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Comparative effects of physostigmine and neostigmine on acquisition and performance of a conditioned avoidance behavior in the rat
Pharmacology Biochemistry and Behavior, Vol. 2, pp. 67--72. ANKHO International Inc., 1974. Printed in the U.S.A.
Comparative Effects of Physostigmine and Neostigmine on Acquisition and Performance of a Conditioned Avoidance Behavior in the Raf J O H N A. R O S E C R A N S 2 A N D E D W A R D F. D O M I N O
Michigan Neuropsychopharmacology Research Program, Department o f Pharmacology, University o f Michigan, A n n Arbor, Michigan 48104
( R e c e i v e d 14 M a r c h 1 9 7 3 )
ROSECRANS, J. A. AND E. F. DOMINO. Comparative effects of physostigmine and neostigmine on acquisition and performance o f a conditioned avoidance behavior in the rat. PHARMAC. BIOCHEM. BEHAV. 2(1) 6 7 - 7 2 , 1974. - The actions of physostigmine (0.10 and 0.15 mg/kg) and neostigmine (0.12 mg/kg) given s.c. on acquisition and performance of conditioned pole jumping were studied in rats receiving either 0.9% saline, ( - ) hyoscyamine or atropine methylnitrate. As estimated by inhibition of acetylcholinesterase (ACHE) activity, physostigmine but not neostigmine entered the rat brain. Even though neostigmine did not significantly alter brain AChE activity in equimolar doses to physostigmine, it depressed both the acquisition and performance of a behavioral task, suggesting a predominant peripheral action. Although physostigmine depressed behavior mainly via a central action, its peripheral actions also play a role in its behavioral effects. Physostigmine
Neosfigmine
Conditioned avoidance
Central and peripheral drug action
effects of the c h o l i n e s t e r a s e i n h i b i t o r s p h y s o s t i g m i n e (a t e r t i a r y a m i n e ) a n d n e o s t i g m i n e (a q u a t e r n a r y a m i n e ) on a c q u i s i t i o n and p e r f o r m a n c e of c o n d i t i o n e d pole j u m p i n g a n d c o r r e l a t e t h e i r b e h a v i o r a l effects w i t h b r a i n AChE activity in t h e rat. T w o c h o l i n e r g i c a n t a g o n i s t s were also used t o s e p a r a t e t h e i r c e n t r a l and p e r i p h e r a l effects. One was ( - ) h y o s c y a m i n e , the active i s o m e r of a t r o p i n e (a t e r t i a r y a m i n e ) a n d the o t h e r m e t h y l a t r o p i n e (a q u a t e r n a r y a m i n e ) . A l t h o u g h similar studies have b e e n r e p o r t e d [3, 4, 7, 8 - 1 0 ] , t h e c o r r e l a t i o n o f b r a i n A c h e activity w i t h b e h a v i o r a l effects o n l y rarely has b e e n done.
P H Y S O S T I G M I N E is a reversible c h o l i n e s t e r a s e i n h i b i t o r ( C h E I ) w h i c h has b e e n useful in s t u d y i n g c h o l i n e r g i c m e c h a n i s m s o f b e h a v i o r [ 1 , 3 , 4, 6 - 1 0 ] . Its p r e s e n c e in t h e c e n t r a l n e r v o u s s y s t e m can b e d e t e r m i n e d i n d i r e c t l y b y its effects o n b r a i n ChE. Because of its reversible n a t u r e , one can m e a s u r e b o t h the o n s e t and r e c o v e r y of its b e h a v i o r a l effects. T h e r e is a good c o r r e l a t i o n b e t w e e n i n h i b i t i o n of b r a i n a c e t y l c h o l i n e s t e r a s e activity (ACHE), an increase in b r a i n a c e t y l c h o l i n e ( A C h ) , a n d b e h a v i o r a l d e p r e s s i o n foll o w i n g p h y s o s t i g m i n e [ 3,8 ]. While this a p p r o a c h a p p e a r s valid in s t u d y i n g t h e behavioral role of b r a i n ACh, t h e r e are p r o b l e m s associated w i t h t h e use o f p h y s o s t i g m i n e and related c o m p o u n d s , Aside f r o m its c e n t r a l effect, p h y s o s t i g m i n e has well k n o w n p e r i p h e r a l actions. In view of t h e fact t h a t the b l o o d - b r a i n barrier tends to exclude quaternary nitrogen compounds, t h e t e r t i a r y and q u a t e r n a r y analogs o f various c h o l i n e r g i c agonists and a n t a g o n i s t s can be useful t o o l s to d e t e r m i n e relative c e n t r a l vs p e r i p h e r a l effects. T h e a d d i t i o n a l m e a s u r e of b r a i n A C h E activity provides f u r t h e r e v i d e n c e t h a t a ChEI has p e n e t r a t e d i n t o t h e c e n t r a l n e r v o u s s y s t e m . The p u r p o s e of t h e p r e s e n t s t u d y was to d e t e r m i n e the
METHOD Male a l b i n o H o l t z m a n rats f r o m 4 5 - 9 0 days of age were used. A c q u i s i t i o n a n d p e r f o r m a n c e studies were c o n d u c t e d utilizing t h e rat pole j u m p r e s p o n s e as described previously [ 8 ] . B o t h i n i t i a t i o n a n d r e c o r d i n g of trials and r e s p o n s e s was a c c o m p l i s h e d a u t o m a t i c a l l y . The CS was a 5 sec b u z z e r w h i c h o v e r l a p p e d a 5 sec US consisting of a 1 ma s h o c k s c r a m b l e d t h r o u g h the grid floor. I n t e r t r i a i periods ranged f r o m 2 0 4 0 sec w i t h a m e a n o f 30 sec. R e s p o n s e s d u r i n g
'Supported in part by USPHS MH-11846. 2Present address: Department of Pharmacology, Medical College of Virginia, Richmond, Virginia. 67
68 t h e i n t e r t r i a l p e r i o d were n o t c o u n t e d . A n i m a l s were allowed t o h a b i t u a t e to t h e a n i m a l r o o m for o n e week p r i o r to testing, d u r i n g w h i c h t i m e t h e y were n u m b e r e d a n d placed in a p r e d e s i g n e d g r o u p utilizing a r a n d o m table. Experim e n t s were c o n d u c t e d in b l o c k s consisting of o n e c o n t r o l a n d five e x p e r i m e n t a l groups o f 12 a n i m a l s each. Saline, ( - ) h y o s c y a m i n e or m e t h y l a t r o p i n e was a d m i n i s t e r e d 30 rain p r i o r t o t h e a d m i n i s t r a t i o n of the ChEIs physostigm i n e a n d n e o s t i g m i n e . Drugs were a d m i n i s t e r e d s.c. Dosage was calculated as base.
Acquisition Studies Prior to t h e initial a v o i d a n c e sessions, naive a n i m a l s were placed in t h e b e h a v i o r a l s i t u a t i o n for a five m i n acclimitization p e r i o d a n d a d m i n i s t e r e d the ChEI. Cholinergic a n t a g o n i s t s were a d m i n i s t e r e d 25 min p r i o r to p l a c e m e n t in the e x p e r i m e n t a l c h a m b e r . T h e r e a f t e r , a n i m a l s received 75 a v o i d a n c e trials. The c r i t e r i o n for l e a r n i n g was n i n e consecutive a v o i d a n c e s out o f 10 trials. All a v o i d a n c e a n d escape data was expressed as p e r c e n t o f 75 trials.
