P~armacology Biochemistry & Behavior, Vol. 3, pp. 1031-1036. Copyright © 1975 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Alcohol, Habituation and the Patterning of Aggressive Responses in a Cichlid Fish ' H A R M A N V. S. P E E K E , S H I R L E Y C. P E E K E , H A R R Y H. A V I S A N D G E O R G E E L L M A N
Laboratory o f Psychobiology, Department o f Psychiatry, University o f California San Francisco CA 94143 ( R e c e i v e d 8 May 1975) PEEKE, H. V. S., S. C. PEEKE, H. H. AVIS, AND G. ELLMAN. Alcohol, habituation and the patterning of aggressive responses in a cichlid fish. PHARMAC. BIOCHEM. BEHAV. 3(6) 1031 1036, 1975. - Territorial cichlids were presented for 30 min with a conspecific male intruder (contained in a clear glass tube). Eight hr prior, 2 groups were administered alcohol (0.15 or 0.30 percent in the aquaria water). A third group served as a control. Three responses were recorded to allow analysis of topographic changes in behavior as well as changes in absolute levels. During the habituation phase, the normal group showed a sequence of long displays, followed by shorter ones as the frequency of attacks increased. The occurrence of threat which gradually gives way to attack is characteristic of the agonistic behavior of this species. In contrast to the controls, the 0.15 percent group was hyperaggressive, while the 0.30 percent group was hypoaggressive. Furthermore, the patterning of responses were abnormal. The 0.15 percent group gave abbreviated threats and more attacks (interpreted as a tendency to attack without warning); whereas, the 0.30 percent group gave many long threat displays, but few attacks. A stimulus specificity test provided strong evidence that the waning found during the initial phase was habituation. Habituation Aggression Stimulus Specificity
Alcohol
Fish
Cichlasoma
T H E p r e s e n t s t u d y was designed t o investigate the effects o f a l c o h o l o n t h e e l i c i t a t i o n a n d h a b i t u a t i o n of aggression in t e r r i t o r i a l male c o n v i c t cichlids. T h e r e are a n u m b e r o f a d v a n t a g e s to t h e use o f this species as a m o d e l for t h e effect o f a l c o h o l on aggression. A f o u n d a t i o n o f data has b e e n p r o v i d e d b y t h e use o f lower v e r t e b r a t e s (especially fish a n d birds) u p o n w h i c h t h e o r i e s o f aggression, territoriality a n d t h e m a i n t e n a n c e of i n t e r p e r s o n a l space have b e e n b~ailt. T h e e x t r a p o l a t i o n of these t h e o r i e s t o h u m a n b e h a v i o r ( n e c e s s i t a t e d b y a p r o s c r i p t i o n against s u c h research w i t h h u m a n s ) has b e e n d o n e w i t h v a r y i n g degrees o f care a n d s o p h i s t i c a t i o n t o yield, on the one h a n d , simplistic a n d p r o b a b l y e r r o n e o u s c o n c e p t i o n s of h u m a n aggression, b u t o n a m o r e s o p h i s t i c a t e d level, guidelines a n d h y p o t h e s e s for f u t u r e research. T h e aggressive a n d t e r r i t o r i a l b e h a v i o r o f the c o n v i c t cichlid has b e e n s t u d i e d e x t e n s i v e l y in the wild [8] a n d in the l a b o r a t o r y [6, 11, 2 1 ] . T h e t i m e course o f territorial e s t a b l i s h m e n t , b r e e d i n g and raising o f fry are well docum e n t e d as are t h e respective roles o f males a n d females at each phase. T h e r e is s u b s t a n t i a l e v i d e n c e t h a t a solitary male ( t h e p r e p a r a t i o n used in the p r e s e n t s t u d y ) will r e m a i n t e r r i t o r i a l for long periods of time, t h u s p r o v i d i n g a relatively c o n s t a n t state of aggression u p o n w h i c h drug effects m a y be s u p e r i m p o s e d . T h e drug t r e a t e d convict cichlid also has a d v a n t a g e s as a p s y c h o p h a r m a c o l o g i c a l tool [ 9 ] . If t h e drug is w a t e r
Nigrofasciatum
Response Topography
