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Actions of repeated injections of LSD and apomorphine on the copulatory response of female rats
Pharmacology Biochemistry & Behavior, Vol. 5, pp. 621--625. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Actions of Repeated Injections of LSD and Apomorphine on the Copulatory Response of Female Rats MONA ELIASSON
Department o f Medical Pharmacology, Box 573, S-751 23 UPPSALA, Sweden (Received 19 August 1976) ELIASSON, M. Actions of repeated in/ections of LSD and apomorphine on the copulatory response of female rats. PHARMAC. BIOCHEM. BEHAV. 5(6) 621-625, 1976. - LSD, a serotonin receptor stimulating agent, inhibits copulatory behavior (lordosis response) in the ovariectomized and estrogen + progesterone treated female rat. The same effect is obtained by apomorphine, a dopamine receptor stimulating compounds. The lordosis has been shown to be dependent on serotonin, but also dopamine has been implicated in its mediation. Tolerance develops to certain responses after repeated injections of LSD and in the present study the influence of apomorphine and LSD was compared, when given in repeated doses. Possible cross-tolerance between the two compounds was also tested on the frequency of lordosis responding in ovariectomized and hormone treated female rats. Tolerance to LSD develops over seven days, while the suppressing influence of apomorphine on lordosis in seven repeated doses is not significantly altered from that of a single dose. No cross-tolerance was observed on the lordosis response with either order of treatments. Repeated doses of LSD did not influence locomotor activity differently from a single dose, while repeated doses of apomorphine enhanced this response in comparison with the effect of an acute dose. These results indicate differential sensitivity to the repeated treatments and further support an interpretation of the LSD effects on lordosis responding to be primarily on serotonergic rather than dopaminergic receptors. LSD
Apomorphine
Lordosis response
Tolerance
single doses o f LSD. One such behavior c o m p o n e n t is the c o p u l a t o r y r e s p o n s e o f the female rat ( l o r d o t i c response). This r e s p o n s e , which consists o f an arching o f the back, head and p e r i n e u m elevation and tail deviation, is d e p e n dent on ovarian h o r m o n e s [31] and is readily elicited in receptive female rats by a m o u n t i n g male. Much evidence is available d e m o n s t r a t i n g i n h i b i t i o n of the lordosis r e s p o n s e to be m e d i a t e d by activation o f s e r o t o n i n and also d o p a m i n e r g i c n e u r o n s in o v a r i e c t o m i z e d female rats t r e a t e d with e s t r o g e n and p r o g e s t e r o n e [19, 20, 2 2 ] . This also seems to be the case w h e n the lordosis response is studied in o v a r i e c t o m i z e d female rats t r e a t e d with r e p e a t e d doses o f e s t r o g e n and n o p r o g e s t e r o n e [11, 28, 3 2 ] . LSD in a r a t h e r small dose (0.05 /ag/kg) lacking significant e f f e c t s on e.g. l o c o m o t o r behavior, inhibits lordosing in female rats receiving estrogen + p r o g e s t e r o n e [ 7 , 8 ] . A p o m o r p h i n e , a c o m p o u n d with d o p a m i n e r e c e p t o r stimulating p r o p e r t i e s [3,9] also has an i n h i b i t o r y action on the lordosis behavior activated by estrogen and p r o g e s t e r o n e [ 8 , 2 2 ] . A n o t h e r d o p a m i n e agonist (ET 495) is r e p o r t e d to have similar effects, w h e n lordosis is induced by estrogen only [ 1 0 ] . The influence of a p o m o r p h i n e was f o u n d to be c o m p l e t e l y
T O L E R A N C E appears in certain behavioral r e s p o n s e s to lysergic acid d i e t h y l a m i d e (LSD). This has b e e n f o u n d in animal as well as h u m a n studies, and is seen in the clinic as well as in the l a b o r a t o r y [ 1, 16, 17, 2 4 ] . When tested in l a b o r a t o r y rats, tolerance to LSD develops for such diverse behaviors as r o p e - c l i m b i n g [12,18] and bar pressing for f o o d r e i n f o r c e m e n t [ 5 , 1 3 ] . On the o t h e r h a n d , in a behavioral p e r f o r m a n c e m a i n t a i n e d by m e a n s o f escape f r o m electric f o o t shock, tolerance to LSD in c o m p a r a b l e doses does a p p a r e n t l y n o t emerge, but e x t i n c t i o n o f the r e s p o n s e is delayed [ 15]. The behavioral m o d i f i c a t i o n s appearing as a c o n s e q u e n c e of r e p e a t e d LSD i n j e c t i o n s are a c c o m p a n i e d by certain biochemical alterations t h a t are related to brain m o n o amines. R e p e a t e d doses o f a m a g n i t u d e c o m p a r a b l e to those used in behavioral studies give an u n c h a n g e d level o f s e r o t o n i n , while t u r n o v e r is significantly increased [ 2 3 ] . Higher doses seem to affect the c a t e c h o l a m i n e levels in a d d i t i o n to t h o s e o f s e r o t o n i n [6, 14, 2 6 ] . LSD is c o n s i d e r e d to be a s e r o t o n i n r e c e p t o r stimulating c o m p o u n d [2,4] and in a c c o r d a n c e with this, f u n c t i o n s m e d i a t e d by s e r o t o n i n have been f o u n d to be i n f l u e n c e d by
1This research was supported by grant No. B75-14X-64-1 l A from the Swedish Medical Research Council to 8engt J. Meyerson. 2Portions of this research was presented at the XIV Scandinavian Congress of Physiology and Pharmacology, Bergen, 1973, and an Abstract appeared in Acta physiol, scand., Suppl. 386, 86, 1973. 3The hormones were generous gifts supplied by N.V. Organon through Erco Ltd., Stockholm. 621
622
ELIASSON
counteracted by pretreatment with a dopamine receptor blocker ( p i m o z i d e ) , while t h e LSD i n d u c e d i n h i b i t i o n of lordosis was o n l y decreased in its d u r a t i o n . This was t a k e n as an i n d i c a t i o n of the differential m e c h a n i s m s of a c t i o n b e h i n d the effects of the t w o drugs on lordosis r e s p o n d i n g
[8]. The p u r p o s e of the present investigation was to f u r t h e r analyze the e x t e n t of d o p a m i n e r g i c relative to s e r o t o n e r g i c i n v o l v e m e n t in the LSD i n d u c e d i n h i b i t i o n of the lordosis response b y c o m p a r i n g it to a p o m o r p h i n e , w h e n b o t h drugs are given in r e p e a t e d doses. The d e v e l o p m e n t of t o l e r a n c e and possible cross-tolerance, m i g h t f u r t h e r elucidate the r e l a t i o n s h i p of these t w o drugs and t h e i r effects on lordosis responding. Lordosis has the advantage, in a d d i t i o n to its k n o w n r e l a t i o n to s e r o t o n i n and d o p a m i n e , of n o t requiring any t r a i n i n g before its m a n i f e s t a t i o n , m a k i n g it particularly suitable for a s t u d y of t o l e r a n c e . METHOD
