Pharmacology Biochemistry & Behavior, Vol. 5, pp. 583--586. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
BRIEF COMMUNICATION The Discriminability of Aspirin in Arthritic and Nonarthritic Rats ALBERT WEISSMAN
Pfizer, Inc., Groton, C T 0 6 3 4 0 (Received 3 S e p t e m b e r 1 9 7 6 )
WEISSMAN, A. The discriminability of aspirin in arthritic and nonarthritic rats. PHARMAC. BIOCHEM. BEHAV. 5(5) 583-586, 1976. - Aspirin, 56 mg/kg IP, was shown to be mildly, but significantly discriminable from saline in a group of 12 nonarthritic rats exposed to a 2-1ever fixed ratio 10 drug discrimination protocol for 56 trials. In a concurrently-tested group of 12 rats made arthritic by injection of Mycobacterium butyricum into a hind paw, the discrimination of aspirin from saline was enhanced. The results exemplify how drug discriminability may vary depending on the pathological state of the subjects exposed to drug-discrimination training. Aspirin
Drug discrimination
Adjuvant arthritis
Operant behavior
S E V E R A L s y s t e m i c a l l y - a d m i n i s t e r e d drugs are k n o w n to serve as discriminative stimuli in a p p r o p r i a t e l y t r a i n e d l a b o r a t o r y animals. This subject has n o t only b e e n freq u e n t l y reviewed [e.g., 2 ] , b u t is t h e f o c u s of active c u r r e n t research in m a n y laboratories. F o r the m o s t part, research to date has b e e n c o n c e r n e d w i t h i d e n t i f y i n g t h o s e drugs t h a t generalize to a d i s c r i m i n a t e d drug, and the specificity of a given drug cue. A m o n g the m a n y p o t e n t i a l o f f s h o o t s of this research area is t h e p r o b l e m of d e t e r m i n i n g states of an organism t h a t favor or disfavor t h e a c q u i s i t i o n of d i s c r i m i n a t i v e p r o p e r t i e s b y a drug. A l t h o u g h f o r m a l studies have n o t b e e n addressed to this p o i n t in h u m a n s , it seems evident t h a t a h u m a n o p i a t e addict s h o u l d be m o r e readily able to discriminate naloxone than a nonaddict and that a human with h e a d a c h e or a r t h r i t i s s h o u l d be m o r e able to discriminate aspirin t h a n a n o n s u f f e r e r . T h e p r e s e n t s t u d y was u n d e r t a k e n as an a t t e m p t to d e m o n s t r a t e w h e t h e r a p o o r l y d i s c r i m i n a t e d drug is b e t t e r d i s c r i m i n a t e d b y rats discriminated by rats s u b j e c t e d to p a t h o l o g i c a l c o n d i t i o n s relieved by t h a t drug. Specifically, it was addressed to t h e d e t e r m i n a t i o n of w h e t h e r or n o t aspirin can serve as a d i s c r i m i n a b l e stimulus, a n d w h e t h e r a c q u i s i t i o n of an aspirin vs saline d i s c r i m i n a t i o n develops m o r e rapidly in a r t h r i t i c t h a n in n o r m a l rats. METHOD
Animals Twenty-four individually housed Sprague-Dawley rats f r o m Charles weighing a b o u t 2 2 0 g o n arrival were liquid f o o d r e i n f o r c e m e n t d u r i n g t h e
a d u l t male a l b i n o River L a b o r a t o r i e s used. T h e y received c o n d i t i o n i n g proce583
dures described below, a n d sufficient s u p p l e m e n t a l P u r i n a rat c h o w a f t e r each session and o n w e e k e n d s to m a i n t a i n t h e i r weights at a b o u t 2 5 0 g t h r o u g h o u t the e x p e r i m e n t . Water was available ad lib in h o m e cages.
Apparatus T r a i n i n g a n d testing were a c c o m p l i s h e d in 6 identical isolated c o m m e r c i a l o p e r a n t c h a m b e r s ( F o r i n g e r t y p e ) p r o g r a m m e d b y e l e c t r o m e c h a n i c a l circuitry. T h e i m p o r t a n t w o r k i n g parts of each c h a m b e r were t w o F o r i n g e r levers m o u n t e d on each side of a m o t o r - d r i v e n dipper, and a h o u s e l i g h t w h i c h r e m a i n e d o n d u r i n g the 15 m i n session. R e i n f o r c e m e n t consisted o f 3 sec p r e s e n t a t i o n of 0.2 cc of C a r n a t i o n Slender, a c o m m e r c i a l liquid diet food consisting of s u c r o s e - s w e e t e n e d skim milk w i t h p r o t e i n , v i t a m i n s a n d minerals added, diluted 1 : 1 w i t h water.