soluble it is easily a d m i n i s t e r e d by a d d i n g it directly to the a q u a r i u m w a t e r a n d t h u s t h e m a i n t e n a n c e of a steady b l o o d a l c o h o l level ( B A L ) is a c c o m p l i s h e d b y m a i n t a i n i n g a given c o n c e n t r a t i o n o f a l c o h o l in t h e a q u a r i u m . It s h o u l d be n o t e d t h a t while t h e level o f b l o o d alcohol is p r o p o r t i o n a l to a q u a r i u m c o n c e n t r a t i o n , the specific level is speciesspecific. U p t a k e studies for the convict cichlid have s h o w n t h a t it will m a i n t a i n an a s y m p t o t i c BAL of 65 p e r c e n t o f t h e a q u a r i u m c o n c e n t r a t i o n a f t e r 6 h r [10] whereas the goldfish m a i n t a i n s an a s y m p t o t i c B A L of 85 p e r c e n t a f t e r a similar l e n g t h o f t i m e [ 17]. Also, like o t h e r species of fish, this p r e p a r a t i o n has n u m e r o u s neurological similarities to m a m m a l s [51. Previous research o n changes in levels o f aggression in fish as a result o f alcohol a d m i n i s t r a t i o n has s h o w n t h a t a m o d e r a t e dose o f alcohol e n h a n c e s aggressiveness whereas a m u c h h i g h e r dose i n h i b i t s the b e h a v i o r [ 1 6 , 1 5 ] . Since u p t a k e data was n o t p r o v i d e d for the species used (Siamese fighting fish) in these t w o studies, dose level can n o t be d e t e r m i n e d . However, similar results were f o u n d by Peeke, et al. [ 10] w h e r e a very low dose (0.07 p e r c e n t alcohol per vol of a q u a r i u m water) had essentially n o effect; a m o d e r a t e dose (0.18 p e r c e n t ) increased t h e f r e q u e n c y a n d d u r a t i o n o f aggressive display a n d f r e q u e n c y of biting; while a t h i r d dose (0.33 p e r c e n t ) i n h i b i t e d all 3 measures, p r o d u c i n g a hypoaggressive b u t o t h e r w i s e n o r m a l fish. Since previous research has s h o w n t h a t various doses
This research was supported in part by Grant No. AA-00280 from ADAMHA and by Mental Health Training Grant No. MH 7082 to the University of California, San Francisco. 1031
1032
PEEKE, PEEKE, AVIS A N D E L L M A N
change initial responsiveness to an aggression-eliciting stimulus, it was r e a s o n e d t h a t the course of h a b i t u a t i o n to such stimuli m i g h t also be changed by alcohol. H a b i t u a t i o n , the waning of response to a r e p e a t e d or c o n s t a n t stimulus, is p e r h a p s a basic f o r m of adaptive m o d i f i c a t i o n while being also one o f t h e least studied learning p h e n o m e n a . R e c e n t l y we have a t t e m p t e d (see also [ 1,4] ) to relate h a b i t u a t i o n to the r e d u c t i o n o f aggressive e n c o u n t e r s b e t w e e n a d j a c e n t l y territorial conspecifics. T h e r e is n o w evidence t h a t h a b i t u a tion of aggressive b e h a v i o r in territorial fish is persistent over t i m e and very s t i m u l u s specific; all are criteria of a process t h a t serves to lower aggression b e t w e e n n e i g h b o r s , b u t m a i n t a i n fighting readiness against i n t r u d e r s [ 12]. Field studies of birds suggest t h a t a similar p h e n o m e n o n o p e r a t e s in t h e i r spacing behavior. T h e r e are several changes in the course of h a b i t u a t i o n that might o c c u r as a result of alcohol. H a b i t u a t i o n m i g h t take place at the n o r m a l rate ( a l t h o u g h from an altered starting p o i n t ) , at an altered rate ( e i t h e r faster or slower) or m i g h t n o t take place at all. An a d d i t i o n a l possibility w o u l d be specific effects u p o n t h e p a t t e r n i n g of the s e q u e n c e of the various aggression behaviors. Such an a l t e r a t i o n in the t o p o g r a p h y of b e h a v i o r w o u l d be particularly i m p o r t a n t since some behaviors serve as signal f u n c t i o n s ( t h r e a t s or warnings) while o t h e r behaviors inflict injury. Several measures of the aggressive s e q u e n c e s of b e h a v i o r are r e p o r t e d here as we feel t h a t changes in the t o p o g r a p h y of b e h a v i o r are as i m p o r t a n t and i n f o r m a t i v e as changes in absolute level or i n t e n s i t y . METHOD
Animals and Apparatus T w e n t y - o n e adult male convict cichlids (Cichlasoma nigrofasciatum) were placed individually in glass aquaria (90 x 30 x 30 cm) c o n t a i n i n g a ceramic pot lying 10 cm f r o m one e n d of the t a n k o n a s u b s t r a t e of coarse gravel. The p o t served as a refuge a n d could be c o n s i d e r e d t h e c e n t e r of a t e r r i t o r y . Air-driven filters m a i n t a i n e d filtration a n d circulation, b u t were d i s c o n n e c t e d w h e n the alcohol was i n t r o d u c e d i n t o the t a n k s for the d u r a t i o n of the e x p e r i m e n t . Water t e m p e r a t u r e was m a i n t a i n e d at 25°C. (-+2°). Fish were fed live brine s h r i m p and dried f o o d daily a n d were allowed to acclimatize to t h e s i t u a t i o n for 5 days before the e x p e r i m e n t began.
