passive a v o i d a n c e b e h a v i o u r . T h e r e m o v a l o f t h e p o s t e r i o r lobe of t h e p i t u i t a r y i n t e r f e r e s w i t h t h e m a i n t e n a n c e of a s h u t t l e b o x a v o i d a n c e r e s p o n s e [ 5 ] , w h i c h is r e s t o r e d b y vasopressin. In i n t a c t rats, vasopressin facilit a t e d t h e p r e s e r v a t i o n o f s u c h learned r e s p o n s e s for a c o n s i d e r a b l e p e r i o d of t i m e [1, 6, 7 ] . T h e c o n s o l i d a t i o n o f an a v o i d a n c e r e s p o n s e is facilitated o n l y if it is a c q u i r e d u n d e r o p t i m a l vasopressin i n f l u e n c e . T h e i n f l u e n c e o f t h e p e p t i d e is also s i t u a t i o n specific [ 3 ] . This e f f e c t of vasopressin m a y b e related to t h e relative ease w i t h w h i c h a given b e h a v i o u r a l r e s p o n s e is elicited w h e n s i t u a t i o n specific cues r e o c c u r . If this is true, it m i g h t b e t h a t an a s s o c i a t i o n exists b e t w e e n e n d o g e n o u s release of vasopressin or related p e p t i d e s a n d specific e n v i r o n m e n t a l cues. In fact, n u m e r o u s s t i m u l i o t h e r t h a n t h o s e c o n c e r n e d w i t h w a t e r h o m e o s t a s i s i n d u c e t h e release o f vasopressin f r o m t h e p o s t e r i o r lobe of t h e p i t u i t a r y [ 1 2 ] . The p r e s e n t e x p e r i m e n t s were t h e r e f o r e designed to
ACTH,_I o
d e t e r m i n e t h e release o f vasopressin d u r i n g aversively m o t i v a t e d passive a v o i d a n c e b e h a v i o u r . In a d d i t i o n , t h e i n f l u e n c e o n vasopressin-release was studied in rats in w h i c h passive a v o i d a n c e l a t e n c y was m o d i f i e d b y p r i o r a d m i n i s t r a t i o n o f a vasopressin related p e p t i d e d e s g l y c i n a m i d e lysine vasopressin ( D G - L V P ) [ 8 ] , and a p e p t i d e related to A C T H (ACTH4_1o) [10]. METHOD Male rats o f an i n b r e d Wistar strain weighing 1 1 0 - 1 2 0 g were used. These were m a i n t a i n e d 5 p e r cage w i t h ad lib f o o d a n d w a t e r a n d t h e lights o n b e t w e e n 5 a.m. a n d 7 p.m. All o b s e r v a t i o n s were m a d e b e t w e e n 9 : 3 0 a.m. a n d 1 2 : 3 0 p.m. The s t e p - t h r o u g h passive a v o i d a n c e p r o c e d u r e a d o p t e d was t h a t of Ader, Weijen and M o l e m a n [ 2 ] . T h e a p p a r a t u s consisted of a 4 0 x 4 0 x 4 0 cm Lucite c h a m b e r w i t h b l a c k wails a n d grid floor. A wire mesh covered elevated r u n w a y , 6 cm wide a n d 25 cm long, e x t e n d e d f r o m o n e side of t h e
J This research was conducted almost completely in Utrecht. E.A.T. was supported by a European Brain and Behaviour Research Grant and by the M.R.C., London. 377
378
T H O M P S O N AND de WIED
c h a m b e r on a level w i t h the grid floor. The a n i m a l was able to e n t e r the s h o c k c h a m b e r f r o m the r u n w a y t h r o u g h a guillotine o p e r a t e d d o o r 6 cm x 6 cm. The s h o c k c h a m b e r r e m a i n e d dark while t h e elevated r u n w a y was i l l u m i n a t e d by a 25 W lamp fixed 40 cm above the r u n w a y . On the first day of a d a p t a t i o n training, the rat was placed inside t h e s h o c k c h a m b e r for 2 m i n a n d t h e n given a single trial. This consisted of placing the animal on the r u n w a y , facing away f r o m t h e d o o r , and m e a s u r i n g the l a t e n c y t o e n t e r the c h a m b e r . The a n i m a l r e m a i n e d in the c h a m b e r 10 sec b e f o r e b e i n g r e t u r n e d to t h e h o m e cage. Three such trials were given on the second day w i t h an intertrial interval of a p p r o x i m a t e l y 2 min. A n y animal giving a l a t e n c y of 30 sec or m o r e was e l i m i n a t e d from the e x p e r i m e n t . A f t e r the t h i r d trial the animals received a scrambled a.c. electric s h o c k t h r o u g h the grid floor. The shock intensities applied were 0.25, 0.50 and 1.00 m A for a 3 sec d u r a t i o n . R e t e n t i o n was tested 24 h r after t h e s h o c k trial w h e n the latency to e n t e r t h e b o x was r e c o r d e d to a m a x i m u m of 300 sec. The p e p t i d e s (30 ug A C T H , _ ~ o or 0.5 ug d e s g l y c i n a m i d e lysine vasopressin in 0.5 ml saline) were a d m i n i s t e r e d s u b c u t a n e o u s l y (in t h e b a c k of t h e n e c k ) 1 hr b e f o r e the first r e t e n t i o n trial. C o n t r o l animals were given saline (0.5 ml) injections at the same time. Some groups of animals were n o t s u b j e c t e d to the electric s h o c k b u t received the same n u m b e r of a d a p t a t i o n trials. In some e x p e r i m e n t s animals were retested 24 hr after the first r e t e n t i o n trial, b u t did n o t receive a s e c o n d injection. This measure of t h e e x t i n c t i o n of the passive a v o i d a n c e condit i o n i n g was t e r m e d the s e c o n d r e t e n t i o n trial. The results are p r e s e n t e d as the latency range, i.e. the u p p e r and lower limits of the latencies r e c o r d e d for each group of animals. I m m e d i a t e l y a f t e r the first or in some e x p e r i m e n t s a f t e r the s e c o n d r e t e n t i o n trial the a n i m a l was a n a e s t h e t i z e d b y e x p o s u r e to e t h e r for a strictly c o n t r o l l e d time period of 45 sec. A 1 - 2 ml sample of eye plexus b l o o d was t a