Performance Studies A n i m a l s were t r e a t e d in this e x p e r i m e n t as in t h e acquis i t i o n s t u d y e x c e p t t h a t t h e y were f u r t h e r t r a i n e d in t w o a d d i t i o n a l a c q u i s i t i o n sessions p r i o r to drug t r e a t m e n t . Prior to the f o u r t h session, a v o i d a n c e b e h a v i o r r a n g e d b e t w e e n 96-+4% ( n = 12) and 98.2-+3% ( n = 14). T r a i n e d animals received e i t h e r m e t h y l a t r o p i n e or ( ) h y o s c y a m i n e in e q u i m o l a r doses a n d were p r e s e n t e d 50 trials. F o l l o w i n g t h e 5 0 t h trial ( 3 0 m i n l a t e r ) animals were r e m o v e d f r o m the s i t u a t i o n a n d a d m i n i s t e r e d e i t h e r p h y s o s t i g m i n e or neostigmine in e q u i m o l a r doses, and r e t u r n e d for an a d d i t i o n a l 100 trials. Drug effects were c o m p a r e d o n the ability to depress a v o i d a n c e b e h a v i o r b y at least 50%. T h e d u r a t i o n of t i m e was d e t e r m i n e d b e t w e e n the period of o n s e t a n d r e c o v e r y o f 50% a v o i d a n c e b e h a v i o r .
Brain A cetylcholinesterase Activity In a s e p a r a t e g r o u p of a n i m a l s A C h E activity was estim a t e d at various t i m e s a f t e r drug a d m i n i s t r a t i o n . F o l l o w i n g sacrifice o f t h e animals b y d e c a p i t a t i o n , the b r a i n s were q u i c k l y r e m o v e d , weighed and h o m o g e n i z e d in 9 v o l u m e s of cold saline (0.9% NaC1). A o n e ml a l i q u o t of b r a i n h o m o g e n a t e was d i l u t e d t o 1:30 w i t h 0.9% saline a n d allowed to e q u i l i b r a t e to a t e m p e r a t u r e of 3 7 . 5 ° C in a w a t e r b a t h . During this p e r i o d (10 min), pH was a d i u s t e d to 7.4. An in vitro c o n c e n t r a t i o n o f A C h (3 x 10 -3 M based o n a 3 0 ml b a t h ) was a d d e d to the assay m e d i u m and its rate o f h y d r o l y s i s was followed utilizing the pH Star m e t h o d described b e f o r e [ 8 ] . T h e rate of ACh h y d r o l y s i s ( m i n u s b l a n k ) was e q u i v a l e n t to , M of N a O H t i t r a t e d . A C h E activity was e x p r e s s e d as t h e uM of A C h h y d r o l y z e d / g / h r . The s u b s t r a t e in dose-response p h y s o s t i g m i n e e x p e r i m e n t s was m e t h a c h o l i n e . This s u b s t r a t e was utilized b e c a u s e , in o u r h a n d s , ACh h y d r o l y s i s rates were curvilinear w h e n A C h E was i n h i b i t e d m o r e t h a n 60% with reversible i n h i b i t o r s . This was n o t so w i t h m e t h a c h o l i n e as rates were linear regardless of i n h i b i t o r or assay e n v i r o n m e n t .
Brain A cetylcholine A t a p p r o p r i a t e t i m e intervals, rats were sacrificed b y d e c a p i t a t i o n , b r a i n tissue r e m o v e d , the c e r e b e l l u m excised a n d t h e right h a l f of t h e c e r e b r u m f r o z e n in i s o p e n t a n e
ROSECRANS AND DOMINO c o n t a i n e d in a b e a k e r s u r r o u n d e d by e t h a n o l and dry ice. T o t a l A C h E was d e t e r m i n e d in the left c e r e b r u m as described above. T o t a l A C h levels in the frozen samples of the r i g h t c e r e b r u m were d e t e r m i n e d using the frog rectus a b d o m i n u s assay as described previously [ 8 ] . RESULTS
Dose Response Relations o f Physostigmine on Rat Brain A cetylch olinesterase and A cetylcholine O u r initial objective was to o b t a i n f u r t h e r i n f o r m a t i o n c o n c e r n i n g the effects of various doses of p h y s o s t i g m i n e on b r a i n A C h a n d A C h E in animals p r e t r e a t e d w i t h 2.1 m g / k g o f m e t h y l a t r o p i n e . The data p r e s e n t e d in Table 1 i n d i c a t e t h a t all doses studied significantly i n c r e a s e d brain ACh a n d r e d u c e d b r a i n A C h E activity. A linear dose r e s p o n s e effect was observed in r e l a t i o n to ACHE. The slope was m u c h m o r e steep w i t h b r a i n A C h a n d leveled off at doses a b o v e 0.25 m g / k g o f p h y s o s t i g m i n e . M a x i m a l s t i m u l a t i o n o f the cholinergic s y s t e m was evid e n c e d at h i g h e r doses (1.0 m g / k g ) as i n d i c a t e d by e x t r e m e m u s c u l a r fasictdation, c h r o m o d a c r y o r r h e a ( b l o o d y tears), salivation and diarrhea. Animals receiving b e t w e e n 0.125 a n d 0 . 2 5 0 m g / k g o f p h y s o s t i g m i n e were n o t overtly affected. This t h e r e f o r e , was a good dose range for s t u d y i n g t h e b e h a v i o r a l effects o f this drug.
TABLE 1 EFFECTS OF PHYSOSTIGMINE ON BRAIN ACh AND AChE LEVELS IN RATS PRETREATED WITH METHYL ATROPINE
Dose (mg/kg)
AChE gmol/g/hr
ACh nmol/g
0
320-+ 7
10.2-+ 1.2
0,125
240 -+ 41 (22%)
15.2 + 1.0 (+49%)
0.250
220 _+ 34 (31%)
24.4 +- 3.9 (+129%)
1.000
176 -+ 26 (-47%)
26.2 + 2.5 (+156%)
4.000
132 + 38 (-59%)
29.4 + 4.0 (+ 194%)
All values are means ± S.E. of 6 animals at each dose of physostigmine. ACh and AChE were obtained from the same animals using one half of each brain for separate assays. Rats were pretreated with methyl atropine (2.1 mg/kg, s.c.) 30 min prior to physostigmine, and were sacrificed 20 min thereafter. The figures in ( ) indicate the change from control. Note that in these methyl atropine treated animals the decrease in AChE was paradoxically less than one would expect. In addition the control levels of brain ACh are also lower than usually reported.
CHOLINERGIC SYSTEM AND BEHAVIOR
69
Effects of ( - ) Hyoscyamine and Methyl Atropine on Aequisition and Performance of Conditioned Pole-/umping ( - ) H y o s c y a m i n e in low doses ( 0 . 3 0 - 0 , 5 0 m g / k g ) h a d little effect o n acquisition. A b i o m o d a l e f f e c t was o b s e r v e d w i t h larger a m o u n t s (Table 2). A dose of 0.75 mg/kg, s.c. significantly facilitated b e h a v i o r , while i n h i b i t i o n was o b s e r v e d at 1.0 mg/kg. T h e drug had n o e f f e c t o n p e r f o r m a n c e of c o n d i t i o n e d a v o i d a n c e r e s p o n s e s in doses u p to 8.0 mg/kg. E q u i m o l a r doses of the q u a t e r n a r y a n t i c h o l i n e r gic drug, m e t h y l a t r o p i n e , had n o e f f e c t o n e i t h e r acquisit i o n or p e r f o r m a n c e of this b e h a v i o r .