Procedure T h r e e groups (n = 7 in e a c h ) were used: 0.0, 0.15 a n d 0.30 p e r c e n t c o n c e n t r a t i o n of e t h a n o l in the water. Based on e t h a n o l u p t a k e studies using similar c o n c e n t r a t i o n s [ 1 0 ] , the level of e t h a n o l in the b l o o d of the fish was e x p e c t e d to r e a c h 65 p e r c e n t of the w a t e r c o n c e n t r a t i o n after a period of 6 hr. These doses were selected to provide a d d i t i o n a l p o i n t s to s u p p l e m e n t previously o b t a i n e d data c o n c e r n e d w i t h the f u n c t i o n relating alcohol c o n c e n t r a t i o n to aggression [ 1 0 ] . The a l c o h o l was i n t r o d u c e d w i t h stirring at 0 8 0 0 in q u a n t i t i e s to c o n f o r m to one of the doses. Water samples were t a k e n after c o m p l e t i o n of the p r o c e d u r e (7 hr) t o c o n f i r m alcohol c o n c e n t r a t i o n s in t h e tanks. Six h o u r s a f t e r i n t r o d u c t i o n of the e t h a n o l , the resident fish was p r e s e n t e d with a live, male conspecific ( c o n f i n e d to a clear glass t u b e , 100 m m alia., filled with water) at a spot 200 m m from t h e e n t r a n c e to the ceramic pot. The
i n t r u d i n g s t i m u l u s was left in place for 30 min ( h a b i t u a t i o n phase) a n d t h e n r e m o v e d . A f t e r a 5 rain period, a different s t i m u l u s fish was p r e s e n t e d in the same m a n n e r for 2.5 min ( s t i m u l u s specificity test phase). An increase in response to this s t i m u l u s following initial d e c r e m e n t in response w o u l d i n d i c a t e t h a t the fish was still capable of vigorous response and would c o n t r o l for fatigue and sensory a d a p t a t i o n , t w o general criteria for d e m o n s t r a t i n g t h a t a d e c r e m e n t in response is truly h a b i t u a t i o n . The e x p e r i m e n t e r was s i t u a t e d in f r o n t of the a q u a r i u m with a h a n d console c o n t a i n i n g b u t t o n s which, w h e n p u s h e d , registered f r e q u e n c y and d u r a t i o n of various responses o n an event recorder. The responses r e c o r d e d were: (1) f r e q u e n c y of displays, where a display was c o u n t e d each time the fish e x t e n d e d gill covers a n d b r a n c h i o s t e g a l m e m b r a n e s : (2) d u r a t i o n of each display: and (3) f r e q u e n c y o f bites at the glass t u b e c o n t a i n i n g the s t i m u l u s fish. T h r e e measures were a n a l y z e d separately and a f o u r t h , average display d u r a t i o n , was calculated from the data on t o t a l display d u r a t i o n and f r e q u e n c y of display. This data yielded a measure of d u r a t i o n of the average t h r e a t display at each time over the course of the experiment. RESULTS This s t u d y had t w o p r i m a r y concerns. T h e first was the effect o f the 3 doses of alcohol on the overall level of r e s p o n d i n g in each measure. Thus, the first set of analyses c o m p a r e the 3 groups of animals on each of the 3 measures. The s e c o n d c o n c e r n was w h e t h e r a given dose of a l c o h o l a f f e c t e d the t o p o g r a p h y of the responses, t h a t is, the i n t e r r e l a t i o n s of t h e responses with respect to ease of elicitation, rate of h a b i t u a t i o n , degree of recovery, etc. Thus, t h e s e c o n d set of analyses provide a m o r e detailed d e s c r i p t i o n of the effects of each dose o f alcohol on the initial level a n d t i m e course of each of the measures. Each measure was g r a p h e d for each drug c o n d i t i o n over the 30 min of h a b i t u a t i o n and the 2.5 min of the stimulus specificity test in Fig. 1. The data o f the 30 rain of the h a b i t u a t i o n phase were divided i n t o twelve 2.5 min blocks.
Overall Effect oJ Dose on Elicitation and Habituation oJ' Aggressive Behaviors Display Jrequeney. A blocks by groups analysis of variance was p e r f o r m e d w h i c h revealed a significant t i m e b l o c k s effect, F ( 1 1 , 1 9 8 ) = 5.58, p < 0 . 0 1 . The blocks effect reflects an overall d o w n w a r d t r e n d in response. E x a m i n a t i o n of the group m e a n s reveal t h a t the 0.15 percent group gave t h e most displays (X = 50.00) followed _by the 0 p e r c e n t ( X = 27.9) a n d t h e 0.30 p e r c e n t group (X =- 12.0). T-tests i n d i c a t e d t h a t all groups differed significantly from each o t h e r (t = 5.36, p < 0 . 0 1 for 0.15 vs 0 p e r c e n t : t = 6.89, p < 0 . 0 1 for 0 vs 0.30 p e r c e n t ; d r = 12 all tests). Average display duration. A blocks by groups analysis of variance d e m o n s t r a t e d a reliable blocks effect (reflecting an overall d i m i n u t i o n in d u r a t i o n of response over time, F ( 1 1 , 1 9 8 ) = 8.83, p < 0 . 0 1 ) and a significant i n t e r a c t i o n b e t w e e n groups a n d blocks F ( 2 2 , 1 9 8 ) = 2.32, p < 0 . 0 1 . This effect a p p e a r e d to be due to absence of waning in the 0.15 p e r c e n t group. To test this s u p p o s i t i o n , the first 3 t i m e b l o c k s were p o o l e d a n d c o m p a r e d w i t h the p o o l e d score for the last 3 blocks by t-tests (for c o r r e l a t e d means) done separately for each group. The n o r m a l group s h o w e d a
ALCOHOL AND AGGRESSION
1033
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difference in trend b e t w e e n groups. To test this supposition, a t-test (correlated means) was p e r f o r m e d between the pooled first 3 and pooled last 3 points for each group. The 0 percent group showed a reliable increase from the start to the finish of the period (t = 2.40, p < 0 . 0 5 , d f = 6). Neither of the alcohol groups showed a reliable change over the course of the habituation phase.