k e n d u r i n g a 20 sec period. This was allowed to clot at 4°C and t h e n centrifuged. The s e r u m was used in a two plus o n e bioassay for AD-activity using a water-loaded alcohol a n a e s t h e t i z e d rat [ 4 ] . F e m a l e Wistar rats weighing 1 2 0 - 1 3 0 g were used. The assay a n i m a l was first lightly a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (±2 mg per rat) a d m i n i s t e r e d via the tail vein. The a n a e s t h e s i a was m a i n t a i n e d by infusing Czaczkes s o l u t i o n (0.3 g % NaC1, 1.67 g % glucose, 2.5% e t h a n o l ) at a rate of 0.2 m l / m i n t h r o u g h the tail vein. A p o l y t h e n e c a n n u l a was i n t r o d u c e d i n t o t h e b l a d d e r via the u r e t h r a and t h e urine flow rate m e a s u r e d over c o n s e c u t i v e 10 rain periods. The rat sera were assayed against s t a n d a r d injections o f Iysine vasopressin given t h r o u g h the tail vein. Each b l o o d sample was used in a d i f f e r e n t assay animal. Statistical analysis was carried o u t on the assays from each g r o u p o f animals and only t h o s e w h i c h gave a correct i n d e x of precision [9] were used. The AD-activity is expressed in m U / m l serum and the m e a n ± the s t a n d a r d error of the m e a n is given for each group. In some assay animals b l o o d pressure was m e a s u r e d s i m u l t a n e o u s l y w i t h urine flow. The carotid artery was c a n n u l a t e d and c o n n e c t e d to a strain-gauge t r a n s d u c e r ( C o n s o l i d a t e d E l e c t r o d y n a m i c s , Type 4 - 3 2 7 ) filled w i t h h e p a r i n i z e d saline and the o u t p u t c o n t i n u o u s l y recorded. RESULTS A n tidiuretic A c tivity
The b l o o d pressure of the assay animals ranged f r o m
1 2 0 - 1 3 0 m m ttg. The i n j e c t i o n of eye plexus serum did n o t p r o d u c e a fall in b l o o d pressure and in several animals there was a rise of b e t w e e n 5 - - 1 0 m m ttg. The decrease in urine flow p r o d u c e d by the serum c a n n o t , t h e r e f o r e , be due to an i n h i b i t o r y a c t i o n of the sample on b l o o d pressure. Van Dyke and Ames [13] have s h o w n t h a t b o t h a q u e o u s pitressin and e n d o g e n o u s vasopressin in urine are inactivated by thioglycollate. We also f o u n d t h a t i n c u b a t i n g the serum sample w i t h freshly p r e p a r e d 0.1M sodium thioglycollate for 1 hr at 37°(? d e s t r o y e d any AD-activity.
TABLE 1 THE EFFECT OF DIFFERENT SHOCK INTENSITIES ON ANTIDIURETIC ACTIVITY
Shock Intensity
1st Retention Trial Latency Range AD-Activity isec) (mU/ml serum)
5
0.34 ± 0.02 (7)
0.25 mA
4
17
0.42 ± 0.02 (7)
0.50 mA
10
300
0.53 ± 0.03 (6t
1.00 mA
20
300
0.63 + 0.02 (6)
The animals were given tile different shock intensities for 3 sec, 24-hr before the 1st retention trial. The mean + S.E.M, of tile AD-Activity and the latency range are given of tile number of animals shown in parenthesis.
The E f f e c t o f D i f f e r e n t S h o c k Intensities
F o u r groups of rats were used. One g r o u p acted as c o n t r o l s and received n o s h o c k , t h e o t h e r s were subjected to s h o c k intensities of e i t h e r 0.25 m A , 0.5 m A , or 1 m A for 3 sec. Blood samples were t a k e n i m m e d i a t e l y after t h e first r e t e n t i o n trial and assayed for a n t i d i u r e t i c activity. The results are s h o w n in Table I. The n u m b e r of animals in the groups were t o o small for a d e q u a t e statistical analysis to be carried o u t o n the latencies. It has b e e n s h o w n , h o w e v e r , b y Ader et al. [1] t h a t altering the s h o c k i n t e n s i t y does p r o d u c e significant changes in the latency. In o u r data there is some overlap in the l a t e n c y ranges b u t the m e d i a n latency increased f r o m 3 sec for the n o n s h o c k e d g r o u p to 6 sec for the 0.25 mA, 85 sec for the 0.5 m A , and 140 sec for t h e 1 m A groups. The AD-activity of the groups s h o w e d a significant increase f r o m 0.34 ±0.02 m U / m l for the n o n s h o c k e d to 0.43 +_0.03 ( p < 0 . 0 1 ) at 0.25 m A and a f u r t h e r increase to 0.53 ~+0.03 ( p < 0 . 0 0 2 5 ) at 0.5 m A , and 0.63 ±0.02 ( p < 0 . 0 1 ) at 1 m A . It is i n t e r e s t i n g to n o t e t h a t animals given s h o c k intensities o f 0.5 m A and 1 m A which gave latencies t h a t fell w i t h i n the range of the 0.25 m A group still s h o w e d a m a r k e d increase in a n t i d i u r e t i c activity w h e n c o m p a r e d to the 0.25 m A s h o c k e d animals. The ETject o.f A C T H ~
~ o oJz A n t i d i u r e t i c A c t i v i t y
A s h o c k i n t e n s i t y of 0.25 m A for a 3 sec d u r a t i o n was
V A S O P R E S S I N AND A V O I D A N C E B E H A V I O U R
379
TABLE 2 ANTIDIURETIC ACTIVITY AND PASSIVE AVOIDANCE LATENCY FOLLOWING TREATMENT WITH ACTH4_10
Injection
Shock Intensity and Duration
Placebo
1st Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2nd Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2 - 5
0.36 ± 0.02 (5)
-
2 - 5
0.36 -+ 0.01 (6)
Placebo
0.25 mA, 3 sec
10 - 21
0.35 +_0.02 (4)
4 - 300
0.35 _+0.03 (4)
30 ~zg ACTH4_10
0.25 mA, 3 sec
102 - 300
0.55 +- 0.02 (7)
6 - 25
0.38 -+ 0.02 (4)
30/~g ACTH4_10
The animals received either placebo or 30 ~g ACTH4_IO in an injection volume of 0.5 ml 1 hr before the 1st retention trial. The latency range and the mean +_S.E.M. of the AD-Activity are given of the number of animals shown in parenthesis.