Animals O v a r i e c t o m i z e d female rats of the Sprague-Dawley strain (Purchased as Specific P a t h o g e n Free, Anticimex, S t o c k h o l m ) were used. They were h o u s e d 5 - 6 to a cage in stainless steel cages, where tap w a t e r and c o m m e r c i a l rat pellets ( A n t i c i m e x 210 or R 3) were available at all times. The a n i m a l r o o m held a c o n s t a n t t e m p e r a t u r e (21 + I°C), had forced v e n t i l a t i o n and c o n t r o l l e d h u m i d i t y . The dayn i g h t cycle was reversed (lights on from 9 p.m. to 9 a.m.). Wistar male rats with considerable sexual e x p e r i e n c e were used for testing of the females. T h e males were h o u s e d in individual cages (40 × 40 × 30 cm), w h i c h also served as test cages, in a d i f f e r e n t section of the animal r o o m . Materials infected. The h o r m o n e s , estradiol b e n z o a t e (EB) and p r o g e s t e r o n e ( b o t h f r o m O r g a n o n ) , were dissolved in olive oil and injected s u b c u t a n e o u s l y . A p o m o r p h i n e and lysergic acid d i e t h y l a m i d e (LSD) t a r t r a t e ( b o t h f r o m Sandoz) were dissolved in saline. LSD was injected intraperitoneally, while a p o m o r p h i n e was given s u b c u t a n e o u s l y . F r e s h s o l u t i o n s were m a d e up of the two latter c o m p o u n d s for every drug test. The doses w h i c h a p p e a r in the t e x t and tables refer to t h e f o r m of the c o m p o u n d s given above. Behavioral observations. Before being used in a drug test each group of 10 12 animals was subjected to a s t a n d a r d test w i t h the regular doses of h o r m o n e s used (10/.zg/kg EB and 0.4 mg p r o g e s t e r o n e 48 hr later) t o asses the level of lordosis r e s p o n d i n g in rela{ion to w h a t is the s t a n d a r d of the l a b o r a t o r y . This is a b o u t 60 80% response in a g r o u p during the h e i g h t of the h o r m o n e effect, 6 - 7 hr a f t e r progesterone. The values before each test are i n d i c a t e d in the tables. The h o r m o n e s were injected at 8 a.m. and testing was p e r f o r m e d b e t w e e n n o o n and 5 p.m. at certain intervals, w h e n t h e h o r m o n e influence is relatively stable. Animals given r e p e a t e d injections of drug were always injected at the same time of the day, i.e. 24 hr apart, with lordosis testing b e g i n n i n g 10 m i n after the last injection. The females were tested individually in the h o m e cages of the males. Only absence or presence of lordosis was scored for each m o u n t and a m a x i m u m of six m o u n t s b y two d i f f e r e n t males was allowed. To be rated as positive by the observer the female had to show two consecutive lordoses with h e a d and p e r i n e u m raised and the tail deviated each time. Thus, the measure for each t r e a t m e n t was t h e percentage females showing lordosis a c c o r d i n g to our
criterion on a particular test. ( F o r f u r t h e r details regarding m e t h o d s see Eliasson and M e y e r s o n ] 7 ] ) . Locomotor exploratory activity. A n o t h e r i n d i c a t i o n of drug effects was the a m o u n t of l o c o m o t o r e x p l o r a t o r y activity e x h i b i t e d during a 10 m i n test. T h e animals were placed one at a t i m e in a testing b o x located o n t o p of an A n i m e x m e t e r ( F a r a d Inc., H~igersten, Sweden), where t h e i r l o c o m o t o r activity was r e c o r d e d a u t o m a t i c a l l y and a c c u m u lated for t h e whole testing period. T h e sensitivity of the a p p a r a t u s was adjusted so t h a t fine m o t o r activity like g r o o m i n g and sniffing was n o t r e c o r d e d . Testing was p e r f o r m e d in a dark and quiet r o o m w i t h no observer present. This t o o k place after r e p e a t e d and single injections of LSD or a p o m o r p h i n e in i n d e p e n d e n t groups. The animals were the same as those p a r t i c i p a t i n g in the lordosis s t u d y and those receiving m u l t i p l e i n j e c t i o n s had received also EB t h e m o r n i n g of the day b e f o r e testing and were scheduled to be tested on lordosis on the following day. (Svensson and T h i e m e [27] give t h e details regarding the testing apparatus). Statistics. Differences b e t w e e n groups of i n d e p e n d e n t animals were a n a l y z e d statistically using the Chi square test corrected for c o n t i n u i t y [ 25 ]. F o r certain t r e a t m e n t s these groups have b e e n c o m b i n e d in the tables for a easier p r e s e n t a t i o n of results. Differences b e t w e e n l o c o m o t o r scores in groups receiving single and groups receiving m u l t i p l e injections were analyzed t h r o u g h the use of the t-test [ 2 9 ] .
RESULTS AS is s h o w n by Table 1, t r e a t m e n t w i t h r e p e a t e d injections of LSD for four days and w i t h testing c o m m e n c ing 10 m i n after the last injection does n o t give any altered r e s p o n s e on the lordosis i n h i b i t i o n test at a dose of 0.10 mg/kg. This is a dose t h a t in a single i n j e c t i o n significantly decreases the p e r c e n t a g e lordosis r e s p o n d i n g females, enh a n c e s startle r e s p o n d i n g and gives some decrease of l o c o m o t o r activity [ 7 , 8 ] . Still after r e p e a t e d injections during four days the general a p p e a r a n c e of t h e animals is more n o r m a l , t h e r e is less piloerection, fewer flight responses to s u d d e n stimuli, b u t ataxia of the hindlegs is still present. The larger doses of 0.25 and 0.50 mg/kg, w h e n a d m i n i s t e r e d for four days, also do n o t a t t e n u a t e the lordosis i n h i b i t o r y effect of LSD in c o m p a r i s o n w i t h single injections. A longer period of daily LSD injections does give an altered response as is evident f r o m Table 2. When LSD, 0. t 0 m g / k g is given daily for a period of seven days, lordosis i n h i b i t i o n tested on t h e last day of i n j e c t i o n with the a p p r o p r i a t e h o r m o n e t r e a t m e n t , is smaller t h a n after a single injection. Significantly m o r e females r e s p o n d w i t h lordosis after m u l t i p l e i n j e c t i o n s t h a n after a single one, w h e n tested at the time of a m a x i m a l effect, 10 rain after injection. The d u r a t i o n of the LSD i n f l u e n c e is altered also, as a significantly lower f r e q u e n c y of lordosis i n h i b i t i o n is o b t a i n e d 40 min a f t e r the LSD injection, w h e n there is a multiple t r e a t m e n t regimen in c o m p a r i s o n with a single t r e a t m e n t (X 2 = 4.88, d f 1 : p < 0 . 0 5 ) . R e p e a t e d t r e a t m e n t s with a p o m o r p h i n e in a dose t h a t significantly inhibits lordosis r e s p o n d i n g after a single injection does n o t a p p e a r to have a n y effect c o m p a r a b l e to t h a t of LSD, w h e n tested u n d e r the same c o n d i t i o n s . The p a t t e r n of lordosis r e s p o n d i n g after seven daily injections of