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J HABITUATION PHASE J
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1 STIMULUS
SPECIFICITY
FIG. 1. Effects of 2 alcohol dosages and control on 3 indicies of aggression over the 12 2½ rain blocks of the habituation phase and the 2Yz rain stimulus specificity test. significant drop (t = 3.15, p < 0 . 0 2 5 , dr= 6) as did the 0.30 percent group (t = 2.75, p < 0 . 0 5 , d f = 6). The 0.15 percent group did not diminish significantly b e t w e e n first and last three blocks. Bites. A blocks by groups analysis of variance for this measure yielded a significant groups effect, F(2,18) = 4.87, p < 0 . 0 5 , a significant blocks effect, F ( 1 1 , 1 9 8 ) = 1.88, p < 0 . 0 5 , and a significant interaction, F ( 2 2 , 1 9 8 ) = 1.73, p < 0 . 0 5 . E x a m i n a t i o n of the group means indicated that, as with the display frequency measure, the 0.15 percent group was most responsive (X = 46.7) with the 0 percent group inte.rmediate (X = 27.3) and the 0.30 percent group least responsive (X = 13.72). T-tests indicated that each group differed significantly from each o t h e r group (t = 4.80, p < 0 . 0 1 for 0.15 vs 0 percent, t = 4.01, p < 0 . 0 1 for 0 vs 0.30 percent, d f = 12 all tests). The significant overall blocks effect reflects a general upward trend in responding, but the significant interaction suggests that there must also be some
With the presentation of a new stimulus after the habituation phase, it was e x p e c t e d that the various responses would return to their prehabituation levels or at least approach those levels. Tests were p e r f o r m e d comparing the last block of the habituation phase with the stimulus specificity test. Display frequency. An analysis of variance yielded a significant increase between the two blocks, F(1,18) = 18.37, p < 0 . 0 1 , as well as a significant difference between groups, F(2,18) = 3.38, p < 0 . 0 1 . The groups effect reflects differences between groups in overall response level and does not add new information. The upward trend of the blocks effect indicates that all 3 groups showed recovery of response, although to differing degrees. Average display duration. There was no groups effect, but a reliable blocks effect, F(1,18) = 13.75, p < 0 . 0 1 , indicating recovery of response during stimulus specificity testing. Bites. This measure yielded results s o m e w h a t different from the others. There was a significant groups effect, F(2,18) = 4.91, p < 0 . 0 5 , and a significant interaction, F(2,18) = 10.80, p < 0 . 0 1 . Inspection of Fig. 1 shows that the 0 and 0.30 percent groups show small decrease, and the 0.15 percent group showed a large increase. The decreases for the 0 and 0.30 percent groups returned t h e m to their initial level of responding. To return responding of the 0.15 percent group to its initial level would have required an increase, and this is what was found; however, the increase that occurred far exceeded the initial and also the peak levels of responding. Inspection of scores for individual animals indicated that 3 of 7 animals in that group responded to the stimulus specificity test by an increase of 1.5 to 2 times their individual peak level for the habituation phase. Three o t h e r animals responded at slightly below peak level and one animal responded far below peak level. Thus, 42 percent of the animals were hyperaggressive as a result of the stimulus specificity test suggesting that the initial exposure may have sensitized those animals to later exposure to an intruding stimulus fish. Comparison with the earlier study. An earlier study whose purpose was to study the initial response to the stimulus rather than habituation, used an identical procedure for 8 min o f stimulus exposure with doses of 0, 0.07, 0.18 and 0.33 percent alcohol. Comparison of the first 8 min of behavior for the groups of the present study provide coml~lementary points in the 8 min dose-response function r e p o r t e d previously [ 10]. Figure 2 presents these c o m b i n e d data expressed as a percentage of the response of the 0 percent alcohol groups. As the figure shows, the present study is quite consistent with the earlier results and serves as a replication of the 1973 study. Effect o f Dose on Response Topography - Habituation Phase Zero percent alcohol group. The long-term waxing and
1034
P E E K E , P E E K E , AVIS A N D E L L M A N
cue) U~ ..a