used for this series o f e x p e r i m e n t s (Table 2). The animals received e i t h e r a p l a c e b o i n j e c t i o n or 30 ug A C T H , _ I o 1 hr b e f o r e the first r e t e n t i o n trial. The act o f giving the i n j e c t i o n had no significant e f f e c t on AD-activity as t h e levels o f the n o n s h o c k e d 0.25 m A groups in Table 1 were similar to t h o s e of the p l a c e b o t r e a t e d n o n s h o c k e d and s h o c k e d groups in Table 2. There was also no significant d i f f e r e n c e b e t w e e n t h e n o n s h o c k e d g r o u p receiving a p l a c e b o i n j e c t i o n and t h o s e given 30 ug A C T H 4 _ j o. It seems, t h e r e f o r e , t h a t 30 ug A C T H , _ I 0 has n o e f f e c t o n the AD-activity o f the serum 1 hr a f t e r the a d m i n i s t r a t i o n . The s h o c k e d animals w h i c h received 30 ug A C T H 4 _ t o s h o w e d a significant ( p < 0 . 0 0 1 ) increase in a n t i d i u r e t i c activity a f t e r the first r e t e n t i o n trial w h e n c o m p a r e d to the p l a c e b o t r e a t e d group. There was also a rise in the latencies o f t h e s h o c k e d ACTH 4_10 t r e a t e d animals above t h o s e o f the p l a c e b o s h o c k e d group. The latencies and the ADactivities o f the first and s e c o n d r e t e n t i o n trials o f the
placebo s h o c k e d animals s h o w e d n o change. The A C T H 4 - 1 0 t r e a t e d animals, h o w e v e r , s h o w e d a significant ( p < 0 . 0 0 5 ) fall in AD-activity f r o m the first r e t e n t i o n trial to the s e c o n d while the latencies also s h o w e d a decrease. All animals e n t e r e d the b o x within a few sec e x c e p t one w h i c h t o o k 300 sec to e n t e r . The values for the s e c o n d r e t e n t i o n trial were n o t significantly d i f f e r e n t f r o m t h o s e o f the p l a c e b o group.
The Effect of Desglycinamide Lysine Vasopressin (DG-L VP) on A ntidiuretic Activity DG-LVP has b e e n s h o w n to be practically devoid o f antidiuretic or pressor activity [ 8 ] . Nevertheless, the n o n s h o c k e d animals s h o w e d a significant ( p < 0 . 0 1 ) increase in AD-activity following DG-LVP t r e a t m e n t (Table 3). The s h o c k e d animals t h a t received DG-LVP 1 h r b e f o r e the first r e t e n t i o n trial s h o w e d a m a r k e d increase in l a t e n c y and also
TABLE 3 ANTIDIURETIC ACTIVITY AND PASSIVE AVOIDANCE LATENCY FOLLOWING TREATMENT WITH DESGLYCINAMIDE LYSINE VASOPRESSIN (DG-LVP)
Injection
Shock Intensity and Duration
1st Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2nd Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
Placebo
-
2 - 27
0.31 +- 0.02 (4)
0.5 ~g DG-LVP
-
3- 5
0.41 -+ 0.02 (4)
Placebo
0.25 mA, 3 sec
11 - 17
0.34 _+0.02 (3)
4 - 6
0.30 _+0.01 (4)
0.5 ,ug DG-LVP
0.25 mA, 3 sec
111 - 300
0.59 -+ 0.02 (7)
191 - 300
0.60 ± 0.01 (6)
The animals received either placebo or 0.5 /sg DG-LVP in an injection volume of 0.5 ml 1 hr before the 1st retention trial. The latency range and the mean _+S.E.M. of the AD-Activity are given of the number of animals shown in parenthesis.