Procedure The p r o c e d u r e was a slight m o d i f i c a t i o n of the 2-bar fixed r a t i o l 0 ( F R 10) d r u g - d i s c r i m i n a t i o n p r o t o c o l first described by C o l p a e r t et al. [3] in t h e i r studies of f e n t a n y l discrimination. F o l l o w i n g f o o d d e p r i v a t i o n to a s u s t a i n e d 2 5 0 g weight, each rat was s h a p e d to bar press, first on a c o n t i n u o u s r e i n f o r c e m e n t C R F ) s c h e d u l e o n each lever, and t h e n o n an F R s c h e d u l e t h a t was rapidly escalated to F R l 0 on each lever. These steps were a c c o m p l i s h e d w i t h a single lever in the cage, for 2 sessions o n t h e left side, and t h e n for 2 a d d i t i o n a l sessions o n the right side, o f t h e dipper. Drug t r a i n i n g was t h e n begun, using the p r o t o c o l of C o l p a e r t et al. [ 3 ] . D e p e n d i n g o n w h e t h e r the rat received aspirin or saline (see b e l o w for details of t r e a t m e n t ) ,
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r e i n f o r c e m e n t was p r o g r a m m e d exclusively o n e i t h e r t h e left or t h e right lever o n t h e F R 10 schedule. O n l y r e s p o n s e s o n t h e left lever were r e i n f o r c e d a f t e r drug administration, and only r e s p o n s e s on the r i g h t lever a f t e r saline a d m i n i s t r a t i o n . Rats were t e s t e d 5 days each w e e k u n d e r t h e a l t e r n a t i n g drug s e q u e n c e s used b y C o l p a e r t et al. [ 3 ] : aspirin-saline-saline-aspirin-aspirin and saline-aspirin-aspirin-saline-saline. To avoid t h e p o s s i b i l i t y t h a t the c o r r e c t lever for rats previously t e s t e d in t h e c h a m b e r s could serve as a cue, a very real possibility based on results of p i l o t studies, t h e s e q u e n c e of aspirin or saline t r e a t m e n t on any day was a l t e r n a t e d as t h e 4 s u b g r o u p s of 6 rats were tested. A f t e r e a c h session of 15 rain, rats were r e t u r n e d to t h e i r h o m e cages a n d fed a q u a n t i t y of P u r i n a lab c h o w equal to the d i f f e r e n c e b e t w e e n t h e i r b o d y weights, d e t e r m i n e d p r i o r to drug t r e a t m e n t , a n d 2 5 0 g. On w e e k e n d s rats were fed 1 O - 1 2 g / d a y of p e l l e t e d lab c h o w , w h i c h m a i n t a i n e d t h e i r weight r e a s o n a b l y c o n s t a n t at a b o u t 250 g. In s u m m a r y , o t h e r t h a n t h e drug t r e a t m e n t s e m p l o y e d , t h e p r i m e m o d i f i c a t i o n s o f the C o l p a e r t et al. [3] t r a i n i n g p r o c e d u r e used in this s t u d y were t h e t y p e of o p e r a n t c h a m b e r s used, the r e i n f o r c i n g stimulus, a n d the m e t h o d of depriving rats. Drug administration. T h r o u g h o u t training, rats received e i t h e r n o r m a l (0.9%) saline or aspirin s u s p e n d e d in n o r m a l saline (56 m g / k g at a v o l u m e o f 5 ml/kg). I n j e c t i o n s were m a d e i n t r a p e r i t o n e a l l y 1 h r p r i o r to e x p o s u r e of the rat to the o p e r a n t c h a m b e r . A f t e r 14 days o f training, a d j u v a n t a r t h r i t i s was prod u c e d in 12 r a n d o m l y c h o s e n rats by a close a p p r o x i m a t i o n of t h e p r o c e d u r e of Walz et al. [ 7 ] , w h i c h consisted of a single i n t r a d e r m a l i n j e c t i o n of 0.75 mg of Mycobacterium butyricum (Difco L a b o r a t o r i e s , D e t r o i t , MI), s u s p e n d e d in p a r a f f i n oil, i n t o the right h i n d p a w . The physiological progress of a d j u v a n t a r t h r i t i s a f t e r this t r e a t m e n t has b e e n described in detail [ 7 ] . T h e r e m a i n i n g 12 rats were u n t r e a t e d . Thus, b e g i n n i n g o n day 15 of training, the 24 rats were s u b d i v i d e d i n t o 2 g r o u p s of 12, an a r t h r i t i c and a n o n a r t h r i t i c group. O t h e r w i s e , d o s i n g a n d t r a i n i n g procedures c o n t i n u e d i d e n t i c a l l y for b o t h groups. Statistical treatment. Choice and r e s p o n s e rate data were coalesced in blocks o f 7 trials, as s h o w n in t h e Results section. F o r individual rats, b l o c k s o f 7 trials always c o n s i s t e d of 4 trials u n d e r o n e t r e a t m e n t c o n d i t i o n (saline or aspirin) a n d 3 trials u n d e r the o t h e r c o n d i t i o n . N o n p a r a m e t r i c statistical tests p e r f o r m e d [5] are i d e n t i f i e d in the Results section. RESULTS
Acquisition o f the Aspirin-Saline Discrimination On average, t h e n o n a r t h r i t i c rats c o n s i s t e n t l y chose t h e c o r r e c t lever for a b o u t 60% of the last 4 blocks o f 7 trials (Fig. 1). T h e c o m b i n e d choice p e r c e n t a g e data f r o m t h e 12 individual rats were significantly g r e a t e r t h a n the 50% level to be e x p e c t e d f r o m r a n d o m choices on blocks 5 t h r o u g h 8 and b l o c k s 7 t h r o u g h 8 ( p < 0 . 0 5 ; b i n o m i a l test) and signify t h a t aspirin, given u n d e r t h e c o n d i t i o n s of this e x p e r i m e n t , was d i s c r i m i n a t e d f r o m saline, a l t h o u g h poorly. T h e p o o r choice p e r f o r m a n c e o n b l o c k 4 has n o a p p a r a n t e x p l a n a tion. Table 1 shows t h a t b e f o r e t h e 56-trial e x p e r i m e n t was c o m p l e t e d , 7 rats in the n o n a r t h r i t i c g r o u p r e a c h e d a criterion of 8 c o r r e c t choices o u t o f 10 successive trials, a n d t h a t 4 r e a c h e d a c r i t e r i o n o f 9 c o r r e c t choices o u t o f 10. No
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BLOCK OF 7 SESSIONS FIG. 1. Mean percent correct lever choice of arthritic and nonarthritic groups as a function of trials in blocks of 7. N equalled 12 in each group until reduced by mortalities (see Table 1). Note that arthritic rats, on average, consistently chose the correct lever more frequently than did nonarthritic rats blocks 5 through 8, but that both groups exceeded change (50%) lever selection on these trials. rats in this g r o u p chose t h e c o r r e c t lever for 10 successive trials. The a r t h r i t i c rats r e a c h e d a greater level of a c c u r a c y t h a n t h e n o n a r t h r i t i c rats, as s h o w n b y the fact t h a t t h e i r m e a n p e r c e n t c o r r e c t choice c o n s i s t e n t l y r e a c h e d a b o u t 7 0 ~ d u r i n g b l o c k s 5 t h r o u g h 8 (Fig. 1). T h e individual choice data were significantly h i g h e r t h a n t h e 50% to be e x p e c t e d f r o m r a n d o m lever selection d u r i n g blocks 5 t h r o u g h 8 a n d 7 t h r o u g h 8 ( p < 0 . 0 1 ; b i n o m i a l test). The choice level was also significantly h i g h e r t h a n the c o r r e c t choice p e r c e n t a g e of n o n a r t h r i t i c rats on b l o c k s 5 t h r o u g h 8 ( p < 0 . 0 5 ; M a n n - W h i t n e y U-test). Even this d i f f e r e n c e p r o b a b l y u n d e r states the t r u e d i f f e r e n c e b e t w e e n the a r t h r i t i c a n d n o n a r thritic rats, since the 2 m o r t a l i t i e s in the a r t h r i t i c g r o u p (Table 1) o c c u r r e d in good rats t h a t rapidly r e a c h e d 8 / 1 0 and 9 / 1 0 c o r r e c t c h o i c e criteria. More rats in the a r t h r i t i c t h a n the n o n a r t h r i t i c g r o u p r e a c h e d the criteria of 8, 9 or 10 c o r r e c t responses o u t of 10 successive trials, as s h o w n in Table 1. T h e difference b e t w e e n t h e n u m b e r s of rats f r o m each g r o u p reaching the 10 o u t of lO criterion ( 0 / 1 2 vs 5 / 1 2 ) was significant ( p < 0 , 0 2 ; Fisher e x a c t p r o b a b i l i t i e s test).