LSD OR APOMORPHINE
AND LORDOSIS TABLE
RESPONSE
623
1
apomorphine at 0.5 m g / k g d o e s n o t d i f f e r s i g n i f i c a n t l y f r o m t h e i n f l u e n c e o f a single i n j e c t i o n at t h i s d o s e . A f t e r a series o f six d a i l y a p o m o r p h i n e injections p r e c e d i n g o n e d o s e o f L S D , t h e r e is a n e f f e c t o f L S D n o t d i f f e r e n t f r o m t h e a c u t e i n f l u e n c e o f t h e s a m e d o s e as s h o w n b y T a b l e 3 A. T h e r e v e r s e o r d e r o f t r e a t m e n t s d o e s n o t give a n y s i g n i f i c a n t r e s u l t s e i t h e r ( T a b l e 3 B): There was an obvious qualitative difference, however, b e t w e e n f e m a l e s r e c e i v i n g a single d o s e o f L S D a n d t h o s e g e t t i n g L S D a f t e r a series o f a p o m o r p h i n e i n j e c t i o n s . T h e latter groups resisting and rejecting the mounting attempts b y t h e m a l e s to a n e x t e n t n o t o b s e r v e d a f t e r o t h e r treatments. R e p e a t e d t r e a t m e n t s w i t h L S D d o n o t s e e m to a f f e c t l o c o m o t o r b e h a v i o r in a n y w a y d i f f e r e n t f r o m a single t r e a t m e n t w i t h a n e q u a l d o s e , as i n d i c a t e d b y t h e r e s u l t s in T a b l e 4 (t = 0 . 8 7 , df 9; NS). A p o r n o r p h i n e , o n t h e o t h e r hand, acts differently under the two treatment regimens, w h e n t e s t e d o n l o c o m o t o r e x p l o r a t i o n . A single d o s e o f apomorphine suppresses the behavior, while six daily
EFFECTS OF SINGLE DOSE AND FOUR REPEATED DOSES OF LSD ON DISPLAY OF LORDOSIS IN THE FEMALE RAT Percentage F e m a l e s Responding preinj. 10 40 90 180 min N
T r e a t m e n t mg/kg Saline L S D 0.10 x LSD0.10x4 L S D 0.25 x L S D 0.25 x L S D 0.50 x L S D 0.50 x
71 67 73 68 52 84 50
1 1" 4 1" 4
75 25 45 0 0 0 0
79 48 73 23 24 0 11
82 92 91 73 82 58 50
71 92 100 73 94 90 89
28 24 11 22 17 31 18
(3)t (2) (1) (4) (2) (3) (2)
*Data taken from Eliasson and M e y e r s o n (7). t N u m b e r s within p a r e n t h e s e s indicate n u m b e r of replications tested.
TABLE2 EFFECTS OF SINGLE AND REPEATED INJECTIONS OF LSD AND APOMORPHINE ON DISPLAY OF LORDOSIS IN THE FEMALE RAT
T r e a t m e n t mg/kg Saline L S D 0.10 x 1 L S D 0.10 x 7 A p o m o r p h i n e 0.5 x 1 A p o m o r p h i n e 0.5 x 7
Percentage F e m a l e s Responding 40/60* 90/120 180 min
Preinj.
10
71 63 83
68 38 72~:
76 54 81~
81 79 88
71 83 94
21 (4)t 24 (2) 36 (3)
33 50
89 92
89 100
---
18 (2) 18 (2)
83 100
N
*LSD was tested 10, 40, 90 and 180 min after the last injection. A p o m o r p h i n e was tested 10, 60 and 120 min after the last injection. Saline controls have been pooled, as there were no differences between groups tested according to either schedule. t S a m e as Table 1. $Significantly different from L S D 0.10 x 1 at p < 0 . 0 5 .
TABLE3 EFFECTS OF REPEATED INJECTIONS OF APOMORPHINE+SINGLE INJECTION OF LSD AND OF REPEATED INJECTIONS OF LSD+SINGLE INJECTION OF APOMORPHINE ON THE FREQUENCY OF LORDOSIS RESPONDING
T r e a t m e n t mg/kg A.
B.
Apomorphine L S D 0.1 Apomorphine L S D 0.1 L S D 0.1 L S D 0.1 x 6 + Apomorphine L S D 0.1 x 6 + Apomorphine Apomorphine Apomorphine
Percentage F e m a l e s Responding 40/60* 90/120 180 min
Preinj.
10
N
75
17
58
83
100
12 ( l ) t
61 82
14 32
64 50
90 86
90 86
28 (3) 22 (2)
0.2
63
50
69
88
--
16 (2)
0.5 0.2 0.5
79 83 85
21 42 40
83 92 60
83 92 95
----
24 (2) 12 (1) 20 (2)
0.2 x 6 + 0.5 x 6+
*tSee Tables 1 and 2.
624
ELIASSON TABLE 4
EFFECTS OF SINGLE AND REPEATED DOSES OF LSD AND APOMORPHINE ON LOCOMOTOR EXPLORATORY ACTIVITY (N = 10) Treatment mg/kg
Average Score _+ SE
Saline LSD 0.10 x I LSD 0. l0 x 6
775.5 635.4 577.9 447.4 1001.2 723.3
Apomorphine 0.5 x 1 Apomorphine 0.5 x 6 Apomorphine 0.5 x 5 + NaCI
+ 32.61 -4- 47.03 + 19.63+ _+ 36.05" _+ 43.18" _+ 77.98 (n - 6)
*Significantly different from saline controls at p <0.05. +Significantly different from saline controls at p<0.01, as tested by Students t-test (Two-tailed). injections preceding testing seem to stimulate it. There is a highly significant difference b e t w e e n these two t r e a t m e n t schedules (t = 6.78, d f 9 ; p < 0.01 ). This e f f e c t is e n h a n c e d by the results o b t a i n e d in the group treated w i t h r e p e a t e d doses of a p o m o r p h i n e and tested after an injection o f saline. S t e r e o t y p e sniffing was observed u n d e r b o t h conditions. L o c o m o t o r behavior in b o t h LSD and a p o m o r p h i n e treated animals differs f r o m the saline group, when the drugs have been given r e p e a t e d l y (t = 3.74, a~f 9; p < 0 . 0 1 and t = 2.98, d f 9; p < 0 . 0 5 , respectively) h o w e v e r , the direction o f this deviation is d i f f e r e n t for each c o m p o u n d . DISCUSSION With r e p e a t e d doses of LSD tolerance to the i n h i b i t o r y influence of this drug on l o r d o t i c r e s p o n d i n g in the o v a r i e c t o m i z e d , estrogen and p r o g e s t e r o n e treated female rat, does appear to develop. There is an a t t e n u a t i o n of the lordosis inhibition and a clear abbreviation of the d u r a t i o n of the LSD effect. A certain length of time seems to be a prerequisite for this action to develop, since tolerance is obtained after seven or m o r e daily injections, but n o t after only four at the same dose. Larger doses of LSD, w h i c h in single injections have a longer d u r a t i o n and a greater effect, are not effective in inducing tolerance over a s h o r t e r period. A p o m o r p h i n e , when c o m p a r e d u n d e r identical c o n d i t i o n s , does n o t s h o w any evidence o f tolerance, there is no m o d i f i c a t i o n of the effect of a single t r e a t m e n t , with the presently used doses and n u m b e r of injections. Tolerance to r e p e a t e d doses of LSD has previously b e e n s h o w n on more c o m p l e x kinds of c o n d i t i o n e d behavior [5, 12, 13, 18, 3 0 ] , where the r e s p o n s e typically is i n t e r r u p t e d by LSD for varying periods o f time. Daily injections and daily testing show this period to be gradually s h o r t e n e d over 3 - 4 or 7 8 days with doses c o m p a r a b l e to the