380
T H O M P S O N A N D de WIED
a significant ( p < 0 . 0 0 1 ) increase in AD-activity above the n o n s h o c k e d DG-LVP treated group. On the s e c o n d retention trial, b o t h the latency range a n d t h e AD-activity r e m a i n e d at levels n o t significantly d i f f e r e n t f r o m those of the first r e t e n t i o n trial. DISCUSSION It is always difficult to be certain t h a t the AD-activity assayed is due to vasopressin b u t the results o f the b l o o d pressure m e a s u r e m e n t s and the fact t h a t t h e activity was d e s t r o y e d by t h i o g l y c o l l a t e seem to suggest t h a t vasopressin itself was b e i n g m e a s u r e d in these studies. A n a e s t h e t i c s affect t h e release of vasopressin [ 11 ] and a l t h o u g h t h e level of anaesthesia was c o n t r o l l e d as far as possible, the results do n o give a measure of the basal level of AD-activity. DG-LVP caused an increase in AD-activity in t h e eye plexus b l o o d of n o n s h o c k e d rats 1 h r after its a d m i n i s t r a tion. This p o l y p e p t i d e is virtually devoid of a n t i d i u r e t i c and pressor activity [8] and was also f o u n d inactive in t h e assay animals used in the p r e s e n t e x p e r i m e n t s . This is n o t well u n d e r s t o o d . T h e r e m a y be some activity in t h e u n a n a e s t h e tized animal t h a t is n o t a p p a r e n t in the assay a n i m a l or the DG-LVP m a y i n t e r f e r e with the release or d e g r a d a t i o n of vasopressin. The results clearly d e m o n s t r a t e t h a t passive a v o i d a n c e is associated w i t h an increased release of vasopressin. The rate of release is related to the i n t e n s i t y of previously aversive stimulus. If avoidance l a t e n c y was increased b y t h e administ r a t i o n of e i t h e r ACTH~ ~o or DG-LVP, AD-activity in
eye plexus b l o o d was c o n s i d e r a b l y a u g m e n t e d . This was f o u n d d u r i n g the first r e t e n t i o n trial for A C T H 4 _ ~o and d u r i n g the first and s e c o n d r e t e n t i o n trials for DG-LVP. The i m m e d i a t e and relatively b r i e f effect of ACTH4__, 0 as o p p o s e d to t h e p r e s e r v a t i o n of the passive a v o i d a n c e response b y DG-LVP [6] is t h e r e f o r e reflected in the discharge o f vasopressin f r o m the p o s t e r i o r lobe of the pituitary. Thus, a t i m e - d e p e n d e n t association b e t w e e n the release of vasopressin and a particular b e h a v i o u r a l response was observed. It m a y be, t h e r e f o r e , t h a t the release of vasopressin or related p e p t i d e s in response to specific e n v i r o n m e n t a l cues is of physiological significance in t h e m a i n t e n a n c e of learned b e h a v i o u r . AD-activity o f eye plexus b l o o d was a p p r o x i m a t e l y the same in ACTH 4_~ 0 and DG-LVP treated rats d u r i n g the first r e t e n t i o n trial while the s u b s e q u e n t a v o i d a n c e latency of the second r e t e n t i o n trial was lower in the ACTtt4 1o animals t h a n the DG-LVP animals. This finding does n o t seem to s u p p o r t o u r c o n c e p t t h a t t h e rate of release of vasopressin d e t e r m i n e s s u b s e q u e n t b e h a v i o u r a l responding. However, vasopressin c o n t e n t was m e a s u r e d only once and the p a t t e r n of release m a y differ m a r k e d l y u n d e r the i n f l u e n c e of e i t h e r p e p t i d e . F u r t h e r e x p e r i m e n t s are n e e d e d t h e r e f o r e to d e t e r m i n e w h e t h e r e n d o g e n o u s vasopressin is of significance in the d e v e l o p m e n t of b e h a v i o u r a l responses to specific e n v i r o n m e n t a l stimuli. ACKNOWLEDGEMENTS We would like to thank Mrs. Wiltemien van Dongen-Ham and Mrs. Dorrit Beyer-van Keulen for their technical assistance.
REFERENCES 1. Ader, R. and D. de Wied. Effects of lysine vasopressin on passive avoidance learning. Psychon. Sci. 2 9 : 4 6 48, 1972. 2. Ader, R., J. A. W. M. Weijnen and P. Moleman. Retention of a passive avoidance response as a function of the intensity and duration of electric shock. Psychon. Sci. 26: 125-129, 1972. 3. Bohus, B., R. Ader and D. de Wied. Effects of vasopressin on active avoidance behaviour. Hormones and Behaviour 3: 191 197, 1972. 4. Czaczkes, J. W., C. R. Kleeman and M. Koenig. Physiologic studies of ADH by its direct measurement in human plasma. J. Clin. Invest. 43: 1625-1640, 1964. 5. de Wied, D. The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. hzt. J. Neuropharmacol. 4 : 1 5 7 167, 1965. 6. de Wied, D. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature 232: 5 8 - 6 0 , 1971. 7. de Wied, D. and B. Bohus. Long term and short term effects on retention of a conditioned avoidance response in rats by treatment with long acting pitressin and c~-MSH. Nature 212: 1484-1486, 1966.
8. de Wied, D., H. M. Greven, S. Lande and A. Witter. Dissociation of the behavioural and endocrine effects of lysine vasopressin by tryptic digestion. Brit. J. Pharmacol. 45: 118 122, 1972. 9. Finney, D. J. ln: Experimental Design and its Statistical Basis. Univ. Chicago Press, 1955. 10. Greven, H. M. and D. de Wied. The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behaviour in rats. Europ. J. Pharmacol. 2 : 1 4 16,1967. 1 I. Heller, H. and M. Ginsburg. Secretion, metabolism and fate of the posterior pituitary hormones. In: The Pituitary Gland edited by G. W. Harris and B. T. Donovan. Berkeley: Univ. California Press, 1966, Vot. 3, p. 333. 12. Nash, F. D. Control of antidiuretic hormone secretion. Introductory remarks. Fed. Proc. 30:1376 1377, 1971. 13. Van Dyke, H. B. and R. G. Ames. Thioglycollate in activation of posterior pituitary antidiuretic principle as determined in the rat. ,°roe. Soc. Exp. Biol. N. Y. 76: 576-.578, 1951.