Alcohol, Habituation and the Patterning of Aggressive Responses in a Cichlid Fish ' H A R M A N V. S. P E E K E , S H I R L E Y C. P E E K E , H A R R Y H. A V I S A N D G E O R G E E L L M A N
Laboratory o f Psychobiology, Department o f Psychiatry, University o f California San Francisco CA 94143 ( R e c e i v e d 8 May 1975) PEEKE, H. V. S., S. C. PEEKE, H. H. AVIS, AND G. ELLMAN. Alcohol, habituation and the patterning of aggressive responses in a cichlid fish. PHARMAC. BIOCHEM. BEHAV. 3(6) 1031 1036, 1975. - Territorial cichlids were presented for 30 min with a conspecific male intruder (contained in a clear glass tube). Eight hr prior, 2 groups were administered alcohol (0.15 or 0.30 percent in the aquaria water). A third group served as a control. Three responses were recorded to allow analysis of topographic changes in behavior as well as changes in absolute levels. During the habituation phase, the normal group showed a sequence of long displays, followed by shorter ones as the frequency of attacks increased. The occurrence of threat which gradually gives way to attack is characteristic of the agonistic behavior of this species. In contrast to the controls, the 0.15 percent group was hyperaggressive, while the 0.30 percent group was hypoaggressive. Furthermore, the patterning of responses were abnormal. The 0.15 percent group gave abbreviated threats and more attacks (interpreted as a tendency to attack without warning); whereas, the 0.30 percent group gave many long threat displays, but few attacks. A stimulus specificity test provided strong evidence that the waning found during the initial phase was habituation. Habituation Aggression Stimulus Specificity
Alcohol
Fish
Cichlasoma
T H E p r e s e n t s t u d y was designed t o investigate the effects o f a l c o h o l o n t h e e l i c i t a t i o n a n d h a b i t u a t i o n of aggression in t e r r i t o r i a l male c o n v i c t cichlids. T h e r e are a n u m b e r o f a d v a n t a g e s to t h e use o f this species as a m o d e l for t h e effect o f a l c o h o l on aggression. A f o u n d a t i o n o f data has b e e n p r o v i d e d b y t h e use o f lower v e r t e b r a t e s (especially fish a n d birds) u p o n w h i c h t h e o r i e s o f aggression, territoriality a n d t h e m a i n t e n a n c e of i n t e r p e r s o n a l space have b e e n b~ailt. T h e e x t r a p o l a t i o n of these t h e o r i e s t o h u m a n b e h a v i o r ( n e c e s s i t a t e d b y a p r o s c r i p t i o n against s u c h research w i t h h u m a n s ) has b e e n d o n e w i t h v a r y i n g degrees o f care a n d s o p h i s t i c a t i o n t o yield, on the one h a n d , simplistic a n d p r o b a b l y e r r o n e o u s c o n c e p t i o n s of h u m a n aggression, b u t o n a m o r e s o p h i s t i c a t e d level, guidelines a n d h y p o t h e s e s for f u t u r e research. T h e aggressive a n d t e r r i t o r i a l b e h a v i o r o f the c o n v i c t cichlid has b e e n s t u d i e d e x t e n s i v e l y in the wild [8] a n d in the l a b o r a t o r y [6, 11, 2 1 ] . T h e t i m e course o f territorial e s t a b l i s h m e n t , b r e e d i n g and raising o f fry are well docum e n t e d as are t h e respective roles o f males a n d females at each phase. T h e r e is s u b s t a n t i a l e v i d e n c e t h a t a solitary male ( t h e p r e p a r a t i o n used in the p r e s e n t s t u d y ) will r e m a i n t e r r i t o r i a l for long periods of time, t h u s p r o v i d i n g a relatively c o n s t a n t state of aggression u p o n w h i c h drug effects m a y be s u p e r i m p o s e d . T h e drug t r e a t e d convict cichlid also has a d v a n t a g e s as a p s y c h o p h a r m a c o l o g i c a l tool [ 9 ] . If t h e drug is w a t e r
Nigrofasciatum
Response Topography
soluble it is easily a d m i n i s t e r e d by a d d i n g it directly to the a q u a r i u m w a t e r a n d t h u s t h e m a i n t e n a n c e of a steady b l o o d a l c o h o l level ( B A L ) is a c c o m p l i s h e d b y m a i n t a i n i n g a given c o n c e n t r a t i o n o f a l c o h o l in t h e a q u a r i u m . It s h o u l d be n o t e d t h a t while t h e level o f b l o o d alcohol is p r o p o r t i o n a l to a q u a r i u m c o n c e n t r a t i o n , the specific level is speciesspecific. U p t a k e studies for the convict cichlid have s h o w n t h a t it will m a i n t a i n an a s y m p t o t i c BAL of 65 p e r c e n t o f t h e a q u a r i u m c o n c e n t r a t i o n a f t e r 6 