presently used one. The results o b t a i n e d on lordosis r e s p o n d i n g are in a g r e e m e n t with these studies in that the maximal disrupting effect is a t t e n u a t e d and the d u r a t i o n o f the effect is decreased by r e p e a t e d injections of LSD. However, four days do not appear to be e n o u g h for tolerance to LSD to develop, w h e n tested on the unlearned lordosis response. In previous investigations p r e t r e a t m e n t with a s e r o t o n i n s y n t h e s i s i n h i b i t o r , PCPA, was f o u n d to have an e n h a n c i n g influence on the LSD suppression of lordosis. I n t e r f e r e n c e with the b i o s y n t h e s i s of catecholamines by p r e t r e a t m e n t with c~-methyl-para-tyrosine in c o m p a r a b l e tests if a n y t h i n g s h o r t e n e d the duration of the LSD effect [ 7 ] . In addition, the p r e v e n t i o n of lordosis inhibition, when induced by a p o m o r p h i n e , by p i m o z i d e , a d o p a m i n e r e c e p t o r blocking agent, did n o t take place when lordosis r e s p o n d i n g was inhibited by LSD. Also here was there an abbreviated effect [ 8 ] . Both o f these findings indicate differential involvements o f d o p a m i n e and serotonin r e c e p t o r s in the effects on lordosis inhibition by a p o m o r p h i n e and LSD, respectively. The influence of p i m o z i d e on the a p o m o r p h i n e effect was i n t e r p r e t e d as a direct influence on d o p a m i n e r e c e p t o r s , however, this does n o t seem to be the case for the action of LSD. The significant lordosis i n h i b i t o r y effects of this c o m p o u n d can be o b t a i n e d also in small doses w i t h o u t any significant effects on e.g. l o c o m o t o r behavior, f u r t h e r indicating t h a t the action o f LSD on lordosis is m o s t likely due to an e f f e c t on serotonergic receptors, mainly. These i n t e r p r e t a t i o n s could be e x t e n d e d to include the present results. When r e c e p t o r s have been stimulated r e p e a t e d l y by LSD there is a difference in sensitivity to the last injection in the series c o m p a r e d to that of a single one, as tested on the lordosis. There is tolerance as the original effect is decreased. That this tolerance to LSD is p r o b a b l y not due to a change in m e t a b o l i s m is suggested by the findings o f Winter [ 3 0 ] . In his study a dose, equivalent to the one inducing tolerance in the present investigation, did give tolerance on a barpressing test. There was also no difference in its effects on brain and liver c o n c e n t r a t i o n s of LSD b e t w e e n groups receiving r e p e a t e d t r e a t m e n t s and groups getting a single injection, nor were these c o n c e n t r a t i o n s of different durations in respective groups. F r e e d m a n and Boggan [ 14] r e p o r t e d similar observations. When a p o m o r p h i n e is tested u n d e r the same c o n d i t i o n s the influence after seven injections on lordosis is not significantly different f r o m that of a single one. That it is possible to m o d i f y a response by repeated injections of a p o m o r p h i n e over a similar time period is indicated by the test of l o c o m o t o r behavior, which was facilitated by repeated apomorphine treatments. Present data taken t o g e t h e r with previous work [7,8] s t r e n g t h e n s the view that lordosis inhibition by LSD is more likely m e d i a t e d by an action primarily on serotonergic than on d o p a m i n e r g i c m e c h a n i s m s .
REFERENCES 1. Abramson, H. A. Tolerance to LSD-25 and a theory of psychoses. In: Neuropharmacology, edited by H. A. Abramson. New York: Josiah Macy, Jr. Foundation, 1956. 2. Aghajanian, G. K., W. E. Foote and M. H. Sheard. Lysergic acid diethylamide: Sensitive neuronal units in the midbrain raphe. Science 16 !: 706-708, 1968. 3. And6n, N. -E., A. Rubenson, K. Fuxe and T. H6kfelt. Evidence for dopaminergic receptor stimulation by apomorphine. J. Pharm. Pharmac. 19: 627-629, 1967.
4. And~n, N. -E., H. Corrodi, K. Fuxe and T. H6kfelt. Evidence for a central 5-hydroxytryptamine receptor stimulation by lysergic acid dietbylamide. Br. J. Pharmac. 34: 1-7, 1968. 5. Appel, J. B. and D. X. Freedman. Tolerance and crosstolerance among psychotominretic drugs. Psychopharmacologia 13: 267-274, 1968. 6. P,oggan, W. O. and D. X. Freedman. LSD tolerance and serotonin metabolism. Fedn Proc. 32: 694, Abstract. 1973.
LSD OR A P O M O R P H I N E AND L O R D O S I S R E S P O N S E
7. Eliasson, M. and B. J. Meyerson. The effects of lysergic acid diethylamide on copulatory behavior in the female rat. Neuropharmacology, in press, 1976. 8. Eliasson, M. and B. J. Meyerson. Comparison of the actions of LSD and apomorphine on the copulatory response in the female rat. Psychopharmacologia, in press, 1976. 9. Ernst, A. M. Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychopharmacologia 10: 316-323, 1967. 10. Everitt, B. J., K. Fuxe and T. H6kfelt. Inhibitory role of dopamine and 5-hydroxytryptamine in the sexual behavior of female rats. Eur. J. Pharmac. 29: 187-191, 1974. 11. Everitt, B. J., K. Fuxe, T. H6kfelt and G. Jonsson. Role of monoamines in the control by hormones of sexual receptivity in the female rat. J. comp. physiol. Psychol. 89: 556-572, 1975. 12. Freedman, D. X., G. K. Aghajanian, B. Rosner and E. M. Ornitz. Patterns of tolerance to lysergic acid diethylamide and mescaline in rats. Science 127:1173-1174, 1958. 13. Freedman, D. X., J. B. Appel, F. R. Hartman and M. E. Molliver. Tolerance to behavioral effects of LSD-25 in rat. J. Pharmac. exp. Ther. 143: 309-313, 1964. 14. Freedman, D. X. and W. O. Boggan. Brain serotonin metabolism after tolerance dosage of LSD. Adv. Biochem. Psychopharmac. 10: 151-157, 1974. 15. Hamilton, C. L. Effects of LSD-25 and amphetamine on a running response in the rat. Archs gen. Psychiat. 2: 104-109, 1960. 16. Isbell, H., H. F. Fraser. A. Wikler and R. E. Belleville. Tolerance to diethylamide of lysergic acid (LSD-25). Fedn Proc. 14: 354, 1955. 17. Isbell, H., R. E. Belleville, H. F. Fraser, A. Wikler and C. R. Logan. Studies on lysergic acid diethylamide (LSD-25). I. Effects in former morphine addicts and development of tolerance during chronic intoxication. A.M.A. Archs Neurol. Psychiat. 76: 468-478, 1956. 18. Mahler, D. J. and F. L. Humoller, Effect of lysergic acid diethylamide and bufotenine on performance of trained rats. Proc. Soc. exp. Biol. Med. 102: 697-701, 1959. 19. Meyerson, B. J. Central nervous monoamines and hormone induced estrous behavior in the spayed rat. Acta physiol. scan& 63: Suppl. 241, 1964.