Effects of Physostigmine and Neostigmine on Acquisition of Conditioned Pole-lumping P h y s o s t i g m i n e in doses of 0.10 a n d 0.15 m g / k g p r e v e n t ed rats f r o m l e a r n i n g this task ( T a b l e 3). This was e v i d e n t in all p a r a m e t e r s used. The effects were dose related i n a s m u c h as 0.15 m g / k g caused a r e d u c t i o n in % a v o i d a n c e a n d escape b e h a v i o r a l m o s t twice t h a t caused b y 0 . 1 0 mg/kg. A f t e r p h y s o s t i g m i n e ( 0 . 1 0 mg/kg, s.c.) a l o n e 73.7 trials were r e q u i r e d for c r i t e r i o n while a f t e r m e t h y l a t r o p i n e plus p h y s o s t i g m i n e 67.0 trials were r e q u i r e d . A n e q u i m o l a r dose of ( ) h y o s c y a m i n e p r o d u c e d a m u c h greater a n t a g o n i s m o f 0 . i 0 m g / k g o f p h y s o s t i g m i n e . I n t e r e s t i n g l y , the larger dose of 0.1 5 m g / k g o f p h y s o s t i g m i n e was n o t as significantly a n t a g o n i z e d b y ( - ) h y o s c y a m i n e . ( - ) H y o s c y a m i n e (1,0 m g / k g ) plus p h y s o s t i g m i n e (0.15 m g / k g ) r e s u l t e d in a n even g r e a t e r s u p p r e s s i o n of a c q u i s i t i o n . Trials to criteria were increased to 62.9 -+ 4.7, while % a v o i d a n c e was decreased to 2 5 . 0 ± 8.5.
TABLE 2 EFFECTS OF VARIOUS DOSES OF ( - ) HYOSCYAMINE ON ACQUISITION OF POLE JUMPING
Dose mg/kg
Trials to Criterion
Mean % Avoidance
Mean % Escape
0
47.1±2.4
57.2±3~0
8.1±1.7
0.30
47.8±5.7
58.5±5.5
17.3±3.9
0.50
46.0+3.3
57.9+4.9
16.6±2.5
0.75
37.9±3.2*
69.1+4,1"
10.3±1.5
1.00
57.9+6.4*
38.0±8.9
10.7±2.9
All values are means -+ S.E. of 12 animals per group. *Significant from saline controls at p<0.05 (Mann-Whitney U Test).
TABLE 3 EFFECTS OF PHYSOSTIGMINE ON ACQUISITION TO THE POLE JUMP RESPONSE
Drug Pretreatment
Dose of Physostigmine
Trials to Criterion
% Avoidance
% Escape
Saline
0.0
47.5 + 3.2
47.3 + 5,3
12.8 + 2.3
Saline
0.10
73.7 ± 1.2"
12.9 ± 4.8*
26.1 + 6.9
Saline
0.15
>75*
3.1 +- 1.5"
14.2 _+7.0
Methyl atropine 1 mg/kg
0.10
67.0 ± 4.7
22.2 +_ 9.4*
20.2 ± 9.9
Methyl atropine 1 mg/kg
0.15
73.3 -+ 4.5*
6.0 ± 3.0
11.7 _+4.7
( - ) Hyoscyamine 0,5 mg/kg
0,10
55.4 _+5.0
42.9 ± I0.2 t
16.8 ± 4.9
(-)Hyoscyamine 0.5 mg/kg
0.15
60.6_+4.1"
35.0±
13.4+_ 3.1
7.5*
All values represent the means -+ S.E. for 12 animals per group. *Significantly different from saline alone, p<0.05 (Mann-Whitney U Test). ~-Significantly different from methyl atropine pretreated animals, p<0.05 (Mann-Whitney U Test). Drug pretreatments were given 30 min before the physostigmine.
70
ROSECRANS AND DOMINO
The effects of n e o s t i g m i n e in a dose e q u i m o l a r to 0.15 m g / k g p h y s o s t i g m i n e , o n t h e o t h e r h a n d , c o u l d n o t be a n t a g o n i z e d b y a n y of t h e p r e t r e a t m e n t s (Table 4). B o t h c h o l i n e r g i c a n t a g o n i s t s a p p e a r e d to alter escape r e s p o n d i n g without affecting % avoidance after neostigmine. However, regardless o f p r e t r e a t m e n t , n o n e of t h e 36 rats given n e o s t i g m i n e were able to learn this task. While these animals were n o t able t o r e a c h criteria (9 o u t of 10 a v o i d a n c e responses), t o t a l % r e s p o n d i n g (% escape + % a v o i d a n c e ) i n d i c a t e d t h a t these rats h a d l e a r n e d the task to s o m e degree. In c o n t r a s t , a n i m a l s receiving e q u i m o l a r doses of p h y s o s t i g m i n e ( 0 . 1 5 0 m g / k g ) m a d e o n e t h i r d less t o t a l responses t h a n t h o s e given n e o s t i g m i n e .
TABLE 5 EFFECTS OF NEO,STIGMINE AND PHYSOSTIGMINE AVOIDANCE PERFORMANCE IN TRAINED RATS
ON
Duration of Behavioral Depression Pretreatment
Neostigmine 0.12 mg/kg
Physostigmine 0.15 mg/kg
Saline
25.4 ± 2.5 (6)
37.3 + 2.4 (6)
Methyl Atropine 2.1 mg/kg
26.5 + 2.9 (18)
14.6 ± 4.5 * (6)
EFFECTS OF NEOSTIGMINE ON ACQUISITION OF THE POLE JUMP RESPONSE
( - ) Hyoscyamine 1.0 mg/kg
21.9 -+ 6.2 (6)
0.07 (6)
Drug Pretreatment
Trials to Criterion
% Avoidance
% Escape
None + saline instead of neostigmine
50.0+3.3
55.3±3.2
17.4±1.6
All values are means _+ S.E. (min) with the number of animals indicated in parentheses. The duration of behavioral depression was calculated from the onset of 50% avoidance to recovery of 50% avoidance. % avoidance was determined at 5 min intervals of each 6 7 trials. *p<0.05 t p < 0.001 ,,
Saline
74.0 ± 0.9
14.5 + 3.0
35.4 ± 6.8
Methyl atropine 1 mg/kg
73.0 ± 1.4
12.2 + 0.7
28.7 ± 7.1
( ) Hyoscyamine 0.5 mg/kg
71.1 + 1.8
17.8 + 3.9
21.0 +_5.4
TABLE 4
Each value represents the mean + S.E. of 12 animals per group. All values obtained with neostigmine were significantly different from saline control data at p<0.001 (Mann-Whitney U Test). Pretreatments were given 30rain before neostigmine, 0.12 mg/kg.
Effects o f Equimolar Doses o f Physostigmine and Neostigmine on Conditioned Pole-jumping Performance and Correlations with Brain A cetylcholinesterase Activity As i n d i c a t e d in Table 5, t h e d u r a t i o n of d e p r e s s i o n of p e r f o r m a n c e o n pole j u m p a v o i d a n c e lasted 37.3 min foll o w i n g 0.15 m g / k g o f p h y s o s t i g m i n e . This effect was r e d u c e d b y 60% w i t h m e t h y l a t r o p i n e a n d was c o m p l e t e l y a n t a g o n i z e d w i t h an e q u i m o l a r dose of ( ) h y o s c y a m i n e . In c o n t r a s t , t h e effects of n e o s t i g m i n e ( 0 . 1 2 m g / k g ) c o u l d n o t b e b l o c k e d b y e i t h e r cholinergic a n t a g o n i s t . N e o s t i g m i n e p r o d u c e d a severe b e h a v i o r a l d e p r e s s i o n w i t h n o e f f e c t o n b r a i n A C h E activity. O n the o t h e r h a n d , the b e h a v i o r a l effects f o l l o w i n g a n e q u i m o l a r dose of p h y s o s t i g m i n e c o r r e l a t e d initially w i t h a r e d u c t i o n o f b r a i n A C h E activity b u t n o t d u r i n g r e c o v e r y as i n d i c a t e d in Fig. 1. The l a t t e r f i n d i n g is q u i t e surprising a n d suggests s o m e a d a p t a t i o n to l o w e r e d brain A C h E activity.
