Physiological changes observed in the goldfish (Carassius auratus) during behavioral arousal and fright

BEHAVIORAL AND NEURALBIOLOGY 29, 255-275 (1980)

Physiological Changes Observed in the Goldfish

(Carassius auratus) during Behavioral Arousal and Fright P. R. LAMING 1 AND G. E. SAVAGE Department of Zoology and Comparative Physiology, Queen Mary College, London E1 4NS Goldfish were placed in a controlled environment and their behavioral and physiological responses to stimuli observed. Various stimuli presented caused behavioral arousal, but in some cases fright behavior was observed. During arousal, cardiac and ventilatory decelerations occurred and there was a decrease in ventilatory amplitude. When fright responses were observed, a brief cardiac deceleration was followed by an acceleration, the ventilatory rate and amplitude increased, and there was an increase in the electromyogram. Three stimuli, the onset of illumination, the passage of a moving black edge across the light source, and the operation of a plunger, were more effective in eliciting physiological arousal responses than a moving spot past the light source, 500 Hz noise, and a small paddle in the aquarium. The physiological changes which occurred on arousal habituated on regular, repeated presentation ot~ the stimulus, habituation not being affected by the interval between stimulus presentations. A close correlation exists between the magnitude of the arousal response of a fish to a novel stimulus and the number of presentations of that stimulus required for habituation of the response. Cardiac and ventilatory changes, associated with behavioral arousal, are considered to be variables, the use of which may aid the study of the phenomenon itself.

Animals respond to environmental stimuli in a variety of ways depending on their physiological state and previous experience. If physiological variables like appetitive state and excitability are controlled and the stimulus is a novel one, two types of behavioral response, which we will classify as arousal and fright, can be seen initially. "Aroused" behavior is in this context synonymous with alerted or orienting behavior, and "fright" is similar to stable, escape, or defensive behavior. "Freezing" behavior can be interpreted in either way depending on whether it is considered as a pause in ongoing behavior (Goodman & Weinberger, 1973) or as a rigid paralysis induced by extreme fright. Although there is a considerable body of information on orienting or 1 Present address: Department of Zoology, The Queen's University of Belfast, Belfast BT7 INN, Northern Ireland. 255 0163-1047/80/060255-21 $02.00/0 Copyright© 1980by AcademicPress, Inc. All rightsof reproductionin any formreserved.

256

LAMING AND SAVAGE

arousal and startle or fright responses in mammals and the physiological correlates of these behaviors, studies on phylogenetically older vertebrates are relatively few. Fish are no exception, though recently much research has been published regarding behavioral responses of fish to biologically significant stimuli like food (Peeke & Peeke, 1972) and conspecifics (Peeke & Peeke, 1970) and the habituation of such responses. The associated behaviors like biting at food and aggressive display are often secondary to orienting responses, the study of which is thus complicated by the appetitive state of the animal. The orienting responses of fish to novel stimuli have been described by Russell (1967) and Savage (1971) as erection of the dorsal fin, small dorsoventral flicks of the caudal fin, and extension and slight movement of the pectoral fins. Work by Laming and Hornby (unpublished) on roach Rutilus rutilus indicates that a change in pectoral fin movement is the most consistent response of fish to a stimulus; even being elicited by weak stimuli. One characteristic of the "orienting" fin responses of fish is that they rarely change the position of the animal in the water, unlike the fright, "tail-flip" or "startle" response of Rogers, Melzack, and Segal (1963). This response consists of a rapid, lateral flexion of the tail fin which propels the animal forward in the water. It is characteristically elicited by strong stimuli like bangs on the aquarium (Rogers et al., 1963) and habituates on repeated presentation of the stimulus, to be replaced by orienting (arousal) responses (Russell, 1967). Fish, as well as responding behaviorally to stimuli, also respond physiologically. Otis, Cerf, and Thomas (1957) reported changes in ventilation and Randall (1966) in heart rate, when stimuli were presented, though no attempt was made to correlate these changes with behavior. Goldfish during behavioral arousal, as described here, also show frequency changes in the EEG (Laming, 1980). The present paper aims to: (i) Describe the physiological correlates of behavioral arousal and fright in goldfish, (ii) determine the effectiveness of various stimuli in eliciting these changes, and (iii) examine the habituation of these physiological changes. METHOD

Animals The 74 goldfish (Carassius auratus) used in these experiments were between 10 and 15 cm in length and obtained from a regular supplier. They were kept in aerated, filtered water at 15° __+ I°C, and fed on alternate days, for at least 1 week before operations.