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20. Meyerson, B. J. The effect of neuropharmacological agents on hormone-activated estrous behaviour in ovariectomized rats. Archs Int. Pharmacodyn. 150: 4-33, 1964. 21. Meyerson, B. J., M. Eliasson, L. Lindstr6m, A. Michanek and A. Ch. S6derlund. Monoamines and female sexual behavior. In: Psychopharmacology, Sexual Disorders and Drug Abuse, edited by T. A. Ban et al. Amsterdam: North-Holland Publishing Company, 1973, pp. 463-473. 22. Meyerson, B. J., H. Carrer and M. Eliasson. 5-Hydroxytryptamine and sexual behavior in the female rat. Adv. Biochem. Psychopharmac. 11: 229-242, 1974. 23. Peters, D.A.V. Comparison of the chronic and acute effects of D-lysergic acid diethylamide (LSD) treatment on rat brain serotonin and norepinephrine. Biochem. Pharmac. 23: 231-237, 1974. 24. Rosenberg, D. E., A. B. Wolbach, Jr., E. J. Miner and H. Isbell. Observations on direct and cross tolerance with LSD and D-amphetamine in man. Psychopharmacologia 5: 1-15, 1963. 25. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 26. Smith, R. C., W. O. Boggan and D. X. Freedman. Effects of single and multiple doses of LSD on endogenous levels of brain tyrosine and catecholamines. Psychopharmacologia 42: 271-276, 1975. 27. Svensson, T. H. and G. Thieme. An investigation of a new instrument to measure motor activity of small animals. Psychopharmacologia 14:157-163, 1969. 28. Ward, I. L., W. R. Crowley, F. P. Zemlan and D. L. Margules. Monoaminergic mediation of female sexual behavior. J. cornp. physiol. PsychoL 88: 53-61, 1975. 29. Winer, B. J. Statistical Principles in Experimental Design. 2nd Ed. New York: McGraw-Hill, 1971. 30. Winter, J. C. Tolerance to a behavioral effect of lysergic acid diethylamide and crosstolerance to mescaline in the rat: Absence of a metabolic component. J. Pharmac. exp. Ther. 178: 625-630, 1971. 31. Young, W. C. The hormones and mating behavior. In: Sex and lnternal secretions, edited by W. C. Young, Vol. 2. Baltimore, Maryland: Williams and Wilkins Co., 1961. 32. Zemlan, F. P., I. L. Ward, W. R. Crowley and D. L. Margules. Activation of lordotic responding in female rats by suppression of serotonergic activity. Science 179:1010-1011, 1973.
Actions of Repeated Injections of LSD and Apomorphine on the Copulatory Response of Female Rats MONA ELIASSON
Department o f Medical Pharmacology, Box 573, S-751 23 UPPSALA, Sweden (Received 19 August 1976) ELIASSON, M. Actions of repeated in/ections of LSD and apomorphine on the copulatory response of female rats. PHARMAC. BIOCHEM. BEHAV. 5(6) 621-625, 1976. - LSD, a serotonin receptor stimulating agent, inhibits copulatory behavior (lordosis response) in the ovariectomized and estrogen + progesterone treated female rat. The same effect is obtained by apomorphine, a dopamine receptor stimulating compounds. The lordosis has been shown to be dependent on serotonin, but also dopamine has been implicated in its mediation. Tolerance develops to certain responses after repeated injections of LSD and in the present study the influence of apomorphine and LSD was compared, when given in repeated doses. Possible cross-tolerance between the two compounds was also tested on the frequency of lordosis responding in ovariectomized and hormone treated female rats. Tolerance to LSD develops over seven days, while the suppressing influence of apomorphine on lordosis in seven repeated doses is not significantly altered from that of a single dose. No cross-tolerance was observed on the lordosis response with either order of treatments. Repeated doses of LSD did not influence locomotor activity differently from a single dose, while repeated doses of apomorphine enhanced this response in comparison with the effect of an acute dose. These results indicate differential sensitivity to the repeated treatments and further support an interpretation of the LSD effects on lordosis responding to be primarily on serotonergic rather than dopaminergic receptors. LSD
Apomorphine
Lordosis response
Tolerance
single doses o f LSD. One such behavior c o m p o n e n t is the c o p u l a t o r y r e s p o n s e o f the female rat ( l o r d o t i c response). This r e s p o n s e , which consists o f an arching o f the back, head and p e r i n e u m elevation and tail deviation, is d e p e n dent on ovarian h o r m o n e s [31] and is readily elicited in receptive female rats by a m o u n t i n g male. Much evidence is available d e m o n s t r a t i n g i n h i b i t i o n of the lordosis r e s p o n s e to be m e d i a t e d by activation o f s e r o t o n i n and also d o p a m i n e r g i c n e u r o n s in o v a r i e c t o m i z e d female rats t r e a t e d with e s t r o g e n and p r o g e s t e r o n e [19, 20, 2 2 ] . This also seems to be the case w h e n the lordosis response is studied in o v a r i e c t o m i z e d female rats t r e a t e d with r e p e a t e d doses o f e s t r o g e n and n o p r o g e s t e r o n e [11, 28, 3 2 ] . LSD in a r a t h e r small dose (0.05 /ag/kg) lacking significant e f f e c t s on e.g. l o c o m o t o r behavior, inhibits lordosing in female rats receiving estrogen + p r o g e s t e r o n e [ 7 , 8 ] . A p o m o r p h i n e , a c o m p o u n d with d o p a m i n e r e c e p t o r stimulating p r o p e r t i e s [3,9] also has an i n h i b i t o r y action on the lordosis behavior activated by estrogen and p r o g e s t e r o n e [ 8 , 2 2 ] . A n o t h e r d o p a m i n e agonist (ET 495) is r e p o r t e d to have similar effects, w h e n lordosis is induced by estrogen only [ 1 0 ] . The influence of a p o m o r p h i n e was f o u n d to be c o m p l e t e l y
T O L E R A N C E appears in certain behavioral r e s p o n s e s to lysergic acid d i e t h y l a m i d e (LSD). This has b e e n f o u n d in animal as well as h u m a n studies, and is seen in the clinic as well as in the l a b o r a t o r y [ 1, 16, 17, 2 4 ] . When tested in l a b o r a t o r y rats, tolerance to LSD develops for such diverse behaviors as r o p e - c l i m b i n g [12,18] and bar pressing for f o o d r e i n f o r c e m e n t [ 5 , 1 3 ] . On the o t h e r h a n d , in a behavioral p e r f o r m a n c e m a i n t a i n e d by m e a n s o f escape f r o m electric f o o t shock, tolerance to LSD in c o m p a r a b l e doses does a p p a r e n t l y n o t emerge, but e x t i n c t i o n o f the r e s p o n s e is delayed [ 15]. The behavioral m o d i f i c a t i o n s appearing as a c o n s e q u e n c e of r e p e a t e d LSD i n j e c t i o n s are a c c o m p a n i e d by certain biochemical alterations t h a t are related to brain m o n o amines. R e p e a t e d doses o f a m a g n i t u d e c o m p a r a b l e to those used in behavioral studies give an u n c h a n g e d level o f s e r o t o n i n , while t u r n o v e r is significantly increased [ 2 3 ] . Higher doses seem to affect the c a t e c h o l a m i n e levels in a d d i t i o n to t h o s e o f s e r o t o n i n [6, 14, 2 6 ] . LSD is c o n s i d e r e d to be a s e r o t o n i n r e c e p t o r stimulating c o m p o u n d [2,4] and in a c c o r d a n c e with this, f u n c t i o n s m e d i a t e d by s e r o t o n i n have been f o u n d to be i n f l u e n c e d by