h r [10] whereas the goldfish m a i n t a i n s an a s y m p t o t i c B A L of 85 p e r c e n t a f t e r a similar l e n g t h o f t i m e [ 17]. Also, like o t h e r species of fish, this p r e p a r a t i o n has n u m e r o u s neurological similarities to m a m m a l s [51. Previous research o n changes in levels o f aggression in fish as a result o f alcohol a d m i n i s t r a t i o n has s h o w n t h a t a m o d e r a t e dose o f alcohol e n h a n c e s aggressiveness whereas a m u c h h i g h e r dose i n h i b i t s the b e h a v i o r [ 1 6 , 1 5 ] . Since u p t a k e data was n o t p r o v i d e d for the species used (Siamese fighting fish) in these t w o studies, dose level can n o t be d e t e r m i n e d . However, similar results were f o u n d by Peeke, et al. [ 10] w h e r e a very low dose (0.07 p e r c e n t alcohol per vol of a q u a r i u m water) had essentially n o effect; a m o d e r a t e dose (0.18 p e r c e n t ) increased t h e f r e q u e n c y a n d d u r a t i o n o f aggressive display a n d f r e q u e n c y of biting; while a t h i r d dose (0.33 p e r c e n t ) i n h i b i t e d all 3 measures, p r o d u c i n g a hypoaggressive b u t o t h e r w i s e n o r m a l fish. Since previous research has s h o w n t h a t various doses
This research was supported in part by Grant No. AA-00280 from ADAMHA and by Mental Health Training Grant No. MH 7082 to the University of California, San Francisco. 1031
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PEEKE, PEEKE, AVIS A N D E L L M A N
change initial responsiveness to an aggression-eliciting stimulus, it was r e a s o n e d t h a t the course of h a b i t u a t i o n to such stimuli m i g h t also be changed by alcohol. H a b i t u a t i o n , the waning of response to a r e p e a t e d or c o n s t a n t stimulus, is p e r h a p s a basic f o r m of adaptive m o d i f i c a t i o n while being also one o f t h e least studied learning p h e n o m e n a . R e c e n t l y we have a t t e m p t e d (see also [ 1,4] ) to relate h a b i t u a t i o n to the r e d u c t i o n o f aggressive e n c o u n t e r s b e t w e e n a d j a c e n t l y territorial conspecifics. T h e r e is n o w evidence t h a t h a b i t u a tion of aggressive b e h a v i o r in territorial fish is persistent over t i m e and very s t i m u l u s specific; all are criteria of a process t h a t serves to lower aggression b e t w e e n n e i g h b o r s , b u t m a i n t a i n fighting readiness against i n t r u d e r s [ 12]. Field studies of birds suggest t h a t a similar p h e n o m e n o n o p e r a t e s in t h e i r spacing behavior. T h e r e are several changes in the course of h a b i t u a t i o n that might o c c u r as a result of alcohol. H a b i t u a t i o n m i g h t take place at the n o r m a l rate ( a l t h o u g h from an altered starting p o i n t ) , at an altered rate ( e i t h e r faster or slower) or m i g h t n o t take place at all. An a d d i t i o n a l possibility w o u l d be specific effects u p o n t h e p a t t e r n i n g of the s e q u e n c e of the various aggression behaviors. Such an a l t e r a t i o n in the t o p o g r a p h y of b e h a v i o r w o u l d be particularly i m p o r t a n t since some behaviors serve as signal f u n c t i o n s ( t h r e a t s or warnings) while o t h e r behaviors inflict injury. Several measures of the aggressive s e q u e n c e s of b e h a v i o r are r e p o r t e d here as we feel t h a t changes in the t o p o g r a p h y of b e h a v i o r are as i m p o r t a n t and i n f o r m a t i v e as changes in absolute level or i n t e n s i t y . METHOD
Animals and Apparatus T w e n t y - o n e adult male convict cichlids (Cichlasoma nigrofasciatum) were placed individually in glass aquaria (90 x 30 x 30 cm) c o n t a i n i n g a ceramic pot lying 10 cm f r o m one e n d of the t a n k o n a s u b s t r a t e of coarse gravel. The p o t served as a refuge a n d could be c o n s i d e r e d t h e c e n t e r of a t e r r i t o r y . Air-driven filters m a i n t a i n e d filtration a n d circulation, b u t were d i s c o n n e c t e d w h e n the alcohol was i n t r o d u c e d i n t o the t a n k s for the d u r a t i o n of the e x p e r i m e n t . Water t e m p e r a t u r e was m a i n t a i n e d at 25°C. (-+2°). Fish were fed live brine s h r i m p and dried f o o d daily a n d were allowed to acclimatize to t h e s i t u a t i o n for 5 days before the e x p e r i m e n t began.
