Behavioural arousal and its habituation in the squirrel fish, Holocentrus rufus: The role of the telecephalon

BEHAVIORAL AND NEURAL BIOLOGY 47, 80--104

(1987)

Behavioural Arousal and Its Habituation in the Squirrel Fish, Holocentrus rufus: The Role of the Telecephalon PETER R . LAMING

Department of Zoology, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland Squirrel fish were captured, maintained for 2 days and then tested for their responses to stimuli in laboratory aquaria. A tap on the side of the aquarium or a shadow moving overhead elicited an arousal response involving a reduction in opercular movements, movement of the pectoral fins, and erection of the spiny dorsal fin. This latter response, when regularly provoked by a moving shadow at 15-s intervals, decreased in duration and habituated. When fish were restricted in a wire mesh trough and the stimulus was the onset of illumination to one eye at 15-s intervals, responses were quantitatively similar although shorter in duration. A series of stimuli delivered to one eye until habituation followed by a series to the other eye reduced the duration of initial responses and the number of trials for habituation of responses to stimuli delivered to the second eye. Responses were equivalent regardless of which eye (left or right) was first presented with stimuli. Section of the telencephalic anterior commissure considerably restricted the transfer of information about experience of stimuli presented to the first eye. Commissural section, combined with unilateral lesions in the posterior-medial telencephalon, severely impaired habituation of responses to the contralateral eye. Lateral lesions had no effect. The results are discussed in relation to the visual projection in Holocentrus and telecephalic involvement in arousal and its habituation in teleosts as a whole. © 1987AcademicPress, Inc.

Fish, like other vertebrates, show responses to stimuli which may fall into two broad categories, those which occur to specific stimuli and are related to particular motivational states, and those which are responses to the novelty of the stimulus situation. In the former category are behaviors such as feeding, courtship, and aggression whereas in the latter This work was supported by a Royal Society Travel Grant in 1981 and in 1984 by a Royal Society Travelling Scholar Award and a NATO Senior Scientist Award. The author is indebted to Professor S. O. E. Ebbesson for organizing and to Dr. M. H. Avila, Dr. E. W. Williams, Dr. D. Y. Shapiro, and Dr. D. Hensley for providing the necessary facilities in Puerto Rico. Dr. Sue Nell of the Zoology Department, Queen's University of Belfast, kindly helped with the histology, and Dr. R. W. Elwood and D. J. Rooney helpfully reviewed the initial draft, for which I am grateful. 80 0163-1047/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

AROUSAL AND HABITUATION IN SQUIRREL FISH

81

are arousal (alertness) and fright (startle, escape) responses. Often these latter responses to nonspecific stimulation are nevertheless associated with species-specific behavior and also may involve a strong spatially oriented (attentional) component. The categorization of behavior into responses to nonspecific stimuli, specifically motivated, or attentional, etc., implies that differing (neuronal) mechanisms may underly their expression. Arousal, defined here as a nondirected response to any novel stimulus, not involving escape or fright, may be one of the most fundamental behavioral responses regulated by the vertebrate brain. Such a suggestion is supported by the existence of definable arousal responses in vertebrates such as fish and amphibia which lack a neocortex. The responses of fish to novel stimuli have been described in Poecilia (Russell, 1967), Carassius (Savage, 1971; Laming & Savage, 1980), Rutilus (Laming & Hornby, 1981) and Halichoeres (Laming & Ebbesson, 1984). The common features of all these descriptions of the behavioral response, were changes in pectoral fin movements and dorsal fin erection. These rarely changed the animal's position in the water. In the species previously observed, these responses were difficult to quantify, though Laming and Ebbesson (1984) reported on the proportions of motor responses associated with arousal which were exhibited to a repeated stimulus by slippery dicks (Halichoeres) during habituation. In general, however, it has been the physiological changes associated with behavioral arousal in fish which have provided some quantitative measure of the response. These physiological correlates of behavioral arousal include cardiac and ventilatory decelerations and a decrease in ventilatory amplitude (Laming & Savage, 1980), a reduction in visceral venous blood flow (Laming & Savage, 1978), and an increase in the frequency and amplitude of the EEG (Laming, 1980). Changes in heart and ventilatory rate have been the most consistently used measures of the magnitude of the response. Both behavioral and physiological arousal responses of fish are made to novel stimuli, which can be of any modality (Laming & Savage, 1980). If similar stimuli are repeatedly presented, these responses habituate. When a quantitative estimation of response magnitude has been used then a linear relationship has been found between the response to the first (novel) stimulus and the number of stimulus presentations, serially given, required for response habituation. This relationship holds for different stimulus modalities (Laming & Savage, 1980) or with differing intensities of the same stimulus (Rooney & Laming, 1986b). The possibility that the teleost telencephalon might be involved in activation or arousal to nonspecific stimuli was developed by Aronson (1963) following the lead of Herrick (1948). Its involvement in arousal and its habituation was first demonstrated by its complete removal in the roach (Rutilus rutilus) using behavioral measures of response (Laming &