BEHAVIORAL AROUSAL IN THE GOLDFISH

257

Surgery All operations took place under 1:10,000 MS222 (methane tricaine sulfonate) anesthesia; fish were perfused continuously as described previously (Savage, 1971).

Electrocardiogram (ECG) ECG electrodes were made and implanted using the methods of Roberts, Wright, and Savage (1973).

Electromyogram (EMG) Tungsten wire (0.029 cm diameter) was straightened and 2.5-cm lengths were sharpened electrolytically until tip diameters of 50-100/zm were obtained. The electrodes and attached leads were then insulated as described by Roberts et al. (1973}. Two such electrodes were embedded in the myotomes, lateral to the vertebral column, 2 mm deep, and 2 cm anterior to the dorsal suture for ECG electrodes. EMG activity was monitored on a Devices M4 polygraph during implantation, and when a manual flick of the fish's tail produced a response on the recorder, the EMG wires were glued to the dorsal suture.

Ventilation Measurement A small area of skin was removed from the skull just posterior to the nares, and a hole was bored through into the buccal cavity, using a dental drill (later inspection confirmed that no nervous damage was caused, since the shaft of the drill passed between the olfactory tracts, which are spread laterally at this point). A length of Portrex tubing (1.25 mm diameter) was inserted through the hole into the buccal cavity, and drawn through the mouth, where its end was flared using a heated scalpel. Once the continuity of the tube had been checked, the tube was pulled back so that the flange fitted against the roof of the buccal cavity. The tube was secured dorsally by gluing it to the skull surface by a mixture of Kodak Eastman 910 adhesive and Simplex dental cement. Water was then passed through the tube with a syringe to clear any minor obstructions.

Buoyancy Compensation Following implantation of ECG and EMG electrodes, and of the ventilatory catheter, and while the fish was under anesthetic, a cube of polystyrene foam was attached to the wires just above the dorsal suture, and cut so that when the animal was placed in water, it was neutrally buoyant. Animals were allowed to recover in individual 60 x 30 cm tanks.

The Experimental Situation Animals were placed in a chamber of 1 cm plastic mesh, 20 cm long, 6 cm wide, and 10 cm deep, which was positioned centrally in a 35 x 35 x

258

LAMING AND SAVAGE

25 cm black plastic tank. The mesh chamber was sufficiently large that fish when at rest did not come into contact with it, but ensured that they were in similar positions for each stimulus. The water in the tank was kept at 15 + I°C by a cooling coil, and was aerated continually. The tank was mounted on sponge rubber, and was situated inside a large 46 × 36 × 36 cm wooden box, painted matt black, whose roof was hinged. The whole apparatus was enclosed in a Weldmesh cage, for electrical shielding purposes. When the fish was in position, the ECG and E M G electrode leads were connected to a Devices M4 polygraph, and the ventilatory catheter was connected to a Bell and Howell pressure transducer, care being taken to exclude air from the system. This transducer also was connected to the polygraph. When satisfactory recordings were obtained, the r o o f of the wooden box was lowered and a sheet of black paper fitted over the hole left at the front of the box. Behavior could be observed through a 2-cmdiameter ~'peephole" in this black paper. Animals were left for 1 hr prior to testing.

The Stimuli Six stimuli were selected which could be remotely operated, and yet mimic naturalistic environmental changes. Identical stimuli had to be presentable consistently and so chemical stimuli were not used. Three types of visual stimulus and three types of pressure wave stimulus were used:

Visual Stimuli Light on. This was the onset of a 60-W striplight, 21 cm long, fixed to the lid of the box, giving an illumination of 2.2 Ix at the fish's head. Other stimuli were presented with this light on. Moving edge. A screen was fixed below the light, and a rotating 22cm-diameter Perspex disk fitted between the light, and a 10 × 5 cm slot in the screen aligned lengthwise above the animal's head. This stimulus was the rotation of a disk consisting of six equal segments, alternately clear and black, at a speed of 0.7 cm/sec, so that a black moving segment traveled across the slot during a stimulus presentation, starting and finishing with a clear field. Moving spot. This stimulus was the presentation o f a 1-cm-diameter black disk replacing the black segments in the previous stimulus. Pressure Stimuli Sound. This was provided by a 3-ohm loudspeaker, situated on the lid of the box. A 500-Hz signal was used, since it was suggested (R. W. Piddington, personal communication) that this frequency represented the lowest threshold of the goldfish audiogram. A h y d r o p h o n e was used to