The Relationship Between the Antidiuretic Activity of Rat Eye Plexus Blood and Passive Avoidance Behaviour E L I Z A B E T H A. T H O M P S O N 1
The Medical Research Council Unit for Metabolic" Studies in Psychiatry, University Department of Psychiatry, Middlewood Hospital, Sheffield, $6 1 TP, England AND D A V I D DE W I E D
The Rudolf Magnus Institute o f Pharmacology, University of Utrecht, Medical Faculty, Utrecht, The Netherlands
(Received 5 F e b r u a r y 1 9 7 3 ) THOMPSON, E. A. AND D. DE WlED. The relationship between the antidiuretic activity of rat eye plexus blood and passive avoidance behaviour. PHYSIOL. BEHAV. 11(3) 3 7 7 - 3 8 0 , 1973.-Antidiuretic activity (AD-activity) of eye plexus blood in rats increases during a 24 hr postshock retention trial of a passive step-through avoidance response. The rate of increase in AD-activity was related to the intensity of the shock to which the animals had been exposed previously. If avoidance latency was increased by the administration of ACTH4_ ~o or desglycinamide lysine vasopressin (DG-LVP) 1 hr prior to the 24 hr postshock retention trial, AD-activity rose concomitantly. The influence of A C T H , _ 1o on avoidance latency which generally has disappeared 24 hr after a single injection in contrast to a single injection of DG-LVP which affects avoidance latency for several days, was reflected in a time dependent rise in AD-activity of eye plexus blood. Passive avoidance behaviour Antidiuretic activity Desglycinamide lysine vasopressin
Eye plexus blood
T H E P O S T E R I O R p i t u i t a r y and vasopressin p r o f o u n d l y a f f e c t a c q u i s i t i o n a n d m a i n t e n a n c e o f aversively s t i m u l a t e d active a n d passive a v o i d a n c e b e h a v i o u r . T h e r e m o v a l o f t h e p o s t e r i o r lobe of t h e p i t u i t a r y i n t e r f e r e s w i t h t h e m a i n t e n a n c e of a s h u t t l e b o x a v o i d a n c e r e s p o n s e [ 5 ] , w h i c h is r e s t o r e d b y vasopressin. In i n t a c t rats, vasopressin facilit a t e d t h e p r e s e r v a t i o n o f s u c h learned r e s p o n s e s for a c o n s i d e r a b l e p e r i o d of t i m e [1, 6, 7 ] . T h e c o n s o l i d a t i o n o f an a v o i d a n c e r e s p o n s e is facilitated o n l y if it is a c q u i r e d u n d e r o p t i m a l vasopressin i n f l u e n c e . T h e i n f l u e n c e o f t h e p e p t i d e is also s i t u a t i o n specific [ 3 ] . This e f f e c t of vasopressin m a y b e related to t h e relative ease w i t h w h i c h a given b e h a v i o u r a l r e s p o n s e is elicited w h e n s i t u a t i o n specific cues r e o c c u r . If this is true, it m i g h t b e t h a t an a s s o c i a t i o n exists b e t w e e n e n d o g e n o u s release of vasopressin or related p e p t i d e s a n d specific e n v i r o n m e n t a l cues. In fact, n u m e r o u s s t i m u l i o t h e r t h a n t h o s e c o n c e r n e d w i t h w a t e r h o m e o s t a s i s i n d u c e t h e release o f vasopressin f r o m t h e p o s t e r i o r lobe of t h e p i t u i t a r y [ 1 2 ] . The p r e s e n t e x p e r i m e n t s were t h e r e f o r e designed to
ACTH,_I o
d e t e r m i n e t h e release o f vasopressin d u r i n g aversively m o t i v a t e d passive a v o i d a n c e b e h a v i o u r . In a d d i t i o n , t h e i n f l u e n c e o n vasopressin-release was studied in rats in w h i c h passive a v o i d a n c e l a t e n c y was m o d i f i e d b y p r i o r a d m i n i s t r a t i o n o f a vasopressin related p e p t i d e d e s g l y c i n a m i d e lysine vasopressin ( D G - L V P ) [ 8 ] , and a p e p t i d e related to A C T H (ACTH4_1o) [10]. METHOD Male rats o f an i n b r e d Wistar strain weighing 1 1 0 - 1 2 0 g were used. These were m a i n t a i n e d 5 p e r cage w i t h ad lib f o o d a n d w a t e r a n d t h e lights o n b e t w e e n 5 a.m. a n d 7 p.m. All o b s e r v a t i o n s were m a d e b e t w e e n 9 : 3 0 a.m. a n d 1 2 : 3 0 p.m. The s t e p - t h r o u g h passive a v o i d a n c e p r o c e d u r e a d o p t e d was t h a t of Ader, Weijen and M o l e m a n [ 2 ] . T h e a p p a r a t u s consisted of a 4 0 x 4 0 x 4 0 cm Lucite c h a m b e r w i t h b l a c k wails a n d grid floor. A wire mesh covered elevated r u n w a y , 6 cm wide a n d 25 cm long, e x t e n d e d f r o m o n e side of t h e
J This research was conducted almost completely in Utrecht. E.A.T. was supported by a European Brain and Behaviour Research Grant and by the M.R.C., London. 377
378
T H O M P S O N AND de WIED