82

PETER R. LAMING

Ennis, 1982) and in the goldfish (Carrasius auratus) using cardiac deceleration as the measure of response (Laming & McKee, 1981). In both cases habituation was severely impaired and there was also some indication of enhanced responsiveness to the novel stimulus. It has since been shown that the deficit in habituation is not due to loss of olfactory influences, as olfactory bulbectomy alone causes no deficit (Rooney & Laming, 1984). Furthermore, discrete unilateral lesions restricted to the posterior telecephalon in the roach still caused a deficit in habituation of behavioral arousal to a tap stimulus (Laming & Hornby, 1981). However, in Betta splendens posterior dorsomedial lesions have been found to cause enhanced habituation of behavioral arousal responses to tap stimuli (Marino-Neto & Sabbatini, 1983). Lesions in the posterior dorsocentral (DC) telencephalon (Murakami, Morita, & Ito, 1983) of goldfish, however, caused deficits in habituation of cardiac arousal responses to the onset of illumination (Rooney & Laming, 1986a). There has been some evidence that lesions near the anterior commissure caused elevated responsiveness and thereby prolonged the time for habituation because of the relationship between initial response magnitude and resistance to habituation (Laming & Hornby, 1981). The teleost telencephalon has thus been shown to be involved both in nonspecific arousal and in its habituation. In order to distinguish the effects of lesions on these two apparently separate regulatory processes the behavioral response must be quantified or a physiological measure involving surgical electrode implantation must be used. The present study wished to avoid the latter contingency and so a species was chosen which appeared to provide a behavioral response which was quantifiable in terms of duration. This species was the squirrel fish, Holocentrus rufus, which also had the advantage of having been the subject of considerable neuroanatomical study especially in relation to the central visual projection (Vanegas & Ebbesson, 1976; Ito, Morita, Sakamoto, & Ueda, 1980; Ebbesson, 1980; Schroeder, Vanegas & Ebbesson, 1980). The squirrel fish, H. rufus (Holocentridae: Beryciformes), is a common fish of the Caribbean and tropical western Atlantic. They are primarily nocturnal predators (Vivien & Peryrot-Clausade, 1974) with well-developed eyes, a large tectum (Vanegas & Ebbesson, 1976), and a well-developed telencephalon (Ito et al., 1980). Like many reef fish, the Holocentridae are very alert animals and their behavior frequently involves changes in the orientation and movement of their fins. These features led to the choice of squirrel fish as subjects for the experiments reported here on the role of the telencephalon in behavioral arousal and its habituation. METHODS

The studies reported here were performed (A) at Culebra Island, Puerto Rico, in 1981 and (B) at Magueyes Marine Laboratory, Puerto Rico, in 1984.

AROUSAL AND HABITUATION IN SQUIRREL FISH

83

(A) Laboratory Observations Squirrel fish, H. rufus, were captured in baited traps by commercial fishermen during July 1981, and maintained for 2 days in an immersed wire mesh cage at Culebra Island prior to use. Individuals used for experiments were 14-18 cm in length and were placed for 24 h in a 0.7 x 0.3 x 0.3-m high outdoor glass aquarium filled with fresh, aerated seawater, shaded from direct sunlight, and with its sides enclosed in beige card. Observations were made of general behavior in both the night and the subsequent dawn after introduction, prior to presentation of stimuli. The weather was uniformly clear in the 2 weeks in which aquarium observations were made.

Experiment I: Components of the behavioral response of H. rufus to novel stimuli. Six fish were used to test the behavioral responses to a moving shadow and a " t a p " stimulus. The shadow was a 10 x 20-cm black card moved over the aquarium at a constant height of 8 cm, passing over the fish on a forward direction at approximately 8 cm/s for 4 s. The tap was delivered by a small wooden hammer falling under gravity. Preliminary observations indicated that stimuli presented every 2 min produced little habituation of responses, so recordings were made every 2 min of responses when taps, moving shadows, and control (no stimulus) conditions were applied in a randomized sequence. Observations were made over two consecutive 5-s periods of the number of opercular and pectoral fin movements for each period, starting at the onset of the stimulus. These were then converted into beats or fin movements per minute. With a separate stimulus presented, the number of movements of the soft dorsal fin and the duration of erection of the spiny dorsal fin was recorded. The average value of the four variables, from three observations per fish, in each of the three stimulus conditions was calculated. Experiment 2: Habituation of the spiny dorsal fin response. In a separate group, nine fish were each presented with the moving shadow stimulus approaching the fish from behind at 15-s intervals. The duration of short latency (2-s) erections of the spiny dorsal fin was recorded as had been the duration of spontaneous erections in a 15-s period, with no stimulus, prior to the experiment. The response was considered to have habituated on the last presentation it was evoked prior to three consecutive stimuli with no response. The original stimulus was then presented in the reverse direction to check for stimulus specificity.