259

BEHAVIORAL AROUSAL IN THE GOLDFISH

record sound levels in water at the position of the fish's head, and an intensity of 119 microbar, or 40 db re 1 microbar, was the level used in the experiments reported here. Paddle. A 40-rpm motor was switched to drive a vertical shaft which entered the water 15 cm from the fish, 30° to the right from the midline in front of the animal. The shaft terminated in two 5-cm-diameter 3-mmthick Clear Perspex disks, separated by three 1.5-cm-high blades of the same material, set at 120° to each other. Plunger. Another pressure stimulus was produced by the movement of a 24-V dc solenoid, connected to a vertical Perspex shaft which entered the water 15 cm from the fish, 30° to the left from the midline in front of the animal. A 5-cm-diameter clear Perspex disk was fitted to the end of the shaft. Operation of the solenoid caused the disk to rise 1.2 cm in 15 msec and immediately to descend the same distance in 20 msec. Preparatory experiments in the dark suggested that visual cues provided by the movement in the water of these clear Perspex devices produced negligible effects.

Observations The following sets of observations were made from the experimental groups summarized in Table 1. (1) The preexperirnental values of the physiological variables. Prior to the presentation of the first in a series of stimuli physiological variables were monitored from 56 fish (groups 1-7), during a 10-sec period, to provide a baseline for changes subsequently observed during stimulus presentation. (2) Physiological responses in relation to behavior. Forty-eight fish in groups of eight were subjected to the six stimuli described (groups 1-6). TABLE 1 A Description of the Experimental Groups Used

Group

Stimulus

1 2 3 4 5 6 7 8 9 l0

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No. of fish

Intertrial interval (sec)

Postoperative recovery (days)

No. of trials, behavior observed

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1 1 1 1 1 1 1 6 1 1

2 2 2 2 2 2 10 2 2 2

No. of trials, physiological observations made 10 10 10 10 10 10 10 10 l0 10

260

LAMING AND SAVAGE

Presentations lasted for 10 sec (except for the plunger which operated once) as preliminary experiments had indicated that the physiological changes associated with behavioral arousal rarely lasted more than 8 sec. Ten presentations of stimuli were made, at 110-sec intervals, and physiological recordings were taken for each presentation. Two of the ten stimulus presentations per fish were selected randomly for behavioral observations to be made, making a total of 96 observations in which behavior could be related to the physiological variables recorded. The limitation on the number of trials for behavioral observation allowed assessment of observer interference. Group 7 fish were used as "controls" undergoing the same time regime without a stimulus being presented, and being observed for behavior on each of the 10 "mock stimulus" periods. The results of physiological measurements for these fish were included regardless of the observed behavior. (3) Stimulus effectiveness in evoking physiological changes. The physiological observations of fish in groups 1-6 were made in the 10 sec before and the 10 sec during the stimulus presentation. Variations in physiological parameters during experiments were expressed as: change = A - B , where A is the value of the parameter before and B the value during stimulus presentation. (A-B)/A was not used because when analyzing a quantity of events, like heart beats, in a fixed time interval, random changes by this method appear as a mean increase. It was therefore considered preferable to describe the normal value of a variable and the value of any change which occurred, separately. The parameters analyzed were heart rate, ventilatory rate, and for both ventilation and EMG, the maximum and minimum amplitudes observed during the 10-sec periods. ECG amplitude changes may be due to changes in the position of the electrodes relative to the heart, hence no such measurements were attempted. EMG values were measured in microvolts, ventilatory pressure in millimeters of Hg. EMG recordings often included a component which was due to the contraction of the ventilatory musculature. Thus a decrease in ventilatory amplitude was often reflected in the EMG record as a decrease in EMG minimum amplitude. Real myotome contractions could be determined by the lack of correlation with ventilatory effort. Two major factors contribute to make some recordings unreadable. Occasionally a fish would swallow air, which on entry into the ventilatory catheter dampens the pressure changes and makes them unmeasurable. In some fish, high impedance in EMG electrodes causes excessive noise in the recordings, and the EMG results from these animals could not therefore be used.

(4) The effect of operational trauma on the physiological responses. One group of five fish (group 8) was left for 6 days after the operation instead of 1 day, to examine the effect of postoperative trauma on the physiological responses to the 'light on' stimulus.