1This research was supported by grant No. B75-14X-64-1 l A from the Swedish Medical Research Council to 8engt J. Meyerson. 2Portions of this research was presented at the XIV Scandinavian Congress of Physiology and Pharmacology, Bergen, 1973, and an Abstract appeared in Acta physiol, scand., Suppl. 386, 86, 1973. 3The hormones were generous gifts supplied by N.V. Organon through Erco Ltd., Stockholm. 621
622
ELIASSON
counteracted by pretreatment with a dopamine receptor blocker ( p i m o z i d e ) , while t h e LSD i n d u c e d i n h i b i t i o n of lordosis was o n l y decreased in its d u r a t i o n . This was t a k e n as an i n d i c a t i o n of the differential m e c h a n i s m s of a c t i o n b e h i n d the effects of the t w o drugs on lordosis r e s p o n d i n g
[8]. The p u r p o s e of the present investigation was to f u r t h e r analyze the e x t e n t of d o p a m i n e r g i c relative to s e r o t o n e r g i c i n v o l v e m e n t in the LSD i n d u c e d i n h i b i t i o n of the lordosis response b y c o m p a r i n g it to a p o m o r p h i n e , w h e n b o t h drugs are given in r e p e a t e d doses. The d e v e l o p m e n t of t o l e r a n c e and possible cross-tolerance, m i g h t f u r t h e r elucidate the r e l a t i o n s h i p of these t w o drugs and t h e i r effects on lordosis responding. Lordosis has the advantage, in a d d i t i o n to its k n o w n r e l a t i o n to s e r o t o n i n and d o p a m i n e , of n o t requiring any t r a i n i n g before its m a n i f e s t a t i o n , m a k i n g it particularly suitable for a s t u d y of t o l e r a n c e . METHOD
Animals O v a r i e c t o m i z e d female rats of the Sprague-Dawley strain (Purchased as Specific P a t h o g e n Free, Anticimex, S t o c k h o l m ) were used. They were h o u s e d 5 - 6 to a cage in stainless steel cages, where tap w a t e r and c o m m e r c i a l rat pellets ( A n t i c i m e x 210 or R 3) were available at all times. The a n i m a l r o o m held a c o n s t a n t t e m p e r a t u r e (21 + I°C), had forced v e n t i l a t i o n and c o n t r o l l e d h u m i d i t y . The dayn i g h t cycle was reversed (lights on from 9 p.m. to 9 a.m.). Wistar male rats with considerable sexual e x p e r i e n c e were used for testing of the females. T h e males were h o u s e d in individual cages (40 × 40 × 30 cm), w h i c h also served as test cages, in a d i f f e r e n t section of the animal r o o m . Materials infected. The h o r m o n e s , estradiol b e n z o a t e (EB) and p r o g e s t e r o n e ( b o t h f r o m O r g a n o n ) , were dissolved in olive oil and injected s u b c u t a n e o u s l y . A p o m o r p h i n e and lysergic acid d i e t h y l a m i d e (LSD) t a r t r a t e ( b o t h f r o m Sandoz) were dissolved in saline. LSD was injected intraperitoneally, while a p o m o r p h i n e was given s u b c u t a n e o u s l y . F r e s h s o l u t i o n s were m a d e up of the two latter c o m p o u n d s for every drug test. The doses w h i c h a p p e a r in the t e x t and tables refer to t h e f o r m of the c o m p o u n d s given above. Behavioral observations. Before being used in a drug test each group of 10 12 animals was subjected to a s t a n d a r d test w i t h the regular doses of h o r m o n e s used (10/.zg/kg EB and 0.4 mg p r o g e s t e r o n e 48 hr later) t o asses the level of lordosis r e s p o n d i n g in rela{ion to w h a t is the s t a n d a r d of the l a b o r a t o r y . This is a b o u t 60 80% response in a g r o u p during the h e i g h t of the h o r m o n e effect, 6 - 7 hr a f t e r progesterone. The values before each test are i n d i c a t e d in the tables. The h o r m o n e s were injected at 8 a.m. and testing was p e r f o r m e d b e t w e e n n o o n and 5 p.m. at certain intervals, w h e n t h e h o r m o n e influence is relatively stable. Animals given r e p e a t e d injections of drug were always injected at the same time of the day, i.e. 24 hr apart, with lordosis testing b e g i n n i n g 10 m i n after the last injection. The females were tested individually in the h o m e cages of the males. Only absence or presence of lordosis was scored for each m o u n t and a m a x i m u m of six m o u n t s b y two d i f f e r e n t males was allowed. To be rated as positive by the observer the female had to show two consecutive lordoses with h e a d and p e r i n e u m raised and the tail deviated each time. Thus, the measure for each t r e a t m e n t was t h e percentage females showing lordosis a c c o r d i n g to our
criterion on a particular test. ( F o r f u r t h e r details regarding m e t h o d s see Eliasson and M e y e r s o n ] 7 ] ) . Locomotor exploratory activity. A n o t h e r i n d i c a t i o n of drug effects was the a m o u n t of l o c o m o t o r e x p l o r a t o r y activity e x h i b i t e d during a 10 m i n test. T h e animals were placed one at a t i m e in a testing b o x located o n t o p of an A n i m e x m e t e r ( F a r a d Inc., H~igersten, Sweden), where t h e i r l o c o m o t o r activity was r e c o r d e d a u t o m a t i c a l l y and a c c u m u lated for t h e whole testing period. T h e sensitivity of the a p p a r a t u s was adjusted so t h a t fine m o t o r activity like g r o o m i n g and sniffing was n o t r e c o r d e d . Testing was p e r f o r m e d in a dark and quiet r o o m w i t h no observer present. This t o o k place after r e p e a t e d and single injections of LSD or a p o m o r p h i n e in i n d e p e n d e n t groups. The animals were the same as those p a r t i c i p a t i n g in the lordosis s t u d y and those receiving m u l t i p l e i n j e c t i o n s had received also EB t h e m o r n i n g of the day b e f o r e testing and were scheduled to be tested on lordosis on the following day. (Svensson and T h i e m e [27] give t h e details regarding the testing apparatus). Statistics. Differences b e t w e e n groups of i n d e p e n d e n t animals were a n a l y z e d statistically using the Chi square test corrected for c o n t i n u i t y [ 25 ]. F o r certain t r e a t m e n t s these groups have b e e n c o m b i n e d in the tables for a easier p r e s e n t a t i o n of results. Differences b e t w e e n l o c o m o t o r scores in groups receiving single and groups receiving m u l t i p l e injections were analyzed t h r o u g h the use of the t-test [ 2 9 ] .