(B) Responses of H. rufus to Unilaterally Presented Stimuli Fish for all subsequent experiments were caught by squid-baited line, at night, in the vicinity of rocky outcrops around Magueyes Island Marine Station, Puerto Rico, in April 1981. After capture, specimens of from 12-18 cm in length were placed in outdoor 0.9 x 0.6 x 0.6-m high aquaria supplied with a flow of filtered seawater. They were maintained

84

PETER R. LAMING

in these aquaria for 2 days prior to use. Each day six individuals were moved to a laboratory where they were maintained in 0.9 × 0.6 × 0.6-m high aquaria filled with clean, aerated seawater both prior to operations and during the subsequent recovery period. The fish were then selected for inclusion in one of five groups representing the pretest treatment they would receive: (1) Normal: fish receiving no surgery; (2) Sham: fish undergoing cranial surgery alone; (3) AC: fish undergoing cranial surgery with the anterio commissure sectioned; (4) Medial: fish undergoing cranial surgery, anterior commissural section, and lesion formation in the medial telencephalon; (5) Lateral: as above, but with lesions in the lateral telencephalon.

Operations Fish were anesthetized in a bath of 1:10,000 MS22 (methane tricaine sulphonate) in sea water until opercular movements ceased. This level of anesthesia was maintained by passing seawater over the gills via the mouth when the gills lost their bright pink color and then reimmersing in MS222 when opercular movement recommenced. For operations the fish was wrapped in moist tissue and held firmly in a simple wooden trough, 2 cm deep, 20 cm long, and 3 cm broad. Fine sharp scissors were inserted through the suture between the frontal bones at the level of the posterior border of the eyes. A 5-mm-diam hole was then cut in the cranial roof at this level. Aspiration of the fatty cerebrospinal fluid then revealed the telencephalon. With sham operations the intracranial fluid was then simply replaced with marine teleost saline and a piece of sterile cork was cut to fill the cranial opening. The cranium was then sealed in with Eastman 910 adhesive activated with Simplex dental cement liquid (Laming, 1980). The fish was then revived by passing a current of fresh sea water through the gills via the mouth. On recommencement of opercular ventilation fish were placed in the stock aquaria for 24-h recovery. Fish on which telencephalic surgery was performed were otherwise treated in an identical fashion. Surgery was carried out while the cranium was open, with a sharp, miniature scalpel having a blade 1 x 3 mm in length. Section of the anterior commissure involved teasing the hemispheres apart with a spatula and then cutting the commissure thus revealed. Commissural section was therefore achieved with minimal damage to the hemispheres. Medial telencephalic lesions were performed after AC section by inserting the point of the scalpel vertically, 3 mm deep into the posterior third of one hemisphere, approximately one-third of the distance from the midline to the lateral border. A longitudinal cut, from 2-4 mm long, was then made while withdrawing the blade. Lateral lesions were made after AC

AROUSAL AND HABITUATION IN SQUIRREL FISH

85

section using a surgical scalpel with a pointed blade. The dorsolateral slice was cut from a point between the midline and lateral border to the ventrolateral border of the telencephalon. The excised portion was then aspirated. After operations the cranium was resealed and the fish revived as previously described.

Apparatus The experimental 0.6 x 0.3 x 0.3-m high aquarium was filled with fresh aerated seawater and a 25 x 4 × 12-cm-high trough of wire mesh (1 cm 2) was suspended a t its center with the longitudinal axis of the trough parallel to the sides of the aquarium. The sides of the aquarium were covered with black card except for a vertical 1.5 x 6-cm slit cut centrally in each side. Identical 40-W bench lights were positioned on each side 0.3 m from these slits so that they shone into the wire mesh cage at the position the fish's head would occupy. A 10 x 10-cm piece of black card was then used to occlude the slits. Observation of the trough was through a piece of one-way plexiglas placed over the end of the aquarium nearest to the observer. The background illumination in the aquarium was then that of a dimly lit room (30 Ix); when one slit was opened it rose to 90 lx at the position of that side of the fish's head.