Effects o f Aspirin and Adluvant on Response Rates Mean r e s p o n s e rate data for the n o n a r t h r i t i c rats (Fig. 2A) s h o w t h a t r e s p o n s e rates increased d u r i n g the first 4 blocks o f 7 trials f r o m a b o u t 4 0 0 4 5 0 to a b o u t 6 5 0 - 7 0 0 t o t a l r e s p o n s e s d u r i n g t h e 15 rain session. C o n s i s t e n t l y over 90% o f these r e s p o n s e s were on the c o r r e c t lever as subjects learned to d i s c r i m i n a t e n o t o n l y t h e drug state, b u t also the response source of the first r e i n f o r c e m e n t . T h r o u g h o u t the e x p e r i m e n t , m e a n F R 10 r e s p o n s e rate u n d e r the aspirin c o n d i t i o n was less t h a n u n d e r the saline c o n d i t i o n in the n o n a r t h r i t i c group. In t h e a r t h r i t i c rats, as is e v i d e n t f r o m a c o m p a r i s o n of Fig. 2A a n d 2B, m e a n response rates following the saline vehicle were c o n s i s t e n t l y l o w e r t h a n in the n o n a r t h r i t i c rats, h o v e r i n g a r o u n d 6 0 0 responses per 15 rain session u n t i l t h e last b l o c k of 7 trials. Such a depressed level of responding could be e x p e c t e d f r o m rats with seriously
D I S C R I M I N A B I L I T Y O F A S P I R I N IN R A T S
585
TABLE 1 DATA ON TRIALS-TO-CRITERION AND MORTALITY AFTER TRAINING ON INTRAPERITONEAL ASPIRIN VS SALINE, AND ON LEVER SELECTION AFTER ORAL ASPIRIN IN INDIVIDUAL RATS
8/10 Non-arthritic Rats Rat #61 62 63 64 65 66 79 80 81 82 83 84 Totals Arthritic Rats Rat #67 68 69 70 71 72 73 74 75 76 77 78 Totals
Trials-to-Criterion* 9/10 10/10
56 >56 >56 >56 >56 36 33 37 >56 56 43 43 7/125
>56 >56 >56 >56 >56 37 34 38 >56 >56 >56 44 4/12:~
>56 >56 >56 >56 >56 >56 >56 >54 >56 >56 >56 >56 0/12§
34 28 35 48 21 30 39 >56 >56 32 48 41 10/12
>56 34 40 >56 >56 31 45 >56 >56 34 49 43 7/12
56 46 43 >56 >56 32 46 >56 >56 >44 56 44 5/12
Day of Death
(58) 54
1/12
35
44
2/12
Lever choice After Oral Aspirint
A A S A S A --A S S A 6/10 A A A A A A -A S A -A S 8/10 A
*Trials until a rat reached the criterion of 8, 9 or 10 correct lever choices on 10 consecutive trials. tLever selection on a trial given 3 days after the termination of acquisition. On this trial rats were given aspirin, 100 mg/kg orally, 1 hr prior to testing, and the lever on which each rat first completed l0 responses was ascertained. A signifies aspirin (left) lever; S signifies saline (right) lever. Sp >0.05 vs arthritic rats: Fisher exact probabilities test. §p>0.02 vs arthritic rats: Fisher exact probabilities test. i n f l a m e d paws, as were a p p a r e n t on inspection. In the arthritic rats, unlike the n o n a r t h r i t i c rats, however, aspirin did n o t depress r e s p o n s e rates as c o m p a r e d with r e s p o n s e rates u n d e r the saline c o n d i t i o n .
Effect o f a Single Probe with Oral Aspirin Following the a c c u m u l a t i o n o f the data r e p o r t e d above, t w o a d d i t i o n a l training trials were run. On the following session all surviving rats (10 in each group) were administered aspirin, 100 m g / k g orally, 1 hr prior t o the test session. Results o f this single generalization p r o b e are s h o w n at the right o f Table 1. It can be seen t h a t 6 / 1 0 n o n a r t h r i t i c rats and 8/10 arthritic rats chose the aspirin lever ( t h a t is, first a c c u m u l a t e d 10 r e s p o n s e s on the aspirin lever), suggesting t h a t systemic, r a t h e r t h a n local effects o f aspirin were the basis for its discrimination. DISCUSSION Aspirin has n o t previously b e e n s t u d i e d for its ability t o serve as a cue in drug d i s c r i m i n a t i o n e x p e r i m e n t s . The p r e s e n t s t u d y d e m o n s t r a t e s t h a t i n t r a p e r i t o n e a l aspirin can
be d i s c r i m i n a t e d f r o m saline in n o n a r t h r i t i c rats during a course o f 56 trials. The d i s c r i m i n a t i o n f o r m e d , while statistically significant, was n o t r o b u s t . On average, nonarthritic rats chose the c o r r e c t lever a b o u t 60% o f the last 28 sessions, and while 7 o u t of 12 rats m e t a criterion o f 8 c o r r e c t choices during 10 c o n s e c u t i v e trials, only 4 o f the 12 rats m e t a 9 o u t o f 10 criterion, and n o n e o f the 12 rats m e t a 10 out o f 10 criterion. By way o f c o n t r a s t , Colpaert and his colleagues, using a very similar p r o t o c o l to the p r e s e n t one, have s h o w n t h a t rats discriminate f e n t a n y l , 0.04 m g / k g sc [3] and a p o m o r p h i n e , 0.16 m g / k g sc [4] to a 10/10 criterion in f e w e r t h a n 40 trials. Elevating the dosage o f i n t r a p e r i t o n e a l aspirin in this t y p e o f e x p e r i m e n t is unlikely t o yield b e t t e r discriminability, since pilot studies u n d e r t a k e n in this l a b o r a t o r y have s h o w n appreciable m o r t a l i t y to result f r o m r e p e a t e d i n t r a p e r i t o n e a l a d m i n i s t r a t i o n o f aspirin at 100 or 75 m g / k g u n d e r a p r o t o c o l similar t o the p r e s e n t one. Even u n d e r the 56 mg/kg dose p r e s e n t l y used, 2 rats died by 58 trials after the o n s e t o f aspirin t r e a t m e n t , given 2 or 3 times weekly. The p r e s e n t s t u d y strongly suggests t h a t aspirin b e c o m e s m o r e discriminable in rats m a d e arthritic by an i n t r a d e r m a l
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FIG. 2. A. Mean response rates (responses/15 min) of nonarthritic rats under saline and aspirin conditions as a function of trials in blocks of 7. N equalled 12 until reduced by mortality (see Table 1). Note that response rate under the aspirin condition is consistently less than response rate under the saline condition. B. As above, except for arthritic rhts. Note that response rate under aspirin and saline conditions were virtually identical.