BEHAVIORAL AROUSAL IN THE GOLDFISH

261

(5) Habituation of physiological responses to the onset of illumination. Changes in physiological responses to the onset of light presented for 10 sec every 110 sec (group 2) were examined. The criterion used for habituation of a physiological response was the first of two consecutive presentations of a stimulus in which a response reached zero or was reversed. (6) The effects of different intertrial intervals on habituation. With the onset of light as the stimulus, the habituation of responses to intertrial intervals of 30, 110, and 300 sec (groups 9, 2, 10, respectively) was examined. (7) Habituation of physiological responses to different stimuli. The habituation of responses to the six different stimuli (groups 1-6) was examined.

RESULTS

(1) Values for the Physiological Variables Monitored, over a lO-sec Period The mean and SEM of values for the number of heart beats and ventilations monitored from 56 fish over 10 see were 7.36 _+ 0.4 and 11.5 +0.35, respectively. This oscillation in ventilatory pressure had a range of amplitude from a minimum of 7.81 -+ 0.72 to a maximum of 13.08 _+ 1.11 mm Hg. EMG potentials ranged from 30 +_ 4.1 to 46.5 _+ 6.7 t~V in amplitude. Fish in group 7, subjected to the " m o c k " stimulus showed no statistically significant changes in behavior or any physiological parameter during this period compared to the previous 10 sec. The extremely small differences obtained between these two periods (A-B) and their variation were subsequently used to compare with responses to the stimuli of groups 1-6.

(2) Physiological Changes Correlated with Behavioral Responses (a) Behavioral arousal (Table 2). Of the 48 fish (groups 1-6) presented with novel stimuli and observed for behavior, only two failed to show arousal on either of the trials for which observations were made. The typical behavioral response of fish was a change in the rate of movement of the pectoral fins and erection of the dorsal fin. Only these criteria were used as indicants of behavioral arousal, as they were the most consistent responses. They were, however, sometimes accompanied by slight dorsoventral undulations of the tail fin and eye movements. On no occasion did any of the fin movements propel the fish more than 1 cm from its original position. On comparison with unstimulated fish (group 7) significant (p < .02) decreases in heart rate (t = 3.1, 52°F) and ventilatory rate (t = 2.65, 52°F) and amplitude (t = 2.79, 52°F) were observed in these behaviorally aroused animals (Table 2). The 10-sec period was adequate to demon-

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strate that these responses declined with time, decreasing to the resting value usually within the 10-sec period. There was a close correlation between cardiac and ventilatory responses during arousal. The 96 observations of behavior included 52 arousal responses, 11 fright responses, and 33 occasions on which no response was observed although on two of the latter physiological responses were seen. Of the 52 behavioral arousals, 37 were accompanied by both cardiac and ventilatory decelerations, and there were 4 cardiac decelerations alone and 9 ventilatory decelerations alone. On two occasions neither response was detectable by the technique of counting heart beats and ventilations. (b) Fright (Table 3). Eleven fish showed fright or "tail-flip" responses to stimuli. The response was characterized by a rapid, violent lateral flexion of the tail which propelled the animal forward until it collided with the front of the mesh trough. The resistance of the trough then usually provoked a continued response for around 15 sec, the animal then returning to a stable, apparently resting condition. Five of these responses were to the plunger, two each of the moving edge and light on, and one each to the paddle and sound. In comparison with behaviorally quiescent fish (Table 3) frightened fish showed increases (p < .05) in the maximum amplitudes of both the EMG (t = 2.14, 16°F) and ventilations (t = 2.26, 17°F) and an increase in ventilatory rate (t = 2.51, 17°F). The situation with respect to the cardiac responses was more complex. An initial, brief bradycardia (1-4 sec) was followed by a tachycardia which could last for up to 30 sec. Over the 10-sec period examined, there was no mean change. An analysis of variance to compare the physiological responses of the three behaviors, fright, arousal, and quiescence, showed significant (p < .01) differences in each of the parameters examined (Table 4). With the exception of heart rate changes, all physiological responses were in the form of an increase during fright and a decrease during arousal.