Experimental Regime Aeration of the aquarium water was stopped and a fish was placed in the wire mesh trough facing away from the observer. It was then left for an hour to adapt to the experimental aquarium. Fish which appeared unduly stressed after 0.5 h were not used. After the 1-h period had elapsed, stimuli were delivered by rapidly raising the black card which occluded the slit on one side for 1 s every 15 ___ 1 s. The magnitude of responses was estimated by the duration in seconds from the onset of either a change in pectoral fin beats or an erection of the spiny dorsal fin to cessation of the last to occur of either of these responses. Habituation was considered to have occurred on the last trial before three consecutive trials with neither response. After these three trials the slit was left occluded and stimulation resumed from the opposite side of the aquarium. Approximately equal numbers of fish in each group obtained stimulation from the left or right sides first. On habituation of the response to the second side the fish was killed in a bath of 1:10,000 MS222, decapitated, and the cork plug sealing the cranium was removed prior to immersion of the head in a labeled vial of 8% Formalin in seawater. The aquarium was then drained and refilled with clean, aerated, seawater prior to testing the next individual. Individuals within treatment groups were randomized in relation to the time of day they were tested. In all, 12 normal fish, 10 with sham operations, 20 with AC sections, and 38 also with telencephalic lesions, were tested.

86

PETER R. LAMING

Histology After 1 week in Formalin, the brains were dissected from the cranium and dehydrated in ethyl and butyl alcohol mixtures, followed by butyl alcohol and chloroform mixtures prior to embedding in paraffin wax. They were then sectioned transversely at 14/zm and stained with hematoxylin-eosin. Serial sections of the telencephalon were examined to determine the position and extent of lesions.

RESULTS (A) Laboratory Observations of the Behavior of the Squirrel Fish

(H. rufus) Specimens of H. rufus appeared to adapt well to the aquarium environment; after introduction they exhibited several minutes exploratory behavior before taking up station in one corner of the aquarium. Here they would actively maintain their position, occasionally, especially at night, making forays into other parts of the aquarium. At night, forays could be initiated by dropping pieces of squid into the aquarium. These were rapidly eaten.

Experiment 1: Components of the Response of H. rufus to an Environmental Stimulus The responses of fish to a tap and a moving shadow presented in random order are summarized in Table 1. Presentation of either stimulus produced a reduction in the number of opercular beats and erection of the spiny dorsal fin. The later response is exhibited in Fig. 1. Pectoral fin movements also decreased when a moving shadow was presented. Experiment 2: Habituation of the Dorsal Fin Erection Response Regular, repeated presentation of a moving shadow at 15-s intervals to nine fish caused the spiny dorsal fin erection response (Fig. 1B) to decrease in duration and ultimately habituate (Fig. 2). The mean duration of the response fell within the SEM of spontaneous responses of nonstimulated fish after 23 trials; however, the mean number of trials to the habituation criterion was 18.22 _+ 2.34 (mean ___ SEM). The response could be reevoked by reversing the direction of the stimulus.

(B) Reponses of Fish to Unilaterally Presented Stimuli General When "caged" fish were presented with repeated rapid changes in illumination caused by occlusion of a unilateral light source they showed more transient arousal responses than when they had been unrestrained and were presented with an overhead moving black card (section A). No fright responses were observed. Fish in the wire trough appeared to

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82.6

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79.3 ± 5.0

72.7 + 5.3

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72.0 ___ 5.8*

1st 5 s

Note. Values significantly different from the control value using a paired test.

Opercular beats (beats/min) Pectoral fin movements (movements/min) Soft dorsal fin m o v e m e n t s (movements/min) Spiny dorsal fin erection (s/10 s)

Response

Moving shadow

Stimulus type

85.3 ± 5.0

77.3 --- 9.0

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2nd 5 s

9.58 ± 0.28**

80.0 _+ 4.2

74.7 ___ 7.5

77.3 ± 5.5*

1st 5 s

Tap

TABLE 1 Responses of Holocentrus, Measured over T w o Consecutive 5-s Periods, to Stimuli Delivered at 2-min Intervals

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PETER R. LAMING

FIG. 1. Behavioral arousal in Holocentrus. Presentation of a novel stimulus to stationary Holocentrus (A) caused erection of the dorsal fin (B).

A R O U S A L A N D H A B I T U A T I O N IN S Q U I R R E L FISH

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adapt well to the relatively confined situation and their responses were qualitatively similar to those described previously. Before reexamining these in detail, however, the different types of data analysis used will be described.

Data Analysis (a) Graphic presentation. As a first approach, the mean duration of response for all fish within a group was plotted for each serial stimulus presentation (Figs. 4-9). The least significant differences (LSDs) (Parker, 1973) for these points provide evidence for the effect of each treatment at the p < .05 level. (b) Two-way analysis of variance (ANOVA). Two-way analyses of variance were applied, with repeated measures on one factor (trials), to compare the effects of different group treatments. To examine the effect of presenting stimuli to different eyes on the same fish a two-way ANOVA with repeated measures on both factors was used. Fma~ and Bartlett's tests were used to check for homogeneity of variance, a fundamental assumption of ANOVA analyses. Where two groups had widely differing rates of habituation, variances were frequently heterogenous owing to low or zero variance after habituation had ensued. Thus, other analyses were needed in these instances. Student's t analyses: The initial response. The duration of responses to novel stimuli was used as an estimate of response magnitude for comparison between treatment group mean and its control group mean using either an unpaired or paired t test as appropriate. Trials to habituation. The mean number of trials to achieve the habituation criterion was compared, between groups, using unpaired Student's t or between eyes using paired t tests as appropriate.