i n j e c t i o n of M y c o b a c t e r i u m b u t y r i c u m i n t o a h i n d p a w t h a n it is in n o n a r t h r i t i c rats. C o n s i s t e n t l y m o r e rats f r o m t h e a r t h r i t i c g r o u p r e a c h e d t h e criteria o f 8, 9 or 10 c o r r e c t r e s p o n s e s o u t of 10 c o n s e c u t i v e trials, a n d t h e d i f f e r e n c e in n u m b e r s of rats f r o m a r t h r i t i c versus n o n a r t h r i t i c groups r e a c h i n g the 10 o u t of 10 c r i t e r i o n was significant. F u r t h e r m o r e , a r t h r i t i c rats, on average, m a d e c o n s i s t e n t l y m o r e c o r r e c t choices ( a b o u t 70%) d u r i n g the final 28 trials of t h e e x p e r i m e n t t h a n did n o n a r t h r i t i c rats. Paw v o l u m e m e a s u r e m e n t s were n o t m a d e in t h e p r e s e n t s t u d y ; h o w e v e r , t h e rats i n j e c t e d w i t h t h e a d j u v a n t s h o w e d obvious e d e m a , first in t h e i n j e c t e d paw, and b e g i n n i n g in a b o u t a w e e k in t h e c o n t r a l a t e r a l a n d f r o n t paws. T h e rats were o b v i o u s l y irritable o n h a n d l i n g . W h e n given in the diet f r o m 14 to 28 days a f t e r a d m i n i s t r a t i o n o f M. b u t y r i c u m , aspirin has b e e n s h o w n to r e d u c e j o i n t d i a m e t e r s at doses of 149 m g / k g a n d a b o v e [ 1 ]. Its EDs 0 was 4 4 0 mg/kg, where EDs o is expressed as t h e dose t h a t very m a r d e d l y reduces j o i n t d i a m e t e r s in 50% of t h e rats [ 1 ] . Aspirin at oral dietary doses as low as 81.3 m g / k g / d a y has also b e e n s h o w n to significantly r e d u c e p a w v o l u m e s of rats m a d e a r t h r i t i c by i n j e c t i o n of M. tuberculosis [ 6 ] . These studies are relevant to t h e p r e s e n t s t u d y in s h o w i n g t h a t aspirin is effective in alleviating this t y p e of a d j u v a n t arthritis, b u t t h e differing p r o t o c o l s ( r o u t e a n d c h r o n i c i t y of t r e a t m e n t ) m a k e direct e x t r a p o l a t i o n of c o n c l u s i o n s difficult. T h e p r e s e n t s t u d y e x e m p l i f i e s t h a t drug d i s c r i m i n a b i l i t y d e p e n d s o n t h e state of t h e o r g a n i s m b e i n g tested: In this e x a m p l e aspirin is b e t t e r d i s c r i m i n a t e d in rats m a d e a r t h r i t i c t h a n in n o n a r t h r i t i c rats. A d d i t i o n a l u n p u b l i s h e d studies in this l a b o r a t o r y , u n s u r p r i s i n g l y , have s h o w n t h a t n a l o x o n e is highly d i s c r i m i n a b l e in rats m a d e c h r o n i c a l l y d e p e n d e n t o n m o r p h i n e , a n d t h a t h a l o p e r i d o l is far b e t t e r d i s c r i m i n a t e d in rats t r e a t e d c h r o n i c a l l y w i t h a m p h e t a m i n e t h a n in n o n d r u g g e d rats. T h e d e s i g n a t i o n of a drug as i n d i s c r i m i n a b l e (or for t h a t m a t t e r , d i s c r i m i n a b l e ) requires d e s c r i p t i o n of t h e a n i m a l s in w h i c h it was tested. ACKNOWLEDGMENT
This study was made possible by the diligent technical assistance provided by Jonathan L. Johnson, Jr. and the computerized data handling support provided by Daniel B. Joseph and Norman J. Stubbs.
REFERENCES 1. Awouters, F., C. J. E. Niemegeers, F. M. Lenaerts and P. A. J. Janssen. The effects of suprofen in rats with Mycobacterium butyricum-induced arthritis. Arznei.-Forsch. 25: 1526-1537, 1975. 2. Barry, H., III. Classification of drugs according to their discriminable effects in rats. Fedn Proc. 33: 1814-1824, 1974. 3. Colpaert, J. C., H. Lal, C. J. E. Niemegeers and P. A. J. Janssen. Investigations on drug produced and subjectively experienced discriminative stimuli. I. The fentanyl cue, a tool to investigate subjectively experienced narcotic drug actions. Life Sci 16: 705-716, 1975. 4. Colpaert, F. C., C. J. E. Niemegeers, J. J. M. D. Kuyps and P. A. J. Janssen. Apomorphine as a discriminative stimulus, and its antagonism by haloperidol. Eur. J. Pharmac. 32: 383-386, 1975.
5.