(3) The Effectiveness of Various Novel Stimuli in Eliciting the Physiological Responses Associated with Behavioral Arousal The mean response of fish to all stimuli, grouped together, was a decrease in heart (t = 2.29, 54°F) and ventilatory rate (t = 2.02, 54°F) which was different (p < .05) from those receiving no stimulus (Table 5). The moving edge and, light on stimuli evoked significant (p < .05) reductions in heart and ventilatory rates and ventilatory amplitude compared to controls, the plunger and sound each evoking reductions in heart rate and ventilatory rate, respectively (Fig. 1). Analysis of variance only showed significant (p < .01) (F = 3.43, 42,5°F) differences between the ventilatory rate changes to different stimuli, but examination of Fig. 1 indicates that whatever physiological change was considered, there was a gradation of

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TABLE 4 Analysis of Variance of Physiological Changes Associated with (1) the Behaviorally Defined Quiescence of the Control Fish, (2) Behavioral Arousal Responses, and (3) Behavioral Fright Responses Variable No. of heart beats No. of ventilations Ventilatory maximum amplitude (mm Hg) Ventilatory minimum amplitude (mm Hg) EMG maximum amplitude (/~V) EMG minimum amplitude (gtV)

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r e s p o n s e to the various stimuli. T w o of the eight fish presented with the plunger as a stimulus and one presented with light onset, exhibited increases in ventilatory rate and amplitude to the novel stimulus.

(4) The Effect of the Postoperative Recovery Period A c o m p a r i s o n b e t w e e n the responses of group 2, given 1-day postoperative r e c o v e r y time, and group 8 given 6 days, b o t h presented with the light on stimulus, revealed no differences in ventilatory rate decrease (t = 0.57, 1 I°F) or amplitude decrease (t = 0.88, 11°F) b e t w e e n the two groups. The 6-day r e c o v e r y period, h o w e v e r , did m a k e the E C G recordings unclear so that only two recordings of the five fish with 6-day recoveries were measureable. The m e a n of the cardiac deceleration of these fish was 2.5 c o m p a r a b l e to that of 2.37 _+ 0.13 in the fish with 1 d a y ' s r e c o v e r y , so 1-day r e c o v e r y periods were used in all other experiments.

(5) Habituation of Cardiac and Ventilatory Changes on the Repeated Presentation of the Light on Stimulus at llO-sec Intertrial Intervals In the previous e x p e r i m e n t s (Section 2 of these results) behavioral arousal responses in fish were shown to be correlated with decreases in heart rate, ventilation rate, and the minimum size of ventilatory pressure changes. D e c r e a s e s in all three m e a s u r e s occurred with the light on stimulus, though these responses declined on regular repetition of the stimulus at 110-sec intertrial intervals (Fig. 2). Cardiac responses (Fig. 2a) d e c r e a s e d rapidly o v e r the first three trials, then m o r e slowly, the group having a m e a n of 4.5 trials to r e a c h the criterion for habituation of two consecutive zero responses. The ventilatory frequency r e s p o n s e c u r v e

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FIG. 1. A histogram of the mean - SEM of changes in (A) number of heart beats, (B) number of ventilations, and (C) minimum ventilatory amplitude (in mm) on the initial presentation of various stimuli. Stimulus types: (1) moving edge, (2) onset of light, (3) the plunger, (4) sound, (5) paddle, (6) moving spot. w a s less regular; a l t h o u g h the c u r v e (Fig. 2b) still indicated a r e s p o n s e after 10 trials, criterion was r e a c h e d after a m e a n o f 3.5 trials, all fish h a v i n g s h o w n t w o c o n s e c u t i v e z e r o r e s p o n s e s b e f o r e 10 trials h a d b e e n given. T h e r e s p o n s e o f the m i n i m u m amplitude o f ventilation o b t a i n e d a m e a n figure o f 4.12 trials to habituation.

(6) The Effect of the Intertrial Interval on Habituation of the Physiological Responses T h e habituation rates o f fish p r e s e n t e d with the o n s e t o f light at intervals o f 30 sec (group 9), 110 sec (group 2), and 300 sec (group 10) w e r e

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BEHAVIORAL AROUSAL IN THE GOLDFISH

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TABLE 6 Number of Trials to Habituation of Physiological Changes, Associated with Arousal, at different Intertrial Intervals Intertrial interval (sec) 30 110 300

No. of animals

Decrease in no. of heartbeats (mean +_ SEM)

Decrease in no. of ventilations (mean-+ SEM)

Decrease in ventilatory amplitude (mean-+ SEM)

5 8 8

2.6 _ 0.79 4.5 +_ 1.02 5.0 _+ 1.24

3.4 _ 0.75 3.5 _+ 1.13 4.0 _+ 0.95

4.4 _ 0.75 4.1 _ 1.34 3.3 +_ 0.6

Note. An analysis of variance of the habituation of each physiologicalchange with df 2,18 gave F values of 1.1, . 1, and .2 for heart rate, ventilatory rate and ventilatory amplitude, respectively. None of these is significant at the p = <.05 level. compared (Table 6) using a one-way analysis of variance (Bailey, 1959). N o effects of intertrial interval on habituation rate were found for any of the physiological responses observed.