90

PETER R. LAMING

The slope of the habituation. Previous work had demonstrated that although habituation itself is curvilinear there is a linear relationship between the magnitude of a fish's response to presentation of the first in a series of stimuli and the number of trials required for that response's habituation (Laming & Savage, 1980; Rooney & Laming, 1986b). Thus, stimuli or treatments which evoke larger responses will extend habituation without changing the slope of the habituation curve. Some surgical treatments, however, can extend habituation without affecting the initial response (Laming & McKee, 1981). Therefore, to examine if habituation itself were affected by treatments, the ratio of initial response magnitude to the number of trials to habituation was calculated for all fish. This gave an estimate of the slope of the habituation curve. The average response for the first two trials was used to estimate initial response magnitude. Plots of the frequency distribution of these ratios indicated that they were normally distributed, and so Student's t tests were used to compare the mean slopes between groups of fish, or between responses to different eyes in the same fish. Effects of the Side on Which Stimuli Were Presented The responses of six normal fish which first received stimuli to the right eye were compared with six which had the left eye stimulated first. There was no effect of the treatment (two-way ANOVA; Table 2). A similar lack of treatment effects obtained when four sham group fish stimulated first on the left were compared with six which were first stimulated on the right (Fig. 3, Table 2). When the second sides stimulated were compared by ANOVA for the two groups of both sham and normal fish, again no laterality was evident (Fig. 3, Table 2). When the mean of the initial response magnitude of comparable groups and the mean number of trials to habituation were compared, there also were no differences found using Student's t at the 0.05 level. The groups used in the ANOVA analyses were all homogeneous and all showed a significant (t9 < 0.01) (Table 2) effect of repeated trials on the response, as they all habituated (Fig. 3). The finding that there was no effect due to which eye (left or right) was first stimulated allowed subsequent analyses to be performed on the grouped data from either eye.

Experience Effects Responses made to the eye first illuminated were compared to those made to the eye illuminated second, using a two-way ANOVA with repeated measures on both factors (Table 2). Experience of the stimulus repeatedly delivered to the "first" eye reduced subsequent initial responses for both normal (Fig. 4) and sham-operated fish (Fig. 5, Table 2). The variances inherent in the treatment effects for these results were, however, heterogenous when tested with Bartlett's test, and so consideration was

Normal, right eye first Sham right eye first Normal right eye second Sham right eye second Normal second eye Sham second eye Sham first eye Sham second eye AC first eye AC second eye AC second eye Ant-med lesion eye Post-med lesion eye Post-lat lesion eye

Treatment 2 1,10 1,8 1,10 1,8 1,209 1,171 1,20 1,20 1,24 1,24 1,285 1,152 1,133 1,95

DF 0.89 0.27 2.13 0.007 82.00 84.50 3.82 2.29 1.01 21.30 6.05 5.77 53.53 14.69

F NS NS NS NS 0.01 0.01 NS NS NS 0.01 0.01 0.05 0.01 0.01

p 8.7 9.4 9.3 11.7 12.9 17.4 16.64 17.54 19.96 12.36 18.14 6.68 3.75 9.65

F

Repeated measures

9,90 9,72 9,90 9,72 9,209 9,171 9,180 9,180 9,216 9,216 9,285 9,152 9,133 9,95

DF

Note. FM, Fmax; B, Bartlett's test; Hom, homogeneity. X, p < 0.01; ,/, p > 0.01. NS, not significant.

Normal, left eye first Sham left eye first Normal left eye second Sham left eye second Normal first eye Sham first eye Normal first eye Normal second eye Sham first eye Sham second eye AC first eye Control eye Control eye Control eye

Treatment 1

Treatment effect

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

p

,/(FM) ,/(FM) ,/(FM) ,/(FM) X(B) X(B) ,/(FM) ,/(FM) ,/(FM) X(FM) X(B) X(B) X(B) X(B)

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TABLE 2 Results of Two-Way ANOVA Analyses to Compare the Effects of Treatments on Arousal Responses and Their Habituation in the Squirrel Fish (Holocentrus refus)