DISCUSSION The basic n e w i n f o r m a t i o n o f this s t u d y is t h a t neostigmine can d r a m a t i c a l l y depress b e h a v i o r in reasonable doses c o m p a r e d to p h y s o s t i g m i n e . This depression is n o t associated w i t h a n y significant changes in brain AChE activity. W i t h o u t measures of brain A C h E activity and the use of selective t e r t i a r y a n d q u a t e r n a r y cholinergic antagonists, o n e m i g h t c o n c l u d e t h a t e q u i m o l a r a m o u n t s of these t w o agonists act to suppress b e h a v i o r b y a similar m e c h a n i s m . ( - ) H y o s c y a m i n e is an effective a n t a g o n i s t of physostigmine in doses w h i c h do n o t affect n e o s t i g m i n e . Alt h o u g h n e o s t i g m i n e did n o t alter brain A C h E activity, it did i m p a i r b o t h a c q u i s i t i o n a n d p e r f o r m a n c e , p r e s u m a b l e by its m a r k e d p e r i p h e r a l actions. In c o n t r a s t , the b e h a v i o r a l supp r e s s a n t effects of p h y s o s t i g m i n e are associated with a decrease in b r a i n A C h E activity and seem to be due primarily to its c e n t r a l actions as has b e e n suggested by others. These results also i n d i c a t e t h a t a c q u i s i t i o n of avoidance b e h a v i o r is a m u c h m o r e sensitive e n d p o i n t t h a n perform a n c e . T h e d e p r e s s a n t effects of p h y s o s t i g m i n e seem to be m o s t clearly c o r r e l a t e d w i t h its c e n t r a l actions in r e d u c i n g b r a i n A C h E activity a n d increasing ACh levels. These findings c o n f i r m a n d e x t e n d o u r earlier studies w h i c h i n d i c a t e an i m p o r t a n t role o f the c h o l i n e r g i c s y s t e m in behavior. One m i g h t have e x p e c t e d t h a t m e t h y l a t r o p i n e would a n t a g o n i z e some of t h e effects of n e o s t i g m i n e on behavior. The data i n d i c a t e it did n o t . Hence, it appears t h a t t h e behavioral e f f e c t s o f n e o s t i g m i n e are primarily ncholinergic. T h e p e r i p h e r a l m-cholinergic effects of neostigm i n e w o u l d b e e x p e c t e d t o b e b l o c k e d by such a p r e t r e a t ment. Another unexpected finding was t h a t with m e t h y l a t r o p i n e p r e t r e a t m e n t p h y s o s t i g m i n e caused f r o m 22 t o 59% i n h i b i t i o n of brain A C h E (see Table 1 ) d e p e n d -
CHOLINERGIC SYSTEM AND BEHAVIOR
IO0
71
NEOSTIGMINE
[
i, (0.12mg/kg)
80
AChE r-n
so
Avoidance ,.; 4 o u~
F
-H
"6 2 0 ¢~ 0
0
I0
20
40
.oo li0., , 20 0
0
,
ii
20
PHYSOSTIGMINE (0.15 mg/kg)
40 Time (rain)
60
90
FIG. 1. Effects of an equimolar dose of neostigmine and physostigmine on brain acetylcholinesterase activity and the performance of pole-jumping avoidance. All animals were pretreated with atropine methyl nitrate (2.1 mg/kg) 30 min prior to a dose of the cholinesterase inhibitor. The dose of neostigmine was 0.12 and physostigmine 0.15 mg/kg, s.c. The behavioral data are given as the mean ± S.E. in groups of 6 - 1 2 animals. Chemical estimations were conducted in 3 - 6 animals at each time interval. Control AChE activity was 859 ± 40uM/g/hr. In the physostigmine treated animals, brain AChE levels were significantly lower than control levels at 20, 40 and 60 min following each dose. Group comparison tests were run comparing each to the control. * p < 0.01, *** p<0.001.
ing u p o n t h e dose given. D o m i n o a n d Olds [3] p r e v i o u s l y r e p o r t e d t h a t relatively small a m o u n t s of p h y s o s t i g m i n e a l o n e (0.1 mg/kg, s.c.) p r o d u c e d a 56% decrease in b r a i n ACHE. It m a y b e t h a t e n o u g h m e t h y l a t r o p i n e was p r e s e n t in t h e b r a i n h o m o g e n a t e s in vitro to partially p r o t e c t A C h E f r o m inhibition by t h e physostigmine. The p o i n t n e e d s to
be e x a m i n e d f u r t h e r for this d i s c r e p a n c y is d i s t u r b i n g . T h e b e h a v i o r a l c o n s e q u e n c e s of an increase in b r a i n A C h are p r i m a r i l y a r e d u c t i o n in r e s p o n d i n g (no-go b e h a v i o r ) in spite o f b e i n g a w a k e [ 2 ] . T h e p r e s e n t findings s u p p o r t t h e d a t a o f G r o s s m a n [5] w h i c h also suggest t h a t the c e n t r a l c h o l i n e r g i c s y s t e m is i n h i b i t o r y to m o s t behaviors.
72
R O S E C R A N S AND DOMINO
REFERENCES 1. Bennett, E. L., M. C. Diamond, D. Krech and M. R. Rosenzweig. Chemical and anatomical plasticity of brain. Science 146: 610-619, 1964. 2. Domino, E. F. Role of the central cholinergic system in wakefulness, fast wave sleep and " N o - G o " behavior. In: The Present Status o f Psychotropic Drugs, edited by A. Cerletti and F. J. Bove. Amsterdam: Excerpta Medica Foundation, 1969, pp. 273-277. 3. Domino, E. F. and M. E. Olds. Cholinergic inhibition of selfstimulation behavior. J. Pharmac. exp. Ther. 164: 202-211, 1968. 4. Goldberg, M. E., H. E. Johnson and J. B. Knaak. Inhibition of discrete avoidance behavior by three anticholinesterase agents. Psychopharmacologia 7 : 7 2 76, 1965. 5. Grossman, S. P. Cholinergic synapses in the limbic system and behavioral inhibition. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 50:315 325, 1972.
6. Michelson, M. J. Pharmacological evidence of the role of acetylcholine in the higher nervous activity of man and animals. Activ. Nerv. Sup. (Praha) 3: 140-147, 1961. 7. Pfeiffer, C. C. and E. H. Jenny. Inhibition of conditioned response and the counteraction of schizophrenia by muscarinic stimulation of the brain. Ann. N. Y. Acad. ScL 66: 753-764, 1957. 8. Rosecrans, J. A., A. T. Dren and E. F. Domino. Effects of physostigmine on rat brain acetylcholine, acetylcholinesterase and conditioned pole jumping. Neuropharmac. 7: 127-134, 1968. 9. Vaillant, G. E. Antagonism between physostigmine and atropine on the behavior of the pigeon. Arch, exp. Path. Pharmak. 248: 406-416, 1964. 10. Vaillant, G. E. A comparison of antagonisms of physostigmine-induced suppression of behavior. J. Pharmacol. exp. Ther. 157: 636-648, 1967.