(7) The Resistance to Habituation of Physiological Responses to Various Stimuli The number of trials required for habituation to o c c u r (Fig. 3), of all fish grouped together, was significantly greater than one for heart rate changes (t = 2.26, 54°F), ventilatory rate changes (t = 2.03, 54°F), and ventilatory amplitude changes (t = 2.13, 54°F) when compared to controls (Table 7). The habituation of changes in all three physiological variables was significantly different from the controls (p < .05) for the moving edge, onset of light, and plunger stimuli. Sound stimuli required longer to habituate heart and ventilatory rate changes, and moving spot and paddle longer to habituate ventilatory amplitude changes than controls. An analysis of variance of the number of trials to habituate a response showed significant differences (p < .05) between the rates of habituation for the different stimuli. There appeared to be a high degree of consistency in the gradation of both the magnitude and resistance to habituation of a response (Figs. 1 and 3, Table 7). This was examined by applying a test for linear correlation (Bailey, 1959) between the magnitude of a response on the first trial and the number of trials for that response to habituate. Using the individual data for all fish, regardless of the stimuli eliciting the response, significant correlations (p < .05) were found for each of the three physiological variables measured. The correlation coefficient for heart rate decreases was 0.52 (t = 4.12, n = 4 6 ° F , p < .001) in relation to the number of trials the response required for habituation. The equivalent coefficient for ventilatory rate responses was 0.60 (t = 5.1, n = 46°F, p < .001) and for ventilatory amplitude responses was 0.35 (t = 2.53, n = 46°F, p < .02).

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In goldfish, two apparently different types of behavioral responses to stimuli, those of " a r o u s a l " (orienting, alerting) and " f r i g h t " (startle, defense, escape), are characterized by similarly different physiological responses. During behavioral arousal, goldfish exhibit cardiac and ventilatory decelerations to about 70% of the resting level and the amplitude of ventilations decreases to 50%. When frightened, typically to intense stimuli like a plunger, the fish exhibit a transient cardiac deceleration, followed b y an acceleration, and an increase in both the rate and amplitude of ventilations. Behavioral arousal is elicited b y all types of stimuli used in this work, though the magnitude of the physiological r e s p o n s e to the novel stimulus

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differs. Stimuli which might be considered "strong," in terms of the change in receivable energy they contained, produce large responses. Stimuli like the onset of light, the moving edge, and the plunger are of this nature, compared to the moving spot, sound, or the paddle which gave comparatively weak responses. The strong stimuli are also more likely to elicit the infrequent fright responses seen in this study. Linearly related to the magnitude of the physiological arousal response to a novel stimulus, is the number of presentations of the stimulus required for habituation of a response to occur. Thus the stimuli which provoke the largest decelerations in heart and ventilatory rate have to be presented most often for these changes to habituate. Although the apparent "strength" of stimuli affected the rate of habituation, the interval between stimulus presentations did not. This does not conform to the findings of many workers in mammals (Bradley, 1957; Gastaut & Bert, 1961) that shorter intervals provide more rapid habituation, though it does conform with predictions of the dual process theory of habituation proposed by Thompson, Groves, Teyler, and Roemer (1973). The physiological changes observed during behavioral arousal in fish, reported here, resemble those reviewed by Lynn (1966) for mammals. Correlations between behavior and physiology are high, since in only 4% of cases does behavioral arousal occur without physiological changes and in another 4% physiological changes occur without behavioral arousal. Subsequent work has shown that physiological changes of this nature, occurring without behavioral arousal, are correlated with EEG changes (Laming, 1976). Sokolov (1960) emphasizes the adaptive nature of the mammalian orientation reaction, as a series of changes designed to enable the animal to gain as much information as possible about the novel stimulus, and to be ready to respond to it. The slowing of cardiac and respiratory activity on arousal in fish appears to be the opposite of what might be expected of an animal preparing for activity. Bradycardias in fish are caused usually by vagal cholinergic action (Randall, 1966; Satchell, 1971) and this might argue for cholinergic mechanisms being involved in arousal. However, if parasympathetic cholinergic factors are involved in promoting the signs of arousal reported here, there would be a decrease in metabolic efficiency since Keys and Bateman (1932) have demonstrated that parasympathetic action increases resistance to flow of blood in the gills. A more likely explanation is that the cardiac and respiratory responses are reflex and secondary in nature. Preparation for action is typically a sympathetic adrenergic reaction, and in such a case there might be vasoconstriction of the visceral but not of the skeletal vessels. Such a change of peripheral resistance could raise blood pressure and Mott (1957) has shown that such an increase can decrease heart rate. Laming and Savage (1978), using an electromagnetic flowmeter, have demonstrated an almost