RESULTS AS is s h o w n by Table 1, t r e a t m e n t w i t h r e p e a t e d injections of LSD for four days and w i t h testing c o m m e n c ing 10 m i n after the last injection does n o t give any altered r e s p o n s e on the lordosis i n h i b i t i o n test at a dose of 0.10 mg/kg. This is a dose t h a t in a single i n j e c t i o n significantly decreases the p e r c e n t a g e lordosis r e s p o n d i n g females, enh a n c e s startle r e s p o n d i n g and gives some decrease of l o c o m o t o r activity [ 7 , 8 ] . Still after r e p e a t e d injections during four days the general a p p e a r a n c e of t h e animals is more n o r m a l , t h e r e is less piloerection, fewer flight responses to s u d d e n stimuli, b u t ataxia of the hindlegs is still present. The larger doses of 0.25 and 0.50 mg/kg, w h e n a d m i n i s t e r e d for four days, also do n o t a t t e n u a t e the lordosis i n h i b i t o r y effect of LSD in c o m p a r i s o n w i t h single injections. A longer period of daily LSD injections does give an altered response as is evident f r o m Table 2. When LSD, 0. t 0 m g / k g is given daily for a period of seven days, lordosis i n h i b i t i o n tested on t h e last day of i n j e c t i o n with the a p p r o p r i a t e h o r m o n e t r e a t m e n t , is smaller t h a n after a single injection. Significantly m o r e females r e s p o n d w i t h lordosis after m u l t i p l e i n j e c t i o n s t h a n after a single one, w h e n tested at the time of a m a x i m a l effect, 10 rain after injection. The d u r a t i o n of the LSD i n f l u e n c e is altered also, as a significantly lower f r e q u e n c y of lordosis i n h i b i t i o n is o b t a i n e d 40 min a f t e r the LSD injection, w h e n there is a multiple t r e a t m e n t regimen in c o m p a r i s o n with a single t r e a t m e n t (X 2 = 4.88, d f 1 : p < 0 . 0 5 ) . R e p e a t e d t r e a t m e n t s with a p o m o r p h i n e in a dose t h a t significantly inhibits lordosis r e s p o n d i n g after a single injection does n o t a p p e a r to have a n y effect c o m p a r a b l e to t h a t of LSD, w h e n tested u n d e r the same c o n d i t i o n s . The p a t t e r n of lordosis r e s p o n d i n g after seven daily injections of
LSD OR APOMORPHINE
AND LORDOSIS TABLE
RESPONSE
623
1
apomorphine at 0.5 m g / k g d o e s n o t d i f f e r s i g n i f i c a n t l y f r o m t h e i n f l u e n c e o f a single i n j e c t i o n at t h i s d o s e . A f t e r a series o f six d a i l y a p o m o r p h i n e injections p r e c e d i n g o n e d o s e o f L S D , t h e r e is a n e f f e c t o f L S D n o t d i f f e r e n t f r o m t h e a c u t e i n f l u e n c e o f t h e s a m e d o s e as s h o w n b y T a b l e 3 A. T h e r e v e r s e o r d e r o f t r e a t m e n t s d o e s n o t give a n y s i g n i f i c a n t r e s u l t s e i t h e r ( T a b l e 3 B): There was an obvious qualitative difference, however, b e t w e e n f e m a l e s r e c e i v i n g a single d o s e o f L S D a n d t h o s e g e t t i n g L S D a f t e r a series o f a p o m o r p h i n e i n j e c t i o n s . T h e latter groups resisting and rejecting the mounting attempts b y t h e m a l e s to a n e x t e n t n o t o b s e r v e d a f t e r o t h e r treatments. R e p e a t e d t r e a t m e n t s w i t h L S D d o n o t s e e m to a f f e c t l o c o m o t o r b e h a v i o r in a n y w a y d i f f e r e n t f r o m a single t r e a t m e n t w i t h a n e q u a l d o s e , as i n d i c a t e d b y t h e r e s u l t s in T a b l e 4 (t = 0 . 8 7 , df 9; NS). A p o r n o r p h i n e , o n t h e o t h e r hand, acts differently under the two treatment regimens, w h e n t e s t e d o n l o c o m o t o r e x p l o r a t i o n . A single d o s e o f apomorphine suppresses the behavior, while six daily
EFFECTS OF SINGLE DOSE AND FOUR REPEATED DOSES OF LSD ON DISPLAY OF LORDOSIS IN THE FEMALE RAT Percentage F e m a l e s Responding preinj. 10 40 90 180 min N
T r e a t m e n t mg/kg Saline L S D 0.10 x LSD0.10x4 L S D 0.25 x L S D 0.25 x L S D 0.50 x L S D 0.50 x
71 67 73 68 52 84 50
1 1" 4 1" 4
75 25 45 0 0 0 0
79 48 73 23 24 0 11
82 92 91 73 82 58 50
71 92 100 73 94 90 89
28 24 11 22 17 31 18
(3)t (2) (1) (4) (2) (3) (2)
*Data taken from Eliasson and M e y e r s o n (7). t N u m b e r s within p a r e n t h e s e s indicate n u m b e r of replications tested.
TABLE2 EFFECTS OF SINGLE AND REPEATED INJECTIONS OF LSD AND APOMORPHINE ON DISPLAY OF LORDOSIS IN THE FEMALE RAT
T r e a t m e n t mg/kg Saline L S D 0.10 x 1 L S D 0.10 x 7 A p o m o r p h i n e 0.5 x 1 A p o m o r p h i n e 0.5 x 7
Percentage F e m a l e s Responding 40/60* 90/120 180 min
Preinj.
10
71 63 83
68 38 72~:
76 54 81~
81 79 88
71 83 94
21 (4)t 24 (2) 36 (3)
33 50
89 92
89 100
---
18 (2) 18 (2)
83 100
N
*LSD was tested 10, 40, 90 and 180 min after the last injection. A p o m o r p h i n e was tested 10, 60 and 120 min after the last injection. Saline controls have been pooled, as there were no differences between groups tested according to either schedule. t S a m e as Table 1. $Significantly different from L S D 0.10 x 1 at p < 0 . 0 5 .
TABLE3 EFFECTS OF REPEATED INJECTIONS OF APOMORPHINE+SINGLE INJECTION OF LSD AND OF REPEATED INJECTIONS OF LSD+SINGLE INJECTION OF APOMORPHINE ON THE FREQUENCY OF LORDOSIS RESPONDING
T r e a t m e n t mg/kg A.