Siegel, S.: Nonparametric Statistics for the Behaviorial Sciences. New York: McGraw-Hill Book Co., 1956. 6. Sofia, R. D., W. Diamantis, R. Gordon, M. Kletzkin, F. M. Berger, J. Edelson, H. Singer and J. F. Douglas. Pharmacology of a new nonsteroidal anti-inflammatory agent 7-chloro-3,3adihydro-2-methyl-2H,9H-isoxazolo-(3,2-b) (1,3)-benzoxazin-9one (W-2395). Eur. J. Pharmac. 26: 5 1 - 6 2 , 1974. 7. Walz, D. T., M. J. Dimartino, J. H. Kuch and W. zuccarello. Adjuvant-induced arthritis in rats. I. Temporal relationship of physiological, biochemical, and hematological parameters. Proc. Soc. exp. Biol. Med. 136: 9 0 6 - 9 1 0 , 1971.
BRIEF COMMUNICATION The Discriminability of Aspirin in Arthritic and Nonarthritic Rats ALBERT WEISSMAN
Pfizer, Inc., Groton, C T 0 6 3 4 0 (Received 3 S e p t e m b e r 1 9 7 6 )
WEISSMAN, A. The discriminability of aspirin in arthritic and nonarthritic rats. PHARMAC. BIOCHEM. BEHAV. 5(5) 583-586, 1976. - Aspirin, 56 mg/kg IP, was shown to be mildly, but significantly discriminable from saline in a group of 12 nonarthritic rats exposed to a 2-1ever fixed ratio 10 drug discrimination protocol for 56 trials. In a concurrently-tested group of 12 rats made arthritic by injection of Mycobacterium butyricum into a hind paw, the discrimination of aspirin from saline was enhanced. The results exemplify how drug discriminability may vary depending on the pathological state of the subjects exposed to drug-discrimination training. Aspirin
Drug discrimination
Adjuvant arthritis
Operant behavior
S E V E R A L s y s t e m i c a l l y - a d m i n i s t e r e d drugs are k n o w n to serve as discriminative stimuli in a p p r o p r i a t e l y t r a i n e d l a b o r a t o r y animals. This subject has n o t only b e e n freq u e n t l y reviewed [e.g., 2 ] , b u t is t h e f o c u s of active c u r r e n t research in m a n y laboratories. F o r the m o s t part, research to date has b e e n c o n c e r n e d w i t h i d e n t i f y i n g t h o s e drugs t h a t generalize to a d i s c r i m i n a t e d drug, and the specificity of a given drug cue. A m o n g the m a n y p o t e n t i a l o f f s h o o t s of this research area is t h e p r o b l e m of d e t e r m i n i n g states of an organism t h a t favor or disfavor t h e a c q u i s i t i o n of d i s c r i m i n a t i v e p r o p e r t i e s b y a drug. A l t h o u g h f o r m a l studies have n o t b e e n addressed to this p o i n t in h u m a n s , it seems evident t h a t a h u m a n o p i a t e addict s h o u l d be m o r e readily able to discriminate naloxone than a nonaddict and that a human with h e a d a c h e or a r t h r i t i s s h o u l d be m o r e able to discriminate aspirin t h a n a n o n s u f f e r e r . T h e p r e s e n t s t u d y was u n d e r t a k e n as an a t t e m p t to d e m o n s t r a t e w h e t h e r a p o o r l y d i s c r i m i n a t e d drug is b e t t e r d i s c r i m i n a t e d b y rats discriminated by rats s u b j e c t e d to p a t h o l o g i c a l c o n d i t i o n s relieved by t h a t drug. Specifically, it was addressed to t h e d e t e r m i n a t i o n of w h e t h e r or n o t aspirin can serve as a d i s c r i m i n a b l e stimulus, a n d w h e t h e r a c q u i s i t i o n of an aspirin vs saline d i s c r i m i n a t i o n develops m o r e rapidly in a r t h r i t i c t h a n in n o r m a l rats. METHOD
Animals Twenty-four individually housed Sprague-Dawley rats f r o m Charles weighing a b o u t 2 2 0 g o n arrival were liquid f o o d r e i n f o r c e m e n t d u r i n g t h e
a d u l t male a l b i n o River L a b o r a t o r i e s used. T h e y received c o n d i t i o n i n g proce583
dures described below, a n d sufficient s u p p l e m e n t a l P u r i n a rat c h o w a f t e r each session and o n w e e k e n d s to m a i n t a i n t h e i r weights at a b o u t 2 5 0 g t h r o u g h o u t the e x p e r i m e n t . Water was available ad lib in h o m e cages.
Apparatus T r a i n i n g a n d testing were a c c o m p l i s h e d in 6 identical isolated c o m m e r c i a l o p e r a n t c h a m b e r s ( F o r i n g e r t y p e ) p r o g r a m m e d b y e l e c t r o m e c h a n i c a l circuitry. T h e i m p o r t a n t w o r k i n g parts of each c h a m b e r were t w o F o r i n g e r levers m o u n t e d on each side of a m o t o r - d r i v e n dipper, and a h o u s e l i g h t w h i c h r e m a i n e d o n d u r i n g the 15 m i n session. R e i n f o r c e m e n t consisted o f 3 sec p r e s e n t a t i o n of 0.2 cc of C a r n a t i o n Slender, a c o m m e r c i a l liquid diet food consisting of s u c r o s e - s w e e t e n e d skim milk w i t h p r o t e i n , v i t a m i n s a n d minerals added, diluted 1 : 1 w i t h water.