Comparative Effects of Physostigmine and Neostigmine on Acquisition and Performance of a Conditioned Avoidance Behavior in the Raf J O H N A. R O S E C R A N S 2 A N D E D W A R D F. D O M I N O
Michigan Neuropsychopharmacology Research Program, Department o f Pharmacology, University o f Michigan, A n n Arbor, Michigan 48104
( R e c e i v e d 14 M a r c h 1 9 7 3 )
ROSECRANS, J. A. AND E. F. DOMINO. Comparative effects of physostigmine and neostigmine on acquisition and performance o f a conditioned avoidance behavior in the rat. PHARMAC. BIOCHEM. BEHAV. 2(1) 6 7 - 7 2 , 1974. - The actions of physostigmine (0.10 and 0.15 mg/kg) and neostigmine (0.12 mg/kg) given s.c. on acquisition and performance of conditioned pole jumping were studied in rats receiving either 0.9% saline, ( - ) hyoscyamine or atropine methylnitrate. As estimated by inhibition of acetylcholinesterase (ACHE) activity, physostigmine but not neostigmine entered the rat brain. Even though neostigmine did not significantly alter brain AChE activity in equimolar doses to physostigmine, it depressed both the acquisition and performance of a behavioral task, suggesting a predominant peripheral action. Although physostigmine depressed behavior mainly via a central action, its peripheral actions also play a role in its behavioral effects. Physostigmine
Neosfigmine
Conditioned avoidance
Central and peripheral drug action
effects of the c h o l i n e s t e r a s e i n h i b i t o r s p h y s o s t i g m i n e (a t e r t i a r y a m i n e ) a n d n e o s t i g m i n e (a q u a t e r n a r y a m i n e ) on a c q u i s i t i o n and p e r f o r m a n c e of c o n d i t i o n e d pole j u m p i n g a n d c o r r e l a t e t h e i r b e h a v i o r a l effects w i t h b r a i n AChE activity in t h e rat. T w o c h o l i n e r g i c a n t a g o n i s t s were also used t o s e p a r a t e t h e i r c e n t r a l and p e r i p h e r a l effects. One was ( - ) h y o s c y a m i n e , the active i s o m e r of a t r o p i n e (a t e r t i a r y a m i n e ) a n d the o t h e r m e t h y l a t r o p i n e (a q u a t e r n a r y a m i n e ) . A l t h o u g h similar studies have b e e n r e p o r t e d [3, 4, 7, 8 - 1 0 ] , t h e c o r r e l a t i o n o f b r a i n A c h e activity w i t h b e h a v i o r a l effects o n l y rarely has b e e n done.
P H Y S O S T I G M I N E is a reversible c h o l i n e s t e r a s e i n h i b i t o r ( C h E I ) w h i c h has b e e n useful in s t u d y i n g c h o l i n e r g i c m e c h a n i s m s o f b e h a v i o r [ 1 , 3 , 4, 6 - 1 0 ] . Its p r e s e n c e in t h e c e n t r a l n e r v o u s s y s t e m can b e d e t e r m i n e d i n d i r e c t l y b y its effects o n b r a i n ChE. Because of its reversible n a t u r e , one can m e a s u r e b o t h the o n s e t and r e c o v e r y of its b e h a v i o r a l effects. T h e r e is a good c o r r e l a t i o n b e t w e e n i n h i b i t i o n of b r a i n a c e t y l c h o l i n e s t e r a s e activity (ACHE), an increase in b r a i n a c e t y l c h o l i n e ( A C h ) , a n d b e h a v i o r a l d e p r e s s i o n foll o w i n g p h y s o s t i g m i n e [ 3,8 ]. While this a p p r o a c h a p p e a r s valid in s t u d y i n g t h e behavioral role of b r a i n ACh, t h e r e are p r o b l e m s associated w i t h t h e use o f p h y s o s t i g m i n e and related c o m p o u n d s , Aside f r o m its c e n t r a l effect, p h y s o s t i g m i n e has well k n o w n p e r i p h e r a l actions. In view of t h e fact t h a t the b l o o d - b r a i n barrier tends to exclude quaternary nitrogen compounds, t h e t e r t i a r y and q u a t e r n a r y analogs o f various c h o l i n e r g i c agonists and a n t a g o n i s t s can be useful t o o l s to d e t e r m i n e relative c e n t r a l vs p e r i p h e r a l effects. T h e a d d i t i o n a l m e a s u r e of b r a i n A C h E activity provides f u r t h e r e v i d e n c e t h a t a ChEI has p e n e t r a t e d i n t o t h e c e n t r a l n e r v o u s s y s t e m . The p u r p o s e of t h e p r e s e n t s t u d y was to d e t e r m i n e the
METHOD Male a l b i n o H o l t z m a n rats f r o m 4 5 - 9 0 days of age were used. A c q u i s i t i o n a n d p e r f o r m a n c e studies were c o n d u c t e d utilizing t h e rat pole j u m p r e s p o n s e as described previously [ 8 ] . B o t h i n i t i a t i o n a n d r e c o r d i n g of trials and r e s p o n s e s was a c c o m p l i s h e d a u t o m a t i c a l l y . The CS was a 5 sec b u z z e r w h i c h o v e r l a p p e d a 5 sec US consisting of a 1 ma s h o c k s c r a m b l e d t h r o u g h the grid floor. I n t e r t r i a i periods ranged f r o m 2 0 4 0 sec w i t h a m e a n o f 30 sec. R e s p o n s e s d u r i n g
'Supported in part by USPHS MH-11846. 2Present address: Department of Pharmacology, Medical College of Virginia, Richmond, Virginia. 67
68 t h e i n t e r t r i a l p e r i o d were n o t c o u n t e d . A n i m a l s were allowed t o h a b i t u a t e to t h e a n i m a l r o o m for o n e week p r i o r to testing, d u r i n g w h i c h t i m e t h e y were n u m b e r e d a n d placed in a p r e d e s i g n e d g r o u p utilizing a r a n d o m table. Experim e n t s were c o n d u c t e d in b l o c k s consisting of o n e c o n t r o l a n d five e x p e r i m e n t a l groups o f 12 a n i m a l s each. Saline, ( - ) h y o s c y a m i n e or m e t h y l a t r o p i n e was a d m i n i s t e r e d 30 rain p r i o r t o t h e a d m i n i s t r a t i o n of the ChEIs physostigm i n e a n d n e o s t i g m i n e . Drugs were a d m i n i s t e r e d s.c. Dosage was calculated as base.
Acquisition Studies Prior to t h e initial a v o i d a n c e sessions, naive a n i m a l s were placed in t h e b e h a v i o r a l s i t u a t i o n for a five m i n acclimitization p e r i o d a n d a d m i n i s t e r e d the ChEI. Cholinergic a n t a g o n i s t s were a d m i n i s t e r e d 25 min p r i o r to p l a c e m e n t in the e x p e r i m e n t a l c h a m b e r . T h e r e a f t e r , a n i m a l s received 75 a v o i d a n c e trials. The c r i t e r i o n for l e a r n i n g was n i n e consecutive a v o i d a n c e s out o f 10 trials. All a v o i d a n c e a n d escape data was expressed as p e r c e n t o f 75 trials.