c h a m b e r on a level w i t h the grid floor. The a n i m a l was able to e n t e r the s h o c k c h a m b e r f r o m the r u n w a y t h r o u g h a guillotine o p e r a t e d d o o r 6 cm x 6 cm. The s h o c k c h a m b e r r e m a i n e d dark while t h e elevated r u n w a y was i l l u m i n a t e d by a 25 W lamp fixed 40 cm above the r u n w a y . On the first day of a d a p t a t i o n training, the rat was placed inside t h e s h o c k c h a m b e r for 2 m i n a n d t h e n given a single trial. This consisted of placing the animal on the r u n w a y , facing away f r o m t h e d o o r , and m e a s u r i n g the l a t e n c y t o e n t e r the c h a m b e r . The a n i m a l r e m a i n e d in the c h a m b e r 10 sec b e f o r e b e i n g r e t u r n e d to t h e h o m e cage. Three such trials were given on the second day w i t h an intertrial interval of a p p r o x i m a t e l y 2 min. A n y animal giving a l a t e n c y of 30 sec or m o r e was e l i m i n a t e d from the e x p e r i m e n t . A f t e r the t h i r d trial the animals received a scrambled a.c. electric s h o c k t h r o u g h the grid floor. The shock intensities applied were 0.25, 0.50 and 1.00 m A for a 3 sec d u r a t i o n . R e t e n t i o n was tested 24 h r after t h e s h o c k trial w h e n the latency to e n t e r t h e b o x was r e c o r d e d to a m a x i m u m of 300 sec. The p e p t i d e s (30 ug A C T H , _ ~ o or 0.5 ug d e s g l y c i n a m i d e lysine vasopressin in 0.5 ml saline) were a d m i n i s t e r e d s u b c u t a n e o u s l y (in t h e b a c k of t h e n e c k ) 1 hr b e f o r e the first r e t e n t i o n trial. C o n t r o l animals were given saline (0.5 ml) injections at the same time. Some groups of animals were n o t s u b j e c t e d to the electric s h o c k b u t received the same n u m b e r of a d a p t a t i o n trials. In some e x p e r i m e n t s animals were retested 24 hr after the first r e t e n t i o n trial, b u t did n o t receive a s e c o n d injection. This measure of t h e e x t i n c t i o n of the passive a v o i d a n c e condit i o n i n g was t e r m e d the s e c o n d r e t e n t i o n trial. The results are p r e s e n t e d as the latency range, i.e. the u p p e r and lower limits of the latencies r e c o r d e d for each group of animals. I m m e d i a t e l y a f t e r the first or in some e x p e r i m e n t s a f t e r the s e c o n d r e t e n t i o n trial the a n i m a l was a n a e s t h e t i z e d b y e x p o s u r e to e t h e r for a strictly c o n t r o l l e d time period of 45 sec. A 1 - 2 ml sample of eye plexus b l o o d was t a k e n d u r i n g a 20 sec period. This was allowed to clot at 4°C and t h e n centrifuged. The s e r u m was used in a two plus o n e bioassay for AD-activity using a water-loaded alcohol a n a e s t h e t i z e d rat [ 4 ] . F e m a l e Wistar rats weighing 1 2 0 - 1 3 0 g were used. The assay a n i m a l was first lightly a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (±2 mg per rat) a d m i n i s t e r e d via the tail vein. The a n a e s t h e s i a was m a i n t a i n e d by infusing Czaczkes s o l u t i o n (0.3 g % NaC1, 1.67 g % glucose, 2.5% e t h a n o l ) at a rate of 0.2 m l / m i n t h r o u g h the tail vein. A p o l y t h e n e c a n n u l a was i n t r o d u c e d i n t o t h e b l a d d e r via the u r e t h r a and t h e urine flow rate m e a s u r e d over c o n s e c u t i v e 10 rain periods. The rat sera were assayed against s t a n d a r d injections o f Iysine vasopressin given t h r o u g h the tail vein. Each b l o o d sample was used in a d i f f e r e n t assay animal. Statistical analysis was carried o u t on the assays from each g r o u p o f animals and only t h o s e w h i c h gave a correct i n d e x of precision [9] were used. The AD-activity is expressed in m U / m l serum and the m e a n ± the s t a n d a r d error of the m e a n is given for each group. In some assay animals b l o o d pressure was m e a s u r e d s i m u l t a n e o u s l y w i t h urine flow. The carotid artery was c a n n u l a t e d and c o n n e c t e d to a strain-gauge t r a n s d u c e r ( C o n s o l i d a t e d E l e c t r o d y n a m i c s , Type 4 - 3 2 7 ) filled w i t h h e p a r i n i z e d saline and the o u t p u t c o n t i n u o u s l y recorded. RESULTS A n tidiuretic A c tivity
The b l o o d pressure of the assay animals ranged f r o m
1 2 0 - 1 3 0 m m ttg. The i n j e c t i o n of eye plexus serum did n o t p r o d u c e a fall in b l o o d pressure and in several animals there was a rise of b e t w e e n 5 - - 1 0 m m ttg. The decrease in urine flow p r o d u c e d by the serum c a n n o t , t h e r e f o r e , be due to an i n h i b i t o r y a c t i o n of the sample on b l o o d pressure. Van Dyke and Ames [13] have s h o w n t h a t b o t h a q u e o u s pitressin and e n d o g e n o u s vasopressin in urine are inactivated by thioglycollate. We also f o u n d t h a t i n c u b a t i n g the serum sample w i t h freshly p r e p a r e d 0.1M sodium thioglycollate for 1 hr at 37°(? d e s t r o y e d any AD-activity.