Procedure The p r o c e d u r e was a slight m o d i f i c a t i o n of the 2-bar fixed r a t i o l 0 ( F R 10) d r u g - d i s c r i m i n a t i o n p r o t o c o l first described by C o l p a e r t et al. [3] in t h e i r studies of f e n t a n y l discrimination. F o l l o w i n g f o o d d e p r i v a t i o n to a s u s t a i n e d 2 5 0 g weight, each rat was s h a p e d to bar press, first on a c o n t i n u o u s r e i n f o r c e m e n t C R F ) s c h e d u l e o n each lever, and t h e n o n an F R s c h e d u l e t h a t was rapidly escalated to F R l 0 on each lever. These steps were a c c o m p l i s h e d w i t h a single lever in the cage, for 2 sessions o n t h e left side, and t h e n for 2 a d d i t i o n a l sessions o n the right side, o f t h e dipper. Drug t r a i n i n g was t h e n begun, using the p r o t o c o l of C o l p a e r t et al. [ 3 ] . D e p e n d i n g o n w h e t h e r the rat received aspirin or saline (see b e l o w for details of t r e a t m e n t ) ,
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WEISSMAN
r e i n f o r c e m e n t was p r o g r a m m e d exclusively o n e i t h e r t h e left or t h e right lever o n t h e F R 10 schedule. O n l y r e s p o n s e s o n t h e left lever were r e i n f o r c e d a f t e r drug administration, and only r e s p o n s e s on the r i g h t lever a f t e r saline a d m i n i s t r a t i o n . Rats were t e s t e d 5 days each w e e k u n d e r t h e a l t e r n a t i n g drug s e q u e n c e s used b y C o l p a e r t et al. [ 3 ] : aspirin-saline-saline-aspirin-aspirin and saline-aspirin-aspirin-saline-saline. To avoid t h e p o s s i b i l i t y t h a t the c o r r e c t lever for rats previously t e s t e d in t h e c h a m b e r s could serve as a cue, a very real possibility based on results of p i l o t studies, t h e s e q u e n c e of aspirin or saline t r e a t m e n t on any day was a l t e r n a t e d as t h e 4 s u b g r o u p s of 6 rats were tested. A f t e r e a c h session of 15 rain, rats were r e t u r n e d to t h e i r h o m e cages a n d fed a q u a n t i t y of P u r i n a lab c h o w equal to the d i f f e r e n c e b e t w e e n t h e i r b o d y weights, d e t e r m i n e d p r i o r to drug t r e a t m e n t , a n d 2 5 0 g. On w e e k e n d s rats were fed 1 O - 1 2 g / d a y of p e l l e t e d lab c h o w , w h i c h m a i n t a i n e d t h e i r weight r e a s o n a b l y c o n s t a n t at a b o u t 250 g. In s u m m a r y , o t h e r t h a n t h e drug t r e a t m e n t s e m p l o y e d , t h e p r i m e m o d i f i c a t i o n s o f the C o l p a e r t et al. [3] t r a i n i n g p r o c e d u r e used in this s t u d y were t h e t y p e of o p e r a n t c h a m b e r s used, the r e i n f o r c i n g stimulus, a n d the m e t h o d of depriving rats. Drug administration. T h r o u g h o u t training, rats received e i t h e r n o r m a l (0.9%) saline or aspirin s u s p e n d e d in n o r m a l saline (56 m g / k g at a v o l u m e o f 5 ml/kg). I n j e c t i o n s were m a d e i n t r a p e r i t o n e a l l y 1 h r p r i o r to e x p o s u r e of the rat to the o p e r a n t c h a m b e r . A f t e r 14 days o f training, a d j u v a n t a r t h r i t i s was prod u c e d in 12 r a n d o m l y c h o s e n rats by a close a p p r o x i m a t i o n of t h e p r o c e d u r e of Walz et al. [ 7 ] , w h i c h consisted of a single i n t r a d e r m a l i n j e c t i o n of 0.75 mg of Mycobacterium butyricum (Difco L a b o r a t o r i e s , D e t r o i t , MI), s u s p e n d e d in p a r a f f i n oil, i n t o the right h i n d p a w . The physiological progress of a d j u v a n t a r t h r i t i s a f t e r this t r e a t m e n t has b e e n described in detail [ 7 ] . T h e r e m a i n i n g 12 rats were u n t r e a t e d . Thus, b e g i n n i n g o n day 15 of training, the 24 rats were s u b d i v i d e d i n t o 2 g r o u p s of 12, an a r t h r i t i c and a n o n a r t h r i t i c group. O t h e r w i s e , d o s i n g a n d t r a i n i n g procedures c o n t i n u e d i d e n t i c a l l y for b o t h groups. Statistical treatment. Choice and r e s p o n s e rate data were coalesced in blocks o f 7 trials, as s h o w n in t h e Results section. F o r individual rats, b l o c k s o f 7 trials always c o n s i s t e d of 4 trials u n d e r o n e t r e a t m e n t c o n d i t i o n (saline or aspirin) a n d 3 trials u n d e r the o t h e r c o n d i t i o n . N o n p a r a m e t r i c statistical tests p e r f o r m e d [5] are i d e n t i f i e d in the Results section. RESULTS
Acquisition o f the Aspirin-Saline Discrimination On average, t h e n o n a r t h r i t i c rats c o n s i s t e n t l y chose t h e c o r r e c t lever for a b o u t 60% of the last 4 blocks o f 7 trials (Fig. 1). T h e c o m b i n e d choice p e r c e n t a g e data f r o m t h e 12 individual rats were significantly g r e a t e r t h a n the 50% level to be e x p e c t e d f r o m r a n d o m choices on blocks 5 t h r o u g h 8 and b l o c k s 7 t h r o u g h 8 ( p < 0 . 0 5 ; b i n o m i a l test) and signify t h a t aspirin, given u n d e r t h e c o n d i t i o n s of this e x p e r i m e n t , was d i s c r i m i n a t e d f r o m saline, a l t h o u g h poorly. T h e p o o r choice p e r f o r m a n c e o n b l o c k 4 has n o a p p a r a n t e x p l a n a tion. Table 1 shows t h a t b e f o r e t h e 56-trial e x p e r i m e n t was c o m p l e t e d , 7 rats in the n o n a r t h r i t i c g r o u p r e a c h e d a criterion of 8 c o r r e c t choices o u t o f 10 successive trials, a n d t h a t 4 r e a c h e d a c r i t e r i o n o f 9 c o r r e c t choices o u t o f 10. No
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1
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2
I
3
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4
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5
I
6
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7
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8
BLOCK OF 7 SESSIONS FIG. 1. Mean percent correct lever choice of arthritic and nonarthritic groups as a function of trials in blocks of 7. N equalled 12 in each group until reduced by mortalities (see Table 1). Note that arthritic rats, on average, consistently chose the correct lever more frequently than did nonarthritic rats blocks 5 through 8, but that both groups exceeded change (50%) lever selection on these trials. rats in this g r o u p chose t h e c o r r e c t lever for 10 successive trials. The a r t h r i t i c rats r e a c h e d a greater level of a c c u r a c y t h a n t h e n o n a r t h r i t i c rats, as s h o w n b y the fact t h a t t h e i r m e a n p e r c e n t c o r r e c t choice c o n s i s t e n t l y r e a c h e d a b o u t 7 0 ~ d u r i n g b l o c k s 5 t h r o u g h 8 (Fig. 1). T h e individual choice data were significantly h i g h e r t h a n t h e 50% to be e x p e c t e d f r o m r a n d o m lever selection d u r i n g blocks 5 t h r o u g h 8 a n d 7 t h r o u g h 8 ( p < 0 . 0 1 ; b i n o m i a l test). The choice level was also significantly h i g h e r t h a n the c o r r e c t choice p e r c e n t a g e of n o n a r t h r i t i c rats on b l o c k s 5 t h r o u g h 8 ( p < 0 . 0 5 ; M a n n - W h i t n e y U-test). Even this d i f f e r e n c e p r o b a b l y u n d e r states the t r u e d i f f e r e n c e b e t w e e n the a r t h r i t i c a n d n o n a r thritic rats, since the 2 m o r t a l i t i e s in the a r t h r i t i c g r o u p (Table 1) o c c u r r e d in good rats t h a t rapidly r e a c h e d 8 / 1 0 and 9 / 1 0 c o r r e c t c h o i c e criteria. More rats in the a r t h r i t i c t h a n the n o n a r t h r i t i c g r o u p r e a c h e d the criteria of 8, 9 or 10 c o r r e c t responses o u t of 10 successive trials, as s h o w n in Table 1. T h e difference b e t w e e n t h e n u m b e r s of rats f r o m each g r o u p reaching the 10 o u t of lO criterion ( 0 / 1 2 vs 5 / 1 2 ) was significant ( p < 0 , 0 2 ; Fisher e x a c t p r o b a b i l i t i e s test).