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given to changes in duration of initial response between treatments and their resistance to habituation. Thus, normal fish showed a decline in duration of initial response to the second set of stimuli (t(11) = 4.3, p < .01). Sham-operated controls showed a similar trend though it just failed significance (t(9) = 2.06, p > .05). Both groups showed a decline in the n u m b e r o f stimuli required for the habituation criterion to be reached. F o r normal fish the number dropped from a mean of 7.9 -_- 1.4 to 3 --+ 0.67 (t(l 1) = 2.95, p < .02) whereas for sham operates it went from 6.3 --- 0.75 to 1.9 --+ 0.43 (t(9) = 7.6, p < .01). Examination of the mean slopes of the habituation curves indicated that the more rapid habituation was not due solely to the lowered duration of initial response, as it increased in steepness in normals from 0.35 +- 0.05 to 0.41 +__ 0.06 (t(10) = 0.581, p = NS) and especially in sham operates where it increased from 0.27 --- 0.05 to 0.43 - 0.05 (t(7) = 4.59, p < .01).

A R O U S A L A N D H A B I T U A T I O N IN S Q U I R R E L F I S H

93

Effects of Operations Sham operations. Fish with sham operations were not significantly different in responsiveness to the pooled data for normal fish described in the previous paragraph. A two-way ANOVA with homogenous variance compared the responses of groups to both first and second series of stimuli (Table 2) and found them nonsignificant. This was also apparent from examination of the graphs of response in normal (Fig. 4) and shamoperated animals (Fig. 5). The sham-operated fish were nevertheless considered as the only valid controls for the effects of AC section. Anterior commissural (AC) section. The anterior commissure in Holocentrus is extremely well developed (Fig. 5A), its ventral portion being largely composed of decussating fibers from the lateral forebrain bundle

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Fx6. 4. Effects of previous experience of unilaterally presented stimuli. N o r m a l Holocentrus unilaterally p r e s e n t e d with a "light o n " stimulus at 15-s intervals to one eye (lst) s h o w e d a progressive decline in the duration of arousal r e s p o n s e s . W h e n a similar stimulus was t h e n p r e s e n t e d to the other eye (2nd) the initial r e s p o n s e was reduced and habituated more rapidly.

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Trials FIG. 5. Effects of operations on responses to unilaterally presented stimuli. A transverse section of the telencephalon of a sham-operated Holocentrus (A) reveals the intact, prominent anterior commissure (AC), the ventral portion of which is composed of decussating fibers of the lateral forebrain bundles (LFB). V, ventral; DL, dorsolateral; DD, dorsodorsal; DC, dorsoventral; DM, dorsomedial telencephalon (after Ito et al., 1980). Sham-operated fish presented with stimuli (B) first to one eye (lst), then to the other (2nd) showed a decline in duration of initial response and a more rapid habituation.

(LFB) (as described by Vanegas & Ito, 1983). Results from fish were only included if at least 80% of the AC was seen to be severed on histological examination (Fig. 6A). The results from the 16 fish which fulfilled this criterion were compared with those of sham-operated fish, with a two-way ANOVA with repeated measures on one factor. No treatment effect was apparent for responses to stimuli presented to the first eye (Table 2) where variances were homogenous. There was, however, a significant difference in responses to the "second" eye (Table 2), though here the variances for both treatment and repeated measure effects were heterogenous. Responses of AC fish to the second eye stimulated

AROUSAL AND HABITUATION IN SQUIRREL FISH B

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FIc. 6. Effects of anterior commissure section on responses to unilaterally presented stimuli. (A) Section of the anterior commissure, AC(s), caused (B) the responses to stimuli presented to the first side (lst) to become similar to those presented to the second (2nd) side. Both initial response levels and habituation were equivalent (NB slightly oblique section). c o m p a r e d to the first (Fig. 6B) did not show the rapid decline evident for s h a m - o p e r a t e d fish (Fig. 5B), though comparing the two sides still gave a significant difference using a t w o - w a y A N O V A with repeated m e a s u r e s on both factors (Table 2). The variances in this analysis were, h o w e v e r , h e t e r o g e n o u s using Bartlett's test and so testing of the initial response duration and number of trials to habituation was again considered as the m o r e a c c e p t a b l e analysis. W h e n initial r e s p o n s e magnitudes were c o m p a r e d b e t w e e n AC and sham groups to the stimulus presented to the first eye, they were equivalent (t(24) = 0.597, p = NS), though AC-sectioned fish showed larger responses than shams to the second eye (t(24) = 2.21, p < .05). Unlike shams, AC-sectioned fish showed no significant drop in duration of initial responses