Procedure T h r e e groups (n = 7 in e a c h ) were used: 0.0, 0.15 a n d 0.30 p e r c e n t c o n c e n t r a t i o n of e t h a n o l in the water. Based on e t h a n o l u p t a k e studies using similar c o n c e n t r a t i o n s [ 1 0 ] , the level of e t h a n o l in the b l o o d of the fish was e x p e c t e d to r e a c h 65 p e r c e n t of the w a t e r c o n c e n t r a t i o n after a period of 6 hr. These doses were selected to provide a d d i t i o n a l p o i n t s to s u p p l e m e n t previously o b t a i n e d data c o n c e r n e d w i t h the f u n c t i o n relating alcohol c o n c e n t r a t i o n to aggression [ 1 0 ] . The a l c o h o l was i n t r o d u c e d w i t h stirring at 0 8 0 0 in q u a n t i t i e s to c o n f o r m to one of the doses. Water samples were t a k e n after c o m p l e t i o n of the p r o c e d u r e (7 hr) t o c o n f i r m alcohol c o n c e n t r a t i o n s in t h e tanks. Six h o u r s a f t e r i n t r o d u c t i o n of the e t h a n o l , the resident fish was p r e s e n t e d with a live, male conspecific ( c o n f i n e d to a clear glass t u b e , 100 m m alia., filled with water) at a spot 200 m m from t h e e n t r a n c e to the ceramic pot. The
i n t r u d i n g s t i m u l u s was left in place for 30 min ( h a b i t u a t i o n phase) a n d t h e n r e m o v e d . A f t e r a 5 rain period, a different s t i m u l u s fish was p r e s e n t e d in the same m a n n e r for 2.5 min ( s t i m u l u s specificity test phase). An increase in response to this s t i m u l u s following initial d e c r e m e n t in response w o u l d i n d i c a t e t h a t the fish was still capable of vigorous response and would c o n t r o l for fatigue and sensory a d a p t a t i o n , t w o general criteria for d e m o n s t r a t i n g t h a t a d e c r e m e n t in response is truly h a b i t u a t i o n . The e x p e r i m e n t e r was s i t u a t e d in f r o n t of the a q u a r i u m with a h a n d console c o n t a i n i n g b u t t o n s which, w h e n p u s h e d , registered f r e q u e n c y and d u r a t i o n of various responses o n an event recorder. The responses r e c o r d e d were: (1) f r e q u e n c y of displays, where a display was c o u n t e d each time the fish e x t e n d e d gill covers a n d b r a n c h i o s t e g a l m e m b r a n e s : (2) d u r a t i o n of each display: and (3) f r e q u e n c y o f bites at the glass t u b e c o n t a i n i n g the s t i m u l u s fish. T h r e e measures were a n a l y z e d separately and a f o u r t h , average display d u r a t i o n , was calculated from the data on t o t a l display d u r a t i o n and f r e q u e n c y of display. This data yielded a measure of d u r a t i o n of the average t h r e a t display at each time over the course of the experiment. RESULTS This s t u d y had t w o p r i m a r y concerns. T h e first was the effect o f the 3 doses of alcohol on the overall level of r e s p o n d i n g in each measure. Thus, the first set of analyses c o m p a r e the 3 groups of animals on each of the 3 measures. The s e c o n d c o n c e r n was w h e t h e r a given dose of a l c o h o l a f f e c t e d the t o p o g r a p h y of the responses, t h a t is, the i n t e r r e l a t i o n s of t h e responses with respect to ease of elicitation, rate of h a b i t u a t i o n , degree of recovery, etc. Thus, t h e s e c o n d set of analyses provide a m o r e detailed d e s c r i p t i o n of the effects of each dose o f alcohol on the initial level a n d t i m e course of each of the measures. Each measure was g r a p h e d for each drug c o n d i t i o n over the 30 min of h a b i t u a t i o n and the 2.5 min of the stimulus specificity test in Fig. 1. The data o f the 30 rain of the h a b i t u a t i o n phase were divided i n t o twelve 2.5 min blocks.
Overall Effect oJ Dose on Elicitation and Habituation oJ' Aggressive Behaviors Display Jrequeney. A blocks by groups analysis of variance was p e r f o r m e d w h i c h revealed a significant t i m e b l o c k s effect, F ( 1 1 , 1 9 8 ) = 5.58, p < 0 . 0 1 . The blocks effect reflects an overall d o w n w a r d t r e n d in response. E x a m i n a t i o n of the group m e a n s reveal t h a t the 0.15 percent group gave t h e most displays (X = 50.00) followed _by the 0 p e r c e n t ( X = 27.9) a n d t h e 0.30 p e r c e n t group (X =- 12.0). T-tests i n d i c a t e d t h a t all groups differed significantly from each o t h e r (t = 5.36, p < 0 . 0 1 for 0.15 vs 0 p e r c e n t : t = 6.89, p < 0 . 0 1 for 0 vs 0.30 p e r c e n t ; d r = 12 all tests). Average display duration. A blocks by groups analysis of variance d e m o n s t r a t e d a reliable blocks effect (reflecting an overall d i m i n u t i o n in d u r a t i o n of response over time, F ( 1 1 , 1 9 8 ) = 8.83, p < 0 . 0 1 ) and a significant i n t e r a c t i o n b e t w e e n groups a n d blocks F ( 2 2 , 1 9 8 ) = 2.32, p < 0 . 0 1 . This effect a p p e a r e d to be due to absence of waning in the 0.15 p e r c e n t group. To test this s u p p o s i t i o n , the first 3 t i m e b l o c k s were p o o l e d a n d c o m p a r e d w i t h the p o o l e d score for the last 3 blocks by t-tests (for c o r r e l a t e d means) done separately for each group. The n o r m a l group s h o w e d a
ALCOHOL AND AGGRESSION
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difference in trend b e t w e e n groups. To test this supposition, a t-test (correlated means) was p e r f o r m e d between the pooled first 3 and pooled last 3 points for each group. The 0 percent group showed a reliable increase from the start to the finish of the period (t = 2.40, p < 0 . 0 5 , d f = 6). Neither of the alcohol groups showed a reliable change over the course of the habituation phase.