Performance Studies A n i m a l s were t r e a t e d in this e x p e r i m e n t as in t h e acquis i t i o n s t u d y e x c e p t t h a t t h e y were f u r t h e r t r a i n e d in t w o a d d i t i o n a l a c q u i s i t i o n sessions p r i o r to drug t r e a t m e n t . Prior to the f o u r t h session, a v o i d a n c e b e h a v i o r r a n g e d b e t w e e n 96-+4% ( n = 12) and 98.2-+3% ( n = 14). T r a i n e d animals received e i t h e r m e t h y l a t r o p i n e or ( ) h y o s c y a m i n e in e q u i m o l a r doses a n d were p r e s e n t e d 50 trials. F o l l o w i n g t h e 5 0 t h trial ( 3 0 m i n l a t e r ) animals were r e m o v e d f r o m the s i t u a t i o n a n d a d m i n i s t e r e d e i t h e r p h y s o s t i g m i n e or neostigmine in e q u i m o l a r doses, and r e t u r n e d for an a d d i t i o n a l 100 trials. Drug effects were c o m p a r e d o n the ability to depress a v o i d a n c e b e h a v i o r b y at least 50%. T h e d u r a t i o n of t i m e was d e t e r m i n e d b e t w e e n the period of o n s e t a n d r e c o v e r y o f 50% a v o i d a n c e b e h a v i o r .
Brain A cetylcholinesterase Activity In a s e p a r a t e g r o u p of a n i m a l s A C h E activity was estim a t e d at various t i m e s a f t e r drug a d m i n i s t r a t i o n . F o l l o w i n g sacrifice o f t h e animals b y d e c a p i t a t i o n , the b r a i n s were q u i c k l y r e m o v e d , weighed and h o m o g e n i z e d in 9 v o l u m e s of cold saline (0.9% NaC1). A o n e ml a l i q u o t of b r a i n h o m o g e n a t e was d i l u t e d t o 1:30 w i t h 0.9% saline a n d allowed to e q u i l i b r a t e to a t e m p e r a t u r e of 3 7 . 5 ° C in a w a t e r b a t h . During this p e r i o d (10 min), pH was a d i u s t e d to 7.4. An in vitro c o n c e n t r a t i o n o f A C h (3 x 10 -3 M based o n a 3 0 ml b a t h ) was a d d e d to the assay m e d i u m and its rate o f h y d r o l y s i s was followed utilizing the pH Star m e t h o d described b e f o r e [ 8 ] . T h e rate of ACh h y d r o l y s i s ( m i n u s b l a n k ) was e q u i v a l e n t to , M of N a O H t i t r a t e d . A C h E activity was e x p r e s s e d as t h e uM of A C h h y d r o l y z e d / g / h r . The s u b s t r a t e in dose-response p h y s o s t i g m i n e e x p e r i m e n t s was m e t h a c h o l i n e . This s u b s t r a t e was utilized b e c a u s e , in o u r h a n d s , ACh h y d r o l y s i s rates were curvilinear w h e n A C h E was i n h i b i t e d m o r e t h a n 60% with reversible i n h i b i t o r s . This was n o t so w i t h m e t h a c h o l i n e as rates were linear regardless of i n h i b i t o r or assay e n v i r o n m e n t .
Brain A cetylcholine A t a p p r o p r i a t e t i m e intervals, rats were sacrificed b y d e c a p i t a t i o n , b r a i n tissue r e m o v e d , the c e r e b e l l u m excised a n d t h e right h a l f of t h e c e r e b r u m f r o z e n in i s o p e n t a n e
ROSECRANS AND DOMINO c o n t a i n e d in a b e a k e r s u r r o u n d e d by e t h a n o l and dry ice. T o t a l A C h E was d e t e r m i n e d in the left c e r e b r u m as described above. T o t a l A C h levels in the frozen samples of the r i g h t c e r e b r u m were d e t e r m i n e d using the frog rectus a b d o m i n u s assay as described previously [ 8 ] . RESULTS
Dose Response Relations o f Physostigmine on Rat Brain A cetylch olinesterase and A cetylcholine O u r initial objective was to o b t a i n f u r t h e r i n f o r m a t i o n c o n c e r n i n g the effects of various doses of p h y s o s t i g m i n e on b r a i n A C h a n d A C h E in animals p r e t r e a t e d w i t h 2.1 m g / k g o f m e t h y l a t r o p i n e . The data p r e s e n t e d in Table 1 i n d i c a t e t h a t all doses studied significantly i n c r e a s e d brain ACh a n d r e d u c e d b r a i n A C h E activity. A linear dose r e s p o n s e effect was observed in r e l a t i o n to ACHE. The slope was m u c h m o r e steep w i t h b r a i n A C h a n d leveled off at doses a b o v e 0.25 m g / k g o f p h y s o s t i g m i n e . M a x i m a l s t i m u l a t i o n o f the cholinergic s y s t e m was evid e n c e d at h i g h e r doses (1.0 m g / k g ) as i n d i c a t e d by e x t r e m e m u s c u l a r fasictdation, c h r o m o d a c r y o r r h e a ( b l o o d y tears), salivation and diarrhea. Animals receiving b e t w e e n 0.125 a n d 0 . 2 5 0 m g / k g o f p h y s o s t i g m i n e were n o t overtly affected. This t h e r e f o r e , was a good dose range for s t u d y i n g t h e b e h a v i o r a l effects o f this drug.
TABLE 1 EFFECTS OF PHYSOSTIGMINE ON BRAIN ACh AND AChE LEVELS IN RATS PRETREATED WITH METHYL ATROPINE
Dose (mg/kg)
AChE gmol/g/hr
ACh nmol/g
0
320-+ 7
10.2-+ 1.2
0,125
240 -+ 41 (22%)
15.2 + 1.0 (+49%)
0.250
220 _+ 34 (31%)
24.4 +- 3.9 (+129%)
1.000
176 -+ 26 (-47%)
26.2 + 2.5 (+156%)
4.000
132 + 38 (-59%)
29.4 + 4.0 (+ 194%)
All values are means ± S.E. of 6 animals at each dose of physostigmine. ACh and AChE were obtained from the same animals using one half of each brain for separate assays. Rats were pretreated with methyl atropine (2.1 mg/kg, s.c.) 30 min prior to physostigmine, and were sacrificed 20 min thereafter. The figures in ( ) indicate the change from control. Note that in these methyl atropine treated animals the decrease in AChE was paradoxically less than one would expect. In addition the control levels of brain ACh are also lower than usually reported.
CHOLINERGIC SYSTEM AND BEHAVIOR
69
Effects of ( - ) Hyoscyamine and Methyl Atropine on Aequisition and Performance of Conditioned Pole-/umping ( - ) H y o s c y a m i n e in low doses ( 0 . 3 0 - 0 , 5 0 m g / k g ) h a d little effect o n acquisition. A b i o m o d a l e f f e c t was o b s e r v e d w i t h larger a m o u n t s (Table 2). A dose of 0.75 mg/kg, s.c. significantly facilitated b e h a v i o r , while i n h i b i t i o n was o b s e r v e d at 1.0 mg/kg. T h e drug had n o e f f e c t o n p e r f o r m a n c e of c o n d i t i o n e d a v o i d a n c e r e s p o n s e s in doses u p to 8.0 mg/kg. E q u i m o l a r doses of the q u a t e r n a r y a n t i c h o l i n e r gic drug, m e t h y l a t r o p i n e , had n o e f f e c t o n e i t h e r acquisit i o n or p e r f o r m a n c e of this b e h a v i o r .