Effects o f Aspirin and Adluvant on Response Rates Mean r e s p o n s e rate data for the n o n a r t h r i t i c rats (Fig. 2A) s h o w t h a t r e s p o n s e rates increased d u r i n g the first 4 blocks o f 7 trials f r o m a b o u t 4 0 0 4 5 0 to a b o u t 6 5 0 - 7 0 0 t o t a l r e s p o n s e s d u r i n g t h e 15 rain session. C o n s i s t e n t l y over 90% o f these r e s p o n s e s were on the c o r r e c t lever as subjects learned to d i s c r i m i n a t e n o t o n l y t h e drug state, b u t also the response source of the first r e i n f o r c e m e n t . T h r o u g h o u t the e x p e r i m e n t , m e a n F R 10 r e s p o n s e rate u n d e r the aspirin c o n d i t i o n was less t h a n u n d e r the saline c o n d i t i o n in the n o n a r t h r i t i c group. In t h e a r t h r i t i c rats, as is e v i d e n t f r o m a c o m p a r i s o n of Fig. 2A a n d 2B, m e a n response rates following the saline vehicle were c o n s i s t e n t l y l o w e r t h a n in the n o n a r t h r i t i c rats, h o v e r i n g a r o u n d 6 0 0 responses per 15 rain session u n t i l t h e last b l o c k of 7 trials. Such a depressed level of responding could be e x p e c t e d f r o m rats with seriously
D I S C R I M I N A B I L I T Y O F A S P I R I N IN R A T S
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TABLE 1 DATA ON TRIALS-TO-CRITERION AND MORTALITY AFTER TRAINING ON INTRAPERITONEAL ASPIRIN VS SALINE, AND ON LEVER SELECTION AFTER ORAL ASPIRIN IN INDIVIDUAL RATS
8/10 Non-arthritic Rats Rat #61 62 63 64 65 66 79 80 81 82 83 84 Totals Arthritic Rats Rat #67 68 69 70 71 72 73 74 75 76 77 78 Totals
Trials-to-Criterion* 9/10 10/10
56 >56 >56 >56 >56 36 33 37 >56 56 43 43 7/125
>56 >56 >56 >56 >56 37 34 38 >56 >56 >56 44 4/12:~
>56 >56 >56 >56 >56 >56 >56 >54 >56 >56 >56 >56 0/12§
34 28 35 48 21 30 39 >56 >56 32 48 41 10/12
>56 34 40 >56 >56 31 45 >56 >56 34 49 43 7/12
56 46 43 >56 >56 32 46 >56 >56 >44 56 44 5/12
Day of Death
(58) 54
1/12
35
44
2/12
Lever choice After Oral Aspirint
A A S A S A --A S S A 6/10 A A A A A A -A S A -A S 8/10 A
*Trials until a rat reached the criterion of 8, 9 or 10 correct lever choices on 10 consecutive trials. tLever selection on a trial given 3 days after the termination of acquisition. On this trial rats were given aspirin, 100 mg/kg orally, 1 hr prior to testing, and the lever on which each rat first completed l0 responses was ascertained. A signifies aspirin (left) lever; S signifies saline (right) lever. Sp >0.05 vs arthritic rats: Fisher exact probabilities test. §p>0.02 vs arthritic rats: Fisher exact probabilities test. i n f l a m e d paws, as were a p p a r e n t on inspection. In the arthritic rats, unlike the n o n a r t h r i t i c rats, however, aspirin did n o t depress r e s p o n s e rates as c o m p a r e d with r e s p o n s e rates u n d e r the saline c o n d i t i o n .
Effect o f a Single Probe with Oral Aspirin Following the a c c u m u l a t i o n o f the data r e p o r t e d above, t w o a d d i t i o n a l training trials were run. On the following session all surviving rats (10 in each group) were administered aspirin, 100 m g / k g orally, 1 hr prior t o the test session. Results o f this single generalization p r o b e are s h o w n at the right o f Table 1. It can be seen t h a t 6 / 1 0 n o n a r t h r i t i c rats and 8/10 arthritic rats chose the aspirin lever ( t h a t is, first a c c u m u l a t e d 10 r e s p o n s e s on the aspirin lever), suggesting t h a t systemic, r a t h e r t h a n local effects o f aspirin were the basis for its discrimination. DISCUSSION Aspirin has n o t previously b e e n s t u d i e d for its ability t o serve as a cue in drug d i s c r i m i n a t i o n e x p e r i m e n t s . The p r e s e n t s t u d y d e m o n s t r a t e s t h a t i n t r a p e r i t o n e a l aspirin can
be d i s c r i m i n a t e d f r o m saline in n o n a r t h r i t i c rats during a course o f 56 trials. The d i s c r i m i n a t i o n f o r m e d , while statistically significant, was n o t r o b u s t . On average, nonarthritic rats chose the c o r r e c t lever a b o u t 60% o f the last 28 sessions, and while 7 o u t of 12 rats m e t a criterion o f 8 c o r r e c t choices during 10 c o n s e c u t i v e trials, only 4 o f the 12 rats m e t a 9 o u t o f 10 criterion, and n o n e o f the 12 rats m e t a 10 out o f 10 criterion. By way o f c o n t r a s t , Colpaert and his colleagues, using a very similar p r o t o c o l to the p r e s e n t one, have s h o w n t h a t rats discriminate f e n t a n y l , 0.04 m g / k g sc [3] and a p o m o r p h i n e , 0.16 m g / k g sc [4] to a 10/10 criterion in f e w e r t h a n 40 trials. Elevating the dosage o f i n t r a p e r i t o n e a l aspirin in this t y p e o f e x p e r i m e n t is unlikely t o yield b e t t e r discriminability, since pilot studies u n d e r t a k e n in this l a b o r a t o r y have s h o w n appreciable m o r t a l i t y to result f r o m r e p e a t e d i n t r a p e r i t o n e a l a d m i n i s t r a t i o n o f aspirin at 100 or 75 m g / k g u n d e r a p r o t o c o l similar t o the p r e s e n t one. Even u n d e r the 56 mg/kg dose p r e s e n t l y used, 2 rats died by 58 trials after the o n s e t o f aspirin t r e a t m e n t , given 2 or 3 times weekly. The p r e s e n t s t u d y strongly suggests t h a t aspirin b e c o m e s m o r e discriminable in rats m a d e arthritic by an i n t r a d e r m a l
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FIG. 2. A. Mean response rates (responses/15 min) of nonarthritic rats under saline and aspirin conditions as a function of trials in blocks of 7. N equalled 12 until reduced by mortality (see Table 1). Note that response rate under the aspirin condition is consistently less than response rate under the saline condition. B. As above, except for arthritic rhts. Note that response rate under aspirin and saline conditions were virtually identical.