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J HABITUATION PHASE J
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1 STIMULUS
SPECIFICITY
FIG. 1. Effects of 2 alcohol dosages and control on 3 indicies of aggression over the 12 2½ rain blocks of the habituation phase and the 2Yz rain stimulus specificity test. significant drop (t = 3.15, p < 0 . 0 2 5 , dr= 6) as did the 0.30 percent group (t = 2.75, p < 0 . 0 5 , d f = 6). The 0.15 percent group did not diminish significantly b e t w e e n first and last three blocks. Bites. A blocks by groups analysis of variance for this measure yielded a significant groups effect, F(2,18) = 4.87, p < 0 . 0 5 , a significant blocks effect, F ( 1 1 , 1 9 8 ) = 1.88, p < 0 . 0 5 , and a significant interaction, F ( 2 2 , 1 9 8 ) = 1.73, p < 0 . 0 5 . E x a m i n a t i o n of the group means indicated that, as with the display frequency measure, the 0.15 percent group was most responsive (X = 46.7) with the 0 percent group inte.rmediate (X = 27.3) and the 0.30 percent group least responsive (X = 13.72). T-tests indicated that each group differed significantly from each o t h e r group (t = 4.80, p < 0 . 0 1 for 0.15 vs 0 percent, t = 4.01, p < 0 . 0 1 for 0 vs 0.30 percent, d f = 12 all tests). The significant overall blocks effect reflects a general upward trend in responding, but the significant interaction suggests that there must also be some
With the presentation of a new stimulus after the habituation phase, it was e x p e c t e d that the various responses would return to their prehabituation levels or at least approach those levels. Tests were p e r f o r m e d comparing the last block of the habituation phase with the stimulus specificity test. Display frequency. An analysis of variance yielded a significant increase between the two blocks, F(1,18) = 18.37, p < 0 . 0 1 , as well as a significant difference between groups, F(2,18) = 3.38, p < 0 . 0 1 . The groups effect reflects differences between groups in overall response level and does not add new information. The upward trend of the blocks effect indicates that all 3 groups showed recovery of response, although to differing degrees. Average display duration. There was no groups effect, but a reliable blocks effect, F(1,18) = 13.75, p < 0 . 0 1 , indicating recovery of response during stimulus specificity testing. Bites. This measure yielded results s o m e w h a t different from the others. There was a significant groups effect, F(2,18) = 4.91, p < 0 . 0 5 , and a significant interaction, F(2,18) = 10.80, p < 0 . 0 1 . Inspection of Fig. 1 shows that the 0 and 0.30 percent groups show small decrease, and the 0.15 percent group showed a large increase. The decreases for the 0 and 0.30 percent groups returned t h e m to their initial level of responding. To return responding of the 0.15 percent group to its initial level would have required an increase, and this is what was found; however, the increase that occurred far exceeded the initial and also the peak levels of responding. Inspection of scores for individual animals indicated that 3 of 7 animals in that group responded to the stimulus specificity test by an increase of 1.5 to 2 times their individual peak level for the habituation phase. Three o t h e r animals responded at slightly below peak level and one animal responded far below peak level. Thus, 42 percent of the animals were hyperaggressive as a result of the stimulus specificity test suggesting that the initial exposure may have sensitized those animals to later exposure to an intruding stimulus fish. Comparison with the earlier study. An earlier study whose purpose was to study the initial response to the stimulus rather than habituation, used an identical procedure for 8 min o f stimulus exposure with doses of 0, 0.07, 0.18 and 0.33 percent alcohol. Comparison of the first 8 min of behavior for the groups of the present study provide coml~lementary points in the 8 min dose-response function r e p o r t e d previously [ 10]. Figure 2 presents these c o m b i n e d data expressed as a percentage of the response of the 0 percent alcohol groups. As the figure shows, the present study is quite consistent with the earlier results and serves as a replication of the 1973 study. Effect o f Dose on Response Topography - Habituation Phase Zero percent alcohol group. The long-term waxing and
1034
P E E K E , P E E K E , AVIS A N D E L L M A N
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