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PETER R. LAMING

to the second eye compared with the first (t(15) = 1.33, p = NS). The number of trials for habituation was also equivalent when the mean for the first eye (7.69 ___ 0.84) was compared with that for the second (7.63 ___ 1.0) (t(15) = 0.06, p = NS). Also, in AC-sectioned fish the slopes of the habituation on repeated stimulation were equivalent for the first (0.27 ± 0.03) and second (0.27 ± 0.06) eyes (t(15) = -0.0011, p = NS). This was in contrast to sham-operated fish where the slope went from 0.27 _+ 0.05 to 0.43 ___ 0.05. The finding that AC-sectioned fish and sham-operated fish had equivalent responses to the first eye stimulated and the largely equivalent responses to both eyes in AC-sectioned fish allowed one side's responses to be used as controls for the other side in experiments where the AC was sectioned and one side was lesioned. Any small "side effect" within AC-sectioned fish was countered by randomizing the sequence of sides to which stimuli were given. Effects oftelencephalic lesions. Histological examination of serial sections of brains revealed the position and extent of lesions, combined with 80% section of the anterior commissure. These were classified into two major groups, those which lay in the lateral half of the telencephalon (lateral lesions) and those which lay medially (medial lesions). This latter group was further subdivided into fish with lesions anterior to a level halfway between the posterior telencephalic border and the AC (anterior medial) and those with lesions posterior to this (posterior medial). Three fish had lesions which crossed the medial/lateral boundary and results of these were not used in analysis. All 23 fish with lesions showed a significant effect of the treatment using a two-way analysis of variance with repeated measures on both factors (Table 2), though the variances of the sets of results proved heterogenous using Bartlett's test. Further examination of the results was therefore needed to determine if these effects were real. In all cases the "lesion" side represents responses to stimuli presented to the eye on the same side as the lesion, though with the assumption of complete crossover in the optic projection, this eye projects to the intact telencephalic hemisphere. Medial lesions. The use of a fine scalpel to make these lesions resulted in damage to a thin slice of tissue in a sagittal plane. The nine fish with anterior medial lesions (Fig. 7A) showed equivalent initial responses to stimuli on both the control and the operated sides (t(8) = 0.36, p = NS; Fig. 7B), though the operation extended the number of trials required for the criterion for habituation to be obtained from 5.77 ± 0.91 to 10.89 ± 1.23 (t(8) = 2.8, p < .05). The slope of the response decline thus decreased from 0.24 ± 0.05 to 0.12 ± 0.02 (t(8) = 2.38, p < .05).

AROUSAL AND HABITUATION IN SQUIRREL FISH B

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FIG. 7. Effects of anterior medial telencephalic lesions on responses to unilaterally presented stimuli. (A) Combined anterior commissural section AC(s) and anterior medial lesions (L) caused (B) a decline in the rate of habituation of responses to the eye on the unlesioned (control) side of the brain. Stimuli presented to the side ipsilateral to the lesion (lesion) habituated normally. Initial responses to both sides were equivalent.

Posterior medial lesions (Fig. 8A) caused no effect on initial responses (t(7) = 1.05, p = NS; Fig. 8). Habituation was, however, considerably delayed, increasing from a mean of 7.38 -+ 1.29 responses to 23.5 + 5.1 responses (t(7) = 3.41, p < .01). The slope of the habituation was thus reduced from 0.27 _ 0.09 to 0.09 _+ 0.02, though high variability led to this change not attaining significance (t(7) = 1.9, p = NS). Lateral lesions. The use of a relatively large scalpel, combined with aspiration of the excised tissue, caused much more telencephalic damage than was the case with medial lesions (Fig. 9A). The damage was largely dorsolateral (areas DM, DL, VL, of Murakami et al. (1983)). The lesion had no effect on the initial response duration (t(5) = 2, p = NS), though it extended the n u m b e r of trims to habituation from 6.17 +_ 0.79 to 10.33

98

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Trials FIc. 8. Effects of posterior dorsomedial telencephalic lesions on responses to unilaterally presented stimuli. (A) Posterior medial lesions (L) in comrnissural-sectioned fish caused (B) slight (nonsignificant) elevation of initial responses and a significant decrease in habituation of responses to stimuli presented to the eye contralateral to the lesion (control) compared to those presented to the side ipsilateral to the lesion (lesion).

--- 2.06 (t(5) = 3.03, p < .05; Fig. 9B). The net effect, however, was no difference in the slope of the habituation from 0.19 --+ 0.03 on the control side and 0.19 + 0.06 on the lesioned side.

DISCUSSION Behavioral Arousal in Holocentrus The initial presentations of a moving shadow stimulus to squirrel fish (H. rufus) described in this study only evoked behavioral arousal and not fright responses. These responses in Holocentrus were a brief (<5s) reduction in opercular beats and a more prolonged (5- to 10-s) decrease in pectoral fin movements, combined with erection of the spiny dorsal fin. These responses are similar to those described previously for goldfish

99

AROUSAL AND HABITUATION IN SQUIRREL FISH

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Fie. 9. Effects of dorsolateral telencephalic lesions on responses to unilaterally presented stimuli. (A) Lesions (L), often extensive, in the dorsolateral telencephalon had no effect on (B) the initial response magnitude nor on the slope of the habituation of responses.