B.
Apomorphine L S D 0.1 Apomorphine L S D 0.1 L S D 0.1 L S D 0.1 x 6 + Apomorphine L S D 0.1 x 6 + Apomorphine Apomorphine Apomorphine
Percentage F e m a l e s Responding 40/60* 90/120 180 min
Preinj.
10
N
75
17
58
83
100
12 ( l ) t
61 82
14 32
64 50
90 86
90 86
28 (3) 22 (2)
0.2
63
50
69
88
--
16 (2)
0.5 0.2 0.5
79 83 85
21 42 40
83 92 60
83 92 95
----
24 (2) 12 (1) 20 (2)
0.2 x 6 + 0.5 x 6+
*tSee Tables 1 and 2.
624
ELIASSON TABLE 4
EFFECTS OF SINGLE AND REPEATED DOSES OF LSD AND APOMORPHINE ON LOCOMOTOR EXPLORATORY ACTIVITY (N = 10) Treatment mg/kg
Average Score _+ SE
Saline LSD 0.10 x I LSD 0. l0 x 6
775.5 635.4 577.9 447.4 1001.2 723.3
Apomorphine 0.5 x 1 Apomorphine 0.5 x 6 Apomorphine 0.5 x 5 + NaCI
+ 32.61 -4- 47.03 + 19.63+ _+ 36.05" _+ 43.18" _+ 77.98 (n - 6)
*Significantly different from saline controls at p <0.05. +Significantly different from saline controls at p<0.01, as tested by Students t-test (Two-tailed). injections preceding testing seem to stimulate it. There is a highly significant difference b e t w e e n these two t r e a t m e n t schedules (t = 6.78, d f 9 ; p < 0.01 ). This e f f e c t is e n h a n c e d by the results o b t a i n e d in the group treated w i t h r e p e a t e d doses of a p o m o r p h i n e and tested after an injection o f saline. S t e r e o t y p e sniffing was observed u n d e r b o t h conditions. L o c o m o t o r behavior in b o t h LSD and a p o m o r p h i n e treated animals differs f r o m the saline group, when the drugs have been given r e p e a t e d l y (t = 3.74, a~f 9; p < 0 . 0 1 and t = 2.98, d f 9; p < 0 . 0 5 , respectively) h o w e v e r , the direction o f this deviation is d i f f e r e n t for each c o m p o u n d . DISCUSSION With r e p e a t e d doses of LSD tolerance to the i n h i b i t o r y influence of this drug on l o r d o t i c r e s p o n d i n g in the o v a r i e c t o m i z e d , estrogen and p r o g e s t e r o n e treated female rat, does appear to develop. There is an a t t e n u a t i o n of the lordosis inhibition and a clear abbreviation of the d u r a t i o n of the LSD effect. A certain length of time seems to be a prerequisite for this action to develop, since tolerance is obtained after seven or m o r e daily injections, but n o t after only four at the same dose. Larger doses of LSD, w h i c h in single injections have a longer d u r a t i o n and a greater effect, are not effective in inducing tolerance over a s h o r t e r period. A p o m o r p h i n e , when c o m p a r e d u n d e r identical c o n d i t i o n s , does n o t s h o w any evidence o f tolerance, there is no m o d i f i c a t i o n of the effect of a single t r e a t m e n t , with the presently used doses and n u m b e r of injections. Tolerance to r e p e a t e d doses of LSD has previously b e e n s h o w n on more c o m p l e x kinds of c o n d i t i o n e d behavior [5, 12, 13, 18, 3 0 ] , where the r e s p o n s e typically is i n t e r r u p t e d by LSD for varying periods o f time. Daily injections and daily testing show this period to be gradually s h o r t e n e d over 3 - 4 or 7 8 days with doses c o m p a r a b l e to the
presently used one. The results o b t a i n e d on lordosis r e s p o n d i n g are in a g r e e m e n t with these studies in that the maximal disrupting effect is a t t e n u a t e d and the d u r a t i o n o f the effect is decreased by r e p e a t e d injections of LSD. However, four days do not appear to be e n o u g h for tolerance to LSD to develop, w h e n tested on the unlearned lordosis response. In previous investigations p r e t r e a t m e n t with a s e r o t o n i n s y n t h e s i s i n h i b i t o r , PCPA, was f o u n d to have an e n h a n c i n g influence on the LSD suppression of lordosis. I n t e r f e r e n c e with the b i o s y n t h e s i s of catecholamines by p r e t r e a t m e n t with c~-methyl-para-tyrosine in c o m p a r a b l e tests if a n y t h i n g s h o r t e n e d the duration of the LSD effect [ 7 ] . In addition, the p r e v e n t i o n of lordosis inhibition, when induced by a p o m o r p h i n e , by p i m o z i d e , a d o p a m i n e r e c e p t o r blocking agent, did n o t take place when lordosis r e s p o n d i n g was inhibited by LSD. Also here was there an abbreviated effect [ 8 ] . Both o f these findings indicate differential involvements o f d o p a m i n e and serotonin r e c e p t o r s in the effects on lordosis inhibition by a p o m o r p h i n e and LSD, respectively. The influence of p i m o z i d e on the a p o m o r p h i n e effect was i n t e r p r e t e d as a direct influence on d o p a m i n e r e c e p t o r s , however, this does n o t seem to be the case for the action of LSD. The significant lordosis i n h i b i t o r y effects of this c o m p o u n d can be o b t a i n e d also in small doses w i t h o u t any significant effects on e.g. l o c o m o t o r behavior, f u r t h e r indicating t h a t the action o f LSD on lordosis is m o s t likely due to an e f f e c t on serotonergic receptors, mainly. These i n t e r p r e t a t i o n s could be e x t e n d e d to include the present results. When r e c e p t o r s have been stimulated r e p e a t e d l y by LSD there is a difference in sensitivity to the last injection in the series c o m p a r e d to that of a single one, as tested on the lordosis. There is tolerance as the original effect is decreased. That this tolerance to LSD is p r o b a b l y not due to a change in m e t a b o l i s m is suggested by the findings o f Winter [ 3 0 ] . In his study a dose, equivalent to the one inducing tolerance in the present investigation, did give tolerance on a barpressing test. There was also no difference in its effects on brain and liver c o n c e n t r a t i o n s of LSD b e t w e e n groups receiving r e p e a t e d t r e a t m e n t s and groups getting a single injection, nor were these c o n c e n t r a t i o n s of different durations in respective groups. F r e e d m a n and Boggan [ 14] r e p o r t e d similar observations. When a p o m o r p h i n e is tested u n d e r the same c o n d i t i o n s the influence after seven injections on lordosis is not significantly different f r o m that of a single one. That it is possible to m o d i f y a response by repeated injections of a p o m o r p h i n e over a similar time period is indicated by the test of l o c o m o t o r behavior, which was facilitated by repeated apomorphine treatments. Present data taken t o g e t h e r with previous work [7,8] s t r e n g t h e n s the view that lordosis inhibition by LSD is more likely m e d i a t e d by an action primarily on serotonergic than on d o p a m i n e r g i c m e c h a n i s m s .
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