TABLE 1 THE EFFECT OF DIFFERENT SHOCK INTENSITIES ON ANTIDIURETIC ACTIVITY
Shock Intensity
1st Retention Trial Latency Range AD-Activity isec) (mU/ml serum)
5
0.34 ± 0.02 (7)
0.25 mA
4
17
0.42 ± 0.02 (7)
0.50 mA
10
300
0.53 ± 0.03 (6t
1.00 mA
20
300
0.63 + 0.02 (6)
The animals were given tile different shock intensities for 3 sec, 24-hr before the 1st retention trial. The mean + S.E.M, of tile AD-Activity and the latency range are given of tile number of animals shown in parenthesis.
The E f f e c t o f D i f f e r e n t S h o c k Intensities
F o u r groups of rats were used. One g r o u p acted as c o n t r o l s and received n o s h o c k , t h e o t h e r s were subjected to s h o c k intensities of e i t h e r 0.25 m A , 0.5 m A , or 1 m A for 3 sec. Blood samples were t a k e n i m m e d i a t e l y after t h e first r e t e n t i o n trial and assayed for a n t i d i u r e t i c activity. The results are s h o w n in Table I. The n u m b e r of animals in the groups were t o o small for a d e q u a t e statistical analysis to be carried o u t o n the latencies. It has b e e n s h o w n , h o w e v e r , b y Ader et al. [1] t h a t altering the s h o c k i n t e n s i t y does p r o d u c e significant changes in the latency. In o u r data there is some overlap in the l a t e n c y ranges b u t the m e d i a n latency increased f r o m 3 sec for the n o n s h o c k e d g r o u p to 6 sec for the 0.25 mA, 85 sec for the 0.5 m A , and 140 sec for t h e 1 m A groups. The AD-activity of the groups s h o w e d a significant increase f r o m 0.34 ±0.02 m U / m l for the n o n s h o c k e d to 0.43 +_0.03 ( p < 0 . 0 1 ) at 0.25 m A and a f u r t h e r increase to 0.53 ~+0.03 ( p < 0 . 0 0 2 5 ) at 0.5 m A , and 0.63 ±0.02 ( p < 0 . 0 1 ) at 1 m A . It is i n t e r e s t i n g to n o t e t h a t animals given s h o c k intensities o f 0.5 m A and 1 m A which gave latencies t h a t fell w i t h i n the range of the 0.25 m A group still s h o w e d a m a r k e d increase in a n t i d i u r e t i c activity w h e n c o m p a r e d to the 0.25 m A s h o c k e d animals. The ETject o.f A C T H ~
~ o oJz A n t i d i u r e t i c A c t i v i t y
A s h o c k i n t e n s i t y of 0.25 m A for a 3 sec d u r a t i o n was
V A S O P R E S S I N AND A V O I D A N C E B E H A V I O U R
379
TABLE 2 ANTIDIURETIC ACTIVITY AND PASSIVE AVOIDANCE LATENCY FOLLOWING TREATMENT WITH ACTH4_10
Injection
Shock Intensity and Duration
Placebo
1st Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2nd Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2 - 5
0.36 ± 0.02 (5)
-
2 - 5
0.36 -+ 0.01 (6)
Placebo
0.25 mA, 3 sec
10 - 21
0.35 +_0.02 (4)
4 - 300
0.35 _+0.03 (4)
30 ~zg ACTH4_10
0.25 mA, 3 sec
102 - 300
0.55 +- 0.02 (7)
6 - 25
0.38 -+ 0.02 (4)
30/~g ACTH4_10
The animals received either placebo or 30 ~g ACTH4_IO in an injection volume of 0.5 ml 1 hr before the 1st retention trial. The latency range and the mean +_S.E.M. of the AD-Activity are given of the number of animals shown in parenthesis.
used for this series o f e x p e r i m e n t s (Table 2). The animals received e i t h e r a p l a c e b o i n j e c t i o n or 30 ug A C T H , _ I o 1 hr b e f o r e the first r e t e n t i o n trial. The act o f giving the i n j e c t i o n had no significant e f f e c t on AD-activity as t h e levels o f the n o n s h o c k e d 0.25 m A groups in Table 1 were similar to t h o s e of the p l a c e b o t r e a t e d n o n s h o c k e d and s h o c k e d groups in Table 2. There was also no significant d i f f e r e n c e b e t w e e n t h e n o n s h o c k e d g r o u p receiving a p l a c e b o i n j e c t i o n and t h o s e given 30 ug A C T H 4 _ j o. It seems, t h e r e f o r e , t h a t 30 ug A C T H , _ I 0 has n o e f f e c t o n the AD-activity o f the serum 1 hr a f t e r the a d m i n i s t r a t i o n . The s h o c k e d animals w h i c h received 30 ug A C T H 4 _ t o s h o w e d a significant ( p < 0 . 0 0 1 ) increase in a n t i d i u r e t i c activity a f t e r the first r e t e n t i o n trial w h e n c o m p a r e d to the p l a c e b o t r e a t e d group. There was also a rise in the latencies o f t h e s h o c k e d ACTH 4_10 t r e a t e d animals above t h o s e o f the p l a c e b o s h o c k e d group. The latencies and the ADactivities o f the first and s e c o n d r e t e n t i o n trials o f the
placebo s h o c k e d animals s h o w e d n o change. The A C T H 4 - 1 0 t r e a t e d animals, h o w e v e r , s h o w e d a significant ( p < 0 . 0 0 5 ) fall in AD-activity f r o m the first r e t e n t i o n trial to the s e c o n d while the latencies also s h o w e d a decrease. All animals e n t e r e d the b o x within a few sec e x c e p t one w h i c h t o o k 300 sec to e n t e r . The values for the s e c o n d r e t e n t i o n trial were n o t significantly d i f f e r e n t f r o m t h o s e o f the p l a c e b o group.