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immediate slowing of visceral flow during behavioral arousal in fish. Work is in progress to see if this does in fact cause an increase in dorsal aortic pressure. Satchell (1971) has reported on cardiac/respiratory synchrony in fish, so the ventilatory changes, generally accompanying the bradycardia, may be due to central rather than peripheral mechanisms. If the autonomic correlates of behavioral arousal, like the bradycardia, are ultimately adrenergic in origin, then their relationship with the fright or startle response becomes interesting. During fright or startle responses in goldfish the EMG amplitude is high, and following a brief bradycardia, a tachycardia is observed; ventilatory rate and amplitude increase. Such responses are adrenergic and seem to differ from those in arousal in intensity not type. For instance, vascular redistribution may cause a bradycardia in mild levels of activation or arousal, but at high levels of activation, the reflex, pressure-mediated bradycardia may be overridden by adrenergic influences on the heart, or by inhibition of the vagus, promoting tachycardia. The combined bradycardia and tachycardia of fright in fish certainly could be interpreted on this basis. Such observations support the suggestion of Gastaut and Roger (1960) that the orientation and defense reactions are part of the same continuum, although differing in intensity. One distinction that has been made between arousal and fright reactions is that the former habituate in around 10 trials whereas the latter may take several days (Moyer, 1963). Information from fish suggests that arousal responses take from two to four trials to habituate, fright taking some five trials with a decreasing intensity of response to be replaced by arousal which itself habituates (Laming, McKee, & Ennis, unpublished). There are few reports of habituation in fish. Welker and Welker (1958) using Eucinostornus gula, found that activity ceased, on introduction of a novel object into the aquarium, gradually increasing to its original level after 10 min. More recent studies by Rogers et al. (1963) using goldfish and Russell (1967) using Poecilia reticulata have shown that the presentation, respectively, of a bang or a light stimulus causes a '~tail flip" response which is thought to be due to Mauthner cell activity. The tail flip response itself does not habituate as rapidly as orientation reactions (Rogers et al., 1963) and this and its very nature reasonably lead to the classification of the reaction as a defensive one. Rogers et al. (1963) also reported an "orientation response" to the same bang stimulus, which did not habituate beyond 50% in 15 days' testing of 10 trials each day. In view of the fact that the same stimulus regularly caused fright reactions, and that high-intensity stimuli, even when they produce orientation rather than fright reactions, habituate only slowly (Sokolov, 1960), it seems unlikely that typical fish orienting behavior was being observed. Similarly Russell (1967) found that a moving shadow stimulus usually elicited a tail flip

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response on early presentations, but that typical orientation responses were observed later, and that these waned gradually, but over a longer period than reported here. The speed of shadow stimulus used by Russell (1967), 48 cm/sec, was chosen to resemble the "order of values quoted for the swimming of fish of a size to prey on Poecilia" so that as for Rogers' stimulus, the effect of presentation would be expected to elicit fright rather than arousal. The use of physiological correlates of behavioral arousal in the habituation study reported here, and the very low proportion of fright responses seen (4%) suggest that it is the habituation of arousal which is being examined. One interesting feature of this habituation is that with the analysis used, no sensitization occurred in the early stimulus presentations as had been found in relation to aggressive displays in B e t t a s p l e n d e n s (Peeke & Peeke, 1970). Sensitization would also be predicted by the "dual process theory of habituation" (Thompson et al., 1973) in which the first stimulus causes sensitization of the animal to the subsequent stimulus, though the sensitization itself also habituates. Thus at the present time evidence for sensitization of the autonomic correlates of arousal in fish is lacking. A more exhaustive examination of the effects of frequency and strength of a stimulus is needed before the evidence leans strongly toward the Thompson et al. (1973) habituation/sensitization dual process theory or the neuronal model concept of Sokolov (1960). The strong correlation between the ongoing behavior and some of the physiological measures examined in this work makes them useful when used together, as indicants of the behavioral state of fish. Thus in telemetry in wild populations (Priede & Young, 1975) and in experiments where observer interference has to be minimal, physiological changes may be used to infer the behavior of the animal. In fish the physiological changes habituate in a manner similar to that shown by arousal responses in mammals. However, in the absence of true cortical structures in fish it is to be expected that the neural mechanisms underlying the orientation reaction and its habituation may be different, for removal of the telencephalon does not abolish the ability to manifest arousal (Savage, 1971) though ongoing work suggests that it may affect its habituation especially in relation to physiological correlates of the behavior. Further studies, on the orientation reaction and its habituation in fish, may clarify these aspects of comparative vertebrate neurobiology.