Effects of Physostigmine and Neostigmine on Acquisition of Conditioned Pole-lumping P h y s o s t i g m i n e in doses of 0.10 a n d 0.15 m g / k g p r e v e n t ed rats f r o m l e a r n i n g this task ( T a b l e 3). This was e v i d e n t in all p a r a m e t e r s used. The effects were dose related i n a s m u c h as 0.15 m g / k g caused a r e d u c t i o n in % a v o i d a n c e a n d escape b e h a v i o r a l m o s t twice t h a t caused b y 0 . 1 0 mg/kg. A f t e r p h y s o s t i g m i n e ( 0 . 1 0 mg/kg, s.c.) a l o n e 73.7 trials were r e q u i r e d for c r i t e r i o n while a f t e r m e t h y l a t r o p i n e plus p h y s o s t i g m i n e 67.0 trials were r e q u i r e d . A n e q u i m o l a r dose of ( ) h y o s c y a m i n e p r o d u c e d a m u c h greater a n t a g o n i s m o f 0 . i 0 m g / k g o f p h y s o s t i g m i n e . I n t e r e s t i n g l y , the larger dose of 0.1 5 m g / k g o f p h y s o s t i g m i n e was n o t as significantly a n t a g o n i z e d b y ( - ) h y o s c y a m i n e . ( - ) H y o s c y a m i n e (1,0 m g / k g ) plus p h y s o s t i g m i n e (0.15 m g / k g ) r e s u l t e d in a n even g r e a t e r s u p p r e s s i o n of a c q u i s i t i o n . Trials to criteria were increased to 62.9 -+ 4.7, while % a v o i d a n c e was decreased to 2 5 . 0 ± 8.5.
TABLE 2 EFFECTS OF VARIOUS DOSES OF ( - ) HYOSCYAMINE ON ACQUISITION OF POLE JUMPING
Dose mg/kg
Trials to Criterion
Mean % Avoidance
Mean % Escape
0
47.1±2.4
57.2±3~0
8.1±1.7
0.30
47.8±5.7
58.5±5.5
17.3±3.9
0.50
46.0+3.3
57.9+4.9
16.6±2.5
0.75
37.9±3.2*
69.1+4,1"
10.3±1.5
1.00
57.9+6.4*
38.0±8.9
10.7±2.9
All values are means -+ S.E. of 12 animals per group. *Significant from saline controls at p<0.05 (Mann-Whitney U Test).
TABLE 3 EFFECTS OF PHYSOSTIGMINE ON ACQUISITION TO THE POLE JUMP RESPONSE
Drug Pretreatment
Dose of Physostigmine
Trials to Criterion
% Avoidance
% Escape
Saline
0.0
47.5 + 3.2
47.3 + 5,3
12.8 + 2.3
Saline
0.10
73.7 ± 1.2"
12.9 ± 4.8*
26.1 + 6.9
Saline
0.15
>75*
3.1 +- 1.5"
14.2 _+7.0
Methyl atropine 1 mg/kg
0.10
67.0 ± 4.7
22.2 +_ 9.4*
20.2 ± 9.9
Methyl atropine 1 mg/kg
0.15
73.3 -+ 4.5*
6.0 ± 3.0
11.7 _+4.7
( - ) Hyoscyamine 0,5 mg/kg
0,10
55.4 _+5.0
42.9 ± I0.2 t
16.8 ± 4.9
(-)Hyoscyamine 0.5 mg/kg
0.15
60.6_+4.1"
35.0±
13.4+_ 3.1
7.5*
All values represent the means -+ S.E. for 12 animals per group. *Significantly different from saline alone, p<0.05 (Mann-Whitney U Test). ~-Significantly different from methyl atropine pretreated animals, p<0.05 (Mann-Whitney U Test). Drug pretreatments were given 30 min before the physostigmine.
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ROSECRANS AND DOMINO
The effects of n e o s t i g m i n e in a dose e q u i m o l a r to 0.15 m g / k g p h y s o s t i g m i n e , o n t h e o t h e r h a n d , c o u l d n o t be a n t a g o n i z e d b y a n y of t h e p r e t r e a t m e n t s (Table 4). B o t h c h o l i n e r g i c a n t a g o n i s t s a p p e a r e d to alter escape r e s p o n d i n g without affecting % avoidance after neostigmine. However, regardless o f p r e t r e a t m e n t , n o n e of t h e 36 rats given n e o s t i g m i n e were able to learn this task. While these animals were n o t able t o r e a c h criteria (9 o u t of 10 a v o i d a n c e responses), t o t a l % r e s p o n d i n g (% escape + % a v o i d a n c e ) i n d i c a t e d t h a t these rats h a d l e a r n e d the task to s o m e degree. In c o n t r a s t , a n i m a l s receiving e q u i m o l a r doses of p h y s o s t i g m i n e ( 0 . 1 5 0 m g / k g ) m a d e o n e t h i r d less t o t a l responses t h a n t h o s e given n e o s t i g m i n e .
TABLE 5 EFFECTS OF NEO,STIGMINE AND PHYSOSTIGMINE AVOIDANCE PERFORMANCE IN TRAINED RATS
ON
Duration of Behavioral Depression Pretreatment
Neostigmine 0.12 mg/kg
Physostigmine 0.15 mg/kg
Saline
25.4 ± 2.5 (6)
37.3 + 2.4 (6)
Methyl Atropine 2.1 mg/kg
26.5 + 2.9 (18)
14.6 ± 4.5 * (6)
EFFECTS OF NEOSTIGMINE ON ACQUISITION OF THE POLE JUMP RESPONSE
( - ) Hyoscyamine 1.0 mg/kg
21.9 -+ 6.2 (6)
0.07 (6)
Drug Pretreatment
Trials to Criterion
% Avoidance
% Escape
None + saline instead of neostigmine
50.0+3.3
55.3±3.2
17.4±1.6
All values are means _+ S.E. (min) with the number of animals indicated in parentheses. The duration of behavioral depression was calculated from the onset of 50% avoidance to recovery of 50% avoidance. % avoidance was determined at 5 min intervals of each 6 7 trials. *p<0.05 t p < 0.001 ,,
Saline
74.0 ± 0.9
14.5 + 3.0
35.4 ± 6.8
Methyl atropine 1 mg/kg
73.0 ± 1.4
12.2 + 0.7
28.7 ± 7.1
( ) Hyoscyamine 0.5 mg/kg
71.1 + 1.8
17.8 + 3.9
21.0 +_5.4
TABLE 4
Each value represents the mean + S.E. of 12 animals per group. All values obtained with neostigmine were significantly different from saline control data at p<0.001 (Mann-Whitney U Test). Pretreatments were given 30rain before neostigmine, 0.12 mg/kg.
Effects o f Equimolar Doses o f Physostigmine and Neostigmine on Conditioned Pole-jumping Performance and Correlations with Brain A cetylcholinesterase Activity As i n d i c a t e d in Table 5, t h e d u r a t i o n of d e p r e s s i o n of p e r f o r m a n c e o n pole j u m p a v o i d a n c e lasted 37.3 min foll o w i n g 0.15 m g / k g o f p h y s o s t i g m i n e . This effect was r e d u c e d b y 60% w i t h m e t h y l a t r o p i n e a n d was c o m p l e t e l y a n t a g o n i z e d w i t h an e q u i m o l a r dose of ( ) h y o s c y a m i n e . In c o n t r a s t , t h e effects of n e o s t i g m i n e ( 0 . 1 2 m g / k g ) c o u l d n o t b e b l o c k e d b y e i t h e r cholinergic a n t a g o n i s t . N e o s t i g m i n e p r o d u c e d a severe b e h a v i o r a l d e p r e s s i o n w i t h n o e f f e c t o n b r a i n A C h E activity. O n the o t h e r h a n d , the b e h a v i o r a l effects f o l l o w i n g a n e q u i m o l a r dose of p h y s o s t i g m i n e c o r r e l a t e d initially w i t h a r e d u c t i o n o f b r a i n A C h E activity b u t n o t d u r i n g r e c o v e r y as i n d i c a t e d in Fig. 1. The l a t t e r f i n d i n g is q u i t e surprising a n d suggests s o m e a d a p t a t i o n to l o w e r e d brain A C h E activity.