(C. auratus) (Laming & Savage, 1980), roach (R. rutilus) (Laming, 1981), and slippery dicks (Laming & Ebbesson, 1984). The changes in position or movements of the fins rarely caused the animal to change its position in the water; indeed a moving fish often stopped moving. They did, however, appear to increase the stability of the animal's position. In this respect the extension of stabilizing fins, like the dorsal fin, can be empirically understood. The reduction in pectoral fin movements may be linked to that of reduced opercular ventilation causing less forward propulsion to compensate for the reduced propulsive effects of expired water. In most fish species previously studied, it was difficult to quantify the magnitude of the behavioral arousal response and so this had to be achieved by quantifying its peripheral physiological correlates. In Holocentrus, however, the overall behavioral response was quantifiable in

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terms of its duration, largely because of the positive change in dorsal fin erection. The onset of this response was abrupt and the subsequent relaxation also evident. Confirmation of the quantifiable nature of this response was evidenced by its smooth habituation.

Arousal Responses to a Monocular Stimulus One of the criteria adopted for arousal responses is that they not be stimulus specific. Thus, they were elicited by a tap, a moving shadow, or the onset of illumination. The last of these stimuli when presented monocularly provoked mild responses of short (2-4 s) duration. This response was equivalent whether left or right eyes received the initial series of stimuli and it habituated after a similar number of trials. There was thus no evidence that the visual system of either side was more sensitive than the other or that stimuli on one side were more likely than those on the other to have a continued effect. When stimuli were presented in a series to one side until habituation occurred, and then subsequently were presented to the other side, there was a profound effect on responses to the second side. Even with the relatively crude measure of duration as response magnitude, when stimuli were presented to the second side the response magnitude declined significantly and habituated far more rapidly. The slope of decline of responses to the second set of stimuli was also greater. This result indicated that interocular transfer of stimulus characteristics was occurring. Alternatively the two eyes may both have been receiving information from each light source despite attempts to make the stimulus monocular.

The Effects of Telencephalic Lesions The effects of anterior commissural section on the responses to stimuli presented first on one side and then on the other confirm that interocular transfer from one telencephalic lobe to the other normally occurs, and that this commissure is important in this transfer in Holocentrus. Indeed, it was also apparent that the initial responses were equivalen t as were the trials to habituation and the slope of habituation for the two sides. The implication is that each telencephalic lobe normally receives information almost exclusively from one eye in Holocentrus. This is surprising because at the level of the midbrain interocular transfer of learned motor responses has been shown to occur via the tectal commissure in other teleosts, as in the cichlid Astronotus ocellatus (Mark, 1966) and the goldfish C. auratus (Mark, Peer, & Steiner, 1973; Ingle & Campbell, 1977). Conditioned cardiac responses have, however, been unaffected by section of the tectal commissure (Yeo & Savage, 1975). Interocular transfer of a conditioned shock avoidance has also been shown to be impaired by posterior eommissural section (Ingle, 1965) as has transfer of color or shape discrimination by section of the diencephalic post-optic

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commissures (Ingle & Campbell, 1977). In the case of this commissure, tectofugal projections traverse it to overlap with the ipsilateral tectothalamic projection (Ebbesson, 1970). Tectofugal fibers in Holocentrus terminate on the ipsilateral nucleus prethalamicus (Ebbesson & Vanegas, 1976), though in goldfish this projection is bilateral (Vanegas & Ito, 1983). This nucleus itself projects to the telencephalon (Ebbesson, 1980). Thus, in Holocentrus, much of the transfer of visual information may occur at the level of the telencephalon. Certainly the anterior commissure is relatively massive in this species (Ito et al., 1980) compared to Carassius. It thus may be more involved with interhemispheric communication than occurs in other species. It should be noted, however, that in four specimens of Carassius, anterior commissural section also has been reported to prevent interocular transfer of a learned discrimination or avoidance (Savage, t969), though Ingle and Campbell (1977) report no effect on shape or color transfer in five similarly operated fish. To the author's knowledge these are the only previous reports of effects of anterior commissural section in teleosts. The minimal interocular transfer of experience of the repeated stimuli in AC-sectioned Holoeentrus found in the present work allowed one side of the brain to be used as a control for lesions on the contralateral side. In order to remove any experiential effect from consideration of the effect of lesions the side which was stimulated first relative to the lesioned side was randomized. The lesion groups were selected on the basis of numbers of animals available with acceptably discrete lesions, knowledge of the visual projection to the telencephalon in Holocentrus, and results of previous experiments with other species. Ipsilateral medial lesions of the telencephalon combined with anterior commissural section caused large deficits in habituation of arousal responses to stimuli presented to the contralateral eye. This was evident both