The Effect of Desglycinamide Lysine Vasopressin (DG-L VP) on A ntidiuretic Activity DG-LVP has b e e n s h o w n to be practically devoid o f antidiuretic or pressor activity [ 8 ] . Nevertheless, the n o n s h o c k e d animals s h o w e d a significant ( p < 0 . 0 1 ) increase in AD-activity following DG-LVP t r e a t m e n t (Table 3). The s h o c k e d animals t h a t received DG-LVP 1 h r b e f o r e the first r e t e n t i o n trial s h o w e d a m a r k e d increase in l a t e n c y and also
TABLE 3 ANTIDIURETIC ACTIVITY AND PASSIVE AVOIDANCE LATENCY FOLLOWING TREATMENT WITH DESGLYCINAMIDE LYSINE VASOPRESSIN (DG-LVP)
Injection
Shock Intensity and Duration
1st Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
2nd Retention Trial Latency Range AD-Activity (sec) (mU/ml serum)
Placebo
-
2 - 27
0.31 +- 0.02 (4)
0.5 ~g DG-LVP
-
3- 5
0.41 -+ 0.02 (4)
Placebo
0.25 mA, 3 sec
11 - 17
0.34 _+0.02 (3)
4 - 6
0.30 _+0.01 (4)
0.5 ,ug DG-LVP
0.25 mA, 3 sec
111 - 300
0.59 -+ 0.02 (7)
191 - 300
0.60 ± 0.01 (6)
The animals received either placebo or 0.5 /sg DG-LVP in an injection volume of 0.5 ml 1 hr before the 1st retention trial. The latency range and the mean _+S.E.M. of the AD-Activity are given of the number of animals shown in parenthesis.
380
T H O M P S O N A N D de WIED
a significant ( p < 0 . 0 0 1 ) increase in AD-activity above the n o n s h o c k e d DG-LVP treated group. On the s e c o n d retention trial, b o t h the latency range a n d t h e AD-activity r e m a i n e d at levels n o t significantly d i f f e r e n t f r o m those of the first r e t e n t i o n trial. DISCUSSION It is always difficult to be certain t h a t the AD-activity assayed is due to vasopressin b u t the results o f the b l o o d pressure m e a s u r e m e n t s and the fact t h a t t h e activity was d e s t r o y e d by t h i o g l y c o l l a t e seem to suggest t h a t vasopressin itself was b e i n g m e a s u r e d in these studies. A n a e s t h e t i c s affect t h e release of vasopressin [ 11 ] and a l t h o u g h t h e level of anaesthesia was c o n t r o l l e d as far as possible, the results do n o give a measure of the basal level of AD-activity. DG-LVP caused an increase in AD-activity in t h e eye plexus b l o o d of n o n s h o c k e d rats 1 h r after its a d m i n i s t r a tion. This p o l y p e p t i d e is virtually devoid of a n t i d i u r e t i c and pressor activity [8] and was also f o u n d inactive in t h e assay animals used in the p r e s e n t e x p e r i m e n t s . This is n o t well u n d e r s t o o d . T h e r e m a y be some activity in t h e u n a n a e s t h e tized animal t h a t is n o t a p p a r e n t in the assay a n i m a l or the DG-LVP m a y i n t e r f e r e with the release or d e g r a d a t i o n of vasopressin. The results clearly d e m o n s t r a t e t h a t passive a v o i d a n c e is associated w i t h an increased release of vasopressin. The rate of release is related to the i n t e n s i t y of previously aversive stimulus. If avoidance l a t e n c y was increased b y t h e administ r a t i o n of e i t h e r ACTH~ ~o or DG-LVP, AD-activity in
eye plexus b l o o d was c o n s i d e r a b l y a u g m e n t e d . This was f o u n d d u r i n g the first r e t e n t i o n trial for A C T H 4 _ ~o and d u r i n g the first and s e c o n d r e t e n t i o n trials for DG-LVP. The i m m e d i a t e and relatively b r i e f effect of ACTH4__, 0 as o p p o s e d to t h e p r e s e r v a t i o n of the passive a v o i d a n c e response b y DG-LVP [6] is t h e r e f o r e reflected in the discharge o f vasopressin f r o m the p o s t e r i o r lobe of the pituitary. Thus, a t i m e - d e p e n d e n t association b e t w e e n the release of vasopressin and a particular b e h a v i o u r a l response was observed. It m a y be, t h e r e f o r e , t h a t the release of vasopressin or related p e p t i d e s in response to specific e n v i r o n m e n t a l cues is of physiological significance in t h e m a i n t e n a n c e of learned b e h a v i o u r . AD-activity o f eye plexus b l o o d was a p p r o x i m a t e l y the same in ACTH 4_~ 0 and DG-LVP treated rats d u r i n g the first r e t e n t i o n trial while the s u b s e q u e n t a v o i d a n c e latency of the second r e t e n t i o n trial was lower in the ACTtt4 1o animals t h a n the DG-LVP animals. This finding does n o t seem to s u p p o r t o u r c o n c e p t t h a t t h e rate of release of vasopressin d e t e r m i n e s s u b s e q u e n t b e h a v i o u r a l responding. However, vasopressin c o n t e n t was m e a s u r e d only once and the p a t t e r n of release m a y differ m a r k e d l y u n d e r the i n f l u e n c e of e i t h e r p e p t i d e . F u r t h e r e x p e r i m e n t s are n e e d e d t h e r e f o r e to d e t e r m i n e w h e t h e r e n d o g e n o u s vasopressin is of significance in the d e v e l o p m e n t of b e h a v i o u r a l responses to specific e n v i r o n m e n t a l stimuli. ACKNOWLEDGEMENTS We would like to thank Mrs. Wiltemien van Dongen-Ham and Mrs. Dorrit Beyer-van Keulen for their technical assistance.
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