DISCUSSION The basic n e w i n f o r m a t i o n o f this s t u d y is t h a t neostigmine can d r a m a t i c a l l y depress b e h a v i o r in reasonable doses c o m p a r e d to p h y s o s t i g m i n e . This depression is n o t associated w i t h a n y significant changes in brain AChE activity. W i t h o u t measures of brain A C h E activity and the use of selective t e r t i a r y a n d q u a t e r n a r y cholinergic antagonists, o n e m i g h t c o n c l u d e t h a t e q u i m o l a r a m o u n t s of these t w o agonists act to suppress b e h a v i o r b y a similar m e c h a n i s m . ( - ) H y o s c y a m i n e is an effective a n t a g o n i s t of physostigmine in doses w h i c h do n o t affect n e o s t i g m i n e . Alt h o u g h n e o s t i g m i n e did n o t alter brain A C h E activity, it did i m p a i r b o t h a c q u i s i t i o n a n d p e r f o r m a n c e , p r e s u m a b l e by its m a r k e d p e r i p h e r a l actions. In c o n t r a s t , the b e h a v i o r a l supp r e s s a n t effects of p h y s o s t i g m i n e are associated with a decrease in b r a i n A C h E activity and seem to be due primarily to its c e n t r a l actions as has b e e n suggested by others. These results also i n d i c a t e t h a t a c q u i s i t i o n of avoidance b e h a v i o r is a m u c h m o r e sensitive e n d p o i n t t h a n perform a n c e . T h e d e p r e s s a n t effects of p h y s o s t i g m i n e seem to be m o s t clearly c o r r e l a t e d w i t h its c e n t r a l actions in r e d u c i n g b r a i n A C h E activity a n d increasing ACh levels. These findings c o n f i r m a n d e x t e n d o u r earlier studies w h i c h i n d i c a t e an i m p o r t a n t role o f the c h o l i n e r g i c s y s t e m in behavior. One m i g h t have e x p e c t e d t h a t m e t h y l a t r o p i n e would a n t a g o n i z e some of t h e effects of n e o s t i g m i n e on behavior. The data i n d i c a t e it did n o t . Hence, it appears t h a t t h e behavioral e f f e c t s o f n e o s t i g m i n e are primarily ncholinergic. T h e p e r i p h e r a l m-cholinergic effects of neostigm i n e w o u l d b e e x p e c t e d t o b e b l o c k e d by such a p r e t r e a t ment. Another unexpected finding was t h a t with m e t h y l a t r o p i n e p r e t r e a t m e n t p h y s o s t i g m i n e caused f r o m 22 t o 59% i n h i b i t i o n of brain A C h E (see Table 1 ) d e p e n d -
CHOLINERGIC SYSTEM AND BEHAVIOR
IO0
71
NEOSTIGMINE
[
i, (0.12mg/kg)
80
AChE r-n
so
Avoidance ,.; 4 o u~
F
-H
"6 2 0 ¢~ 0
0
I0
20
40
.oo li0., , 20 0
0
,
ii
20
PHYSOSTIGMINE (0.15 mg/kg)
40 Time (rain)
60
90
FIG. 1. Effects of an equimolar dose of neostigmine and physostigmine on brain acetylcholinesterase activity and the performance of pole-jumping avoidance. All animals were pretreated with atropine methyl nitrate (2.1 mg/kg) 30 min prior to a dose of the cholinesterase inhibitor. The dose of neostigmine was 0.12 and physostigmine 0.15 mg/kg, s.c. The behavioral data are given as the mean ± S.E. in groups of 6 - 1 2 animals. Chemical estimations were conducted in 3 - 6 animals at each time interval. Control AChE activity was 859 ± 40uM/g/hr. In the physostigmine treated animals, brain AChE levels were significantly lower than control levels at 20, 40 and 60 min following each dose. Group comparison tests were run comparing each to the control. * p < 0.01, *** p<0.001.
ing u p o n t h e dose given. D o m i n o a n d Olds [3] p r e v i o u s l y r e p o r t e d t h a t relatively small a m o u n t s of p h y s o s t i g m i n e a l o n e (0.1 mg/kg, s.c.) p r o d u c e d a 56% decrease in b r a i n ACHE. It m a y b e t h a t e n o u g h m e t h y l a t r o p i n e was p r e s e n t in t h e b r a i n h o m o g e n a t e s in vitro to partially p r o t e c t A C h E f r o m inhibition by t h e physostigmine. The p o i n t n e e d s to
be e x a m i n e d f u r t h e r for this d i s c r e p a n c y is d i s t u r b i n g . T h e b e h a v i o r a l c o n s e q u e n c e s of an increase in b r a i n A C h are p r i m a r i l y a r e d u c t i o n in r e s p o n d i n g (no-go b e h a v i o r ) in spite o f b e i n g a w a k e [ 2 ] . T h e p r e s e n t findings s u p p o r t t h e d a t a o f G r o s s m a n [5] w h i c h also suggest t h a t the c e n t r a l c h o l i n e r g i c s y s t e m is i n h i b i t o r y to m o s t behaviors.
72
R O S E C R A N S AND DOMINO
REFERENCES 1. Bennett, E. L., M. C. Diamond, D. Krech and M. R. Rosenzweig. Chemical and anatomical plasticity of brain. Science 146: 610-619, 1964. 2. Domino, E. F. Role of the central cholinergic system in wakefulness, fast wave sleep and " N o - G o " behavior. In: The Present Status o f Psychotropic Drugs, edited by A. Cerletti and F. J. Bove. Amsterdam: Excerpta Medica Foundation, 1969, pp. 273-277. 3. Domino, E. F. and M. E. Olds. Cholinergic inhibition of selfstimulation behavior. J. Pharmac. exp. Ther. 164: 202-211, 1968. 4. Goldberg, M. E., H. E. Johnson and J. B. Knaak. Inhibition of discrete avoidance behavior by three anticholinesterase agents. Psychopharmacologia 7 : 7 2 76, 1965. 5. Grossman, S. P. Cholinergic synapses in the limbic system and behavioral inhibition. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 50:315 325, 1972.
6. Michelson, M. J. Pharmacological evidence of the role of acetylcholine in the higher nervous activity of man and animals. Activ. Nerv. Sup. (Praha) 3: 140-147, 1961. 7. Pfeiffer, C. C. and E. H. Jenny. Inhibition of conditioned response and the counteraction of schizophrenia by muscarinic stimulation of the brain. Ann. N. Y. Acad. ScL 66: 753-764, 1957. 8. Rosecrans, J. A., A. T. Dren and E. F. Domino. Effects of physostigmine on rat brain acetylcholine, acetylcholinesterase and conditioned pole jumping. Neuropharmac. 7: 127-134, 1968. 9. Vaillant, G. E. Antagonism between physostigmine and atropine on the behavior of the pigeon. Arch, exp. Path. Pharmak. 248: 406-416, 1964. 10. Vaillant, G. E. A comparison of antagonisms of physostigmine-induced suppression of behavior. J. Pharmacol. exp. Ther. 157: 636-648, 1967.