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Gastaut, H., & Roger, A. (1960) Les mechanismes de l'activite nerveuse superieure du niveau des grandes structures fonctionelles du cerveau. Electroencephalography and Clinical Neurophysiology Supplement, 13. Goodman, D. A., & Weinberger, N. M. (1973). Habituation in 'lower' tetrapod vertebrates. Amphibia as vertebrate model systems. In H. V. S. Peeke and M. J. Herz (Eds.), Habituation, Vol. 1. New York: Academic Press. Keys, A., & Bateman, J. B. (1932). Branchial responses to adrenaline and pitressin in the eel. Biological Bulletin, 63, 327-336. Laming, P. R. (1976). Physiological Correlates of Behavioral Arousal in the Goldfish (Carassius auratus). Ph.D. thesis, University of London. Laming, P. R. (1980). Electroencephalographic studies on arousal in the goldfish, Carassius auratus. Journal of Comparative and Physiological Psychology, 94, 238-254. Laming, P. R., & Savage, G. E. (1978). Flow changes in visceral blood vessels of the chub (Leuciscus cephalus) during behavioral arousal. Comparative Biochemistry and Physiology, A59 (3), 291-293. Lynn, R. (1966). Attention, Arousal and the Orientation Reaction. London: Pergamon. Mott, J. C. (1957). The cardiovascular system. In M. E. Browne (Ed.), The Physiology of Fishes, Vol. 1, pp. 81-105. New York: Academic Press. Moyer, K. E. (1963). Startle response habituation over trials and days, and sex and strain differences. Journal of Comparative and Physiological Psychology, 56, 863-865. Otis, L. S., Cerf, J. A., & Thomas, G. J. (1957). Conditioned inhibition of respiration and heart rate in the Goldfish. Science, 126, 263-264. Peeke, H. V. S., & Peeke, S. C. (1970). Habituation of aggressive responses in the Siamese fighting fish (Betta splendens). Behavior, 36, 232-245. Peeke, H. V. S., & Peeke, S. C. (1972). Habituation, reinforcement and recovery of predatory responses in two species of fish (Carassius auratus and Macropodus opercularis). Animal Behaviour, 20, 268-273. Priede, I. G., & Young, A. Y. (1975). The ultrasonic telemetry of cardiac rhythms of wild brown trout (Salmo trutta) as an indicator of bio-energetics and behaviour. Journal of Fish Biology, 10, 299-318. Randall, D. J. (1966). The nervous control of cardiac activity in the trench (Tinca tinca) and the goldfish (Carassius auratus). Physiological Zoology, 34, 185-192. Roberts, M. G., Wright, D. E., & Savage, G. E. (1973). A technique for obtaining the electrocardiogram of fish. Comparative Biochemistry and Physiology, 44A, 665-668. Rogers, W. L., Melzack, R., & Segal, J. R. (1963). "Tail flip response" in goldfish. Journal of Comparative and Physiological Psychology, 56~ 917-923. Russell, E. M. (1967). Changes in the behaviour of Lebistes reticularis upon repeated shadow stimulus. Animal Behaviour, 15, 574-585. Satchell, G. H. (1971). Circulation in Fishes. Cambridge: Cambridge Univ. Press. Savage, G. E. (1971). Behavioural effects of electrical stimulation of the telencephalon of the goldfish, Carassius auratus. Animal Behaviour, 19, 661-668. Sokolov, E. N. (1960). Neuronal models and the orienting reflex. In M. A. Brazier (Ed.), The Central Nervous System and Behavior, pp. 187-276. New York: Macy. Thompson, R. F., Groves, P. M., Teyler, T. J., & Roemer, R. A. (1973). A dual-process theory of habituation: Theory and behavior. In H. V. S. Peeke and M. J. Herz (Eds.), Habituation, Vol. 1. New York: Academic Press. Welker, W. I., & Welker, J. (1958). Activity and habituation ofEucinostomus gula. Ecology, 39, 283-288.