i n j e c t i o n of M y c o b a c t e r i u m b u t y r i c u m i n t o a h i n d p a w t h a n it is in n o n a r t h r i t i c rats. C o n s i s t e n t l y m o r e rats f r o m t h e a r t h r i t i c g r o u p r e a c h e d t h e criteria o f 8, 9 or 10 c o r r e c t r e s p o n s e s o u t of 10 c o n s e c u t i v e trials, a n d t h e d i f f e r e n c e in n u m b e r s of rats f r o m a r t h r i t i c versus n o n a r t h r i t i c groups r e a c h i n g the 10 o u t of 10 c r i t e r i o n was significant. F u r t h e r m o r e , a r t h r i t i c rats, on average, m a d e c o n s i s t e n t l y m o r e c o r r e c t choices ( a b o u t 70%) d u r i n g the final 28 trials of t h e e x p e r i m e n t t h a n did n o n a r t h r i t i c rats. Paw v o l u m e m e a s u r e m e n t s were n o t m a d e in t h e p r e s e n t s t u d y ; h o w e v e r , t h e rats i n j e c t e d w i t h t h e a d j u v a n t s h o w e d obvious e d e m a , first in t h e i n j e c t e d paw, and b e g i n n i n g in a b o u t a w e e k in t h e c o n t r a l a t e r a l a n d f r o n t paws. T h e rats were o b v i o u s l y irritable o n h a n d l i n g . W h e n given in the diet f r o m 14 to 28 days a f t e r a d m i n i s t r a t i o n o f M. b u t y r i c u m , aspirin has b e e n s h o w n to r e d u c e j o i n t d i a m e t e r s at doses of 149 m g / k g a n d a b o v e [ 1 ]. Its EDs 0 was 4 4 0 mg/kg, where EDs o is expressed as t h e dose t h a t very m a r d e d l y reduces j o i n t d i a m e t e r s in 50% of t h e rats [ 1 ] . Aspirin at oral dietary doses as low as 81.3 m g / k g / d a y has also b e e n s h o w n to significantly r e d u c e p a w v o l u m e s of rats m a d e a r t h r i t i c by i n j e c t i o n of M. tuberculosis [ 6 ] . These studies are relevant to t h e p r e s e n t s t u d y in s h o w i n g t h a t aspirin is effective in alleviating this t y p e of a d j u v a n t arthritis, b u t t h e differing p r o t o c o l s ( r o u t e a n d c h r o n i c i t y of t r e a t m e n t ) m a k e direct e x t r a p o l a t i o n of c o n c l u s i o n s difficult. T h e p r e s e n t s t u d y e x e m p l i f i e s t h a t drug d i s c r i m i n a b i l i t y d e p e n d s o n t h e state of t h e o r g a n i s m b e i n g tested: In this e x a m p l e aspirin is b e t t e r d i s c r i m i n a t e d in rats m a d e a r t h r i t i c t h a n in n o n a r t h r i t i c rats. A d d i t i o n a l u n p u b l i s h e d studies in this l a b o r a t o r y , u n s u r p r i s i n g l y , have s h o w n t h a t n a l o x o n e is highly d i s c r i m i n a b l e in rats m a d e c h r o n i c a l l y d e p e n d e n t o n m o r p h i n e , a n d t h a t h a l o p e r i d o l is far b e t t e r d i s c r i m i n a t e d in rats t r e a t e d c h r o n i c a l l y w i t h a m p h e t a m i n e t h a n in n o n d r u g g e d rats. T h e d e s i g n a t i o n of a drug as i n d i s c r i m i n a b l e (or for t h a t m a t t e r , d i s c r i m i n a b l e ) requires d e s c r i p t i o n of t h e a n i m a l s in w h i c h it was tested. ACKNOWLEDGMENT
This study was made possible by the diligent technical assistance provided by Jonathan L. Johnson, Jr. and the computerized data handling support provided by Daniel B. Joseph and Norman J. Stubbs.
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5.
Siegel, S.: Nonparametric Statistics for the Behaviorial Sciences. New York: McGraw-Hill Book Co., 1956. 6. Sofia, R. D., W. Diamantis, R. Gordon, M. Kletzkin, F. M. Berger, J. Edelson, H. Singer and J. F. Douglas. Pharmacology of a new nonsteroidal anti-inflammatory agent 7-chloro-3,3adihydro-2-methyl-2H,9H-isoxazolo-(3,2-b) (1,3)-benzoxazin-9one (W-2395). Eur. J. Pharmac. 26: 5 1 - 6 2 , 1974. 7. Walz, D. T., M. J. Dimartino, J. H. Kuch and W. zuccarello. Adjuvant-induced arthritis in rats. I. Temporal relationship of physiological, biochemical, and hematological parameters. Proc. Soc. exp. Biol. Med. 136: 9 0 6 - 9 1 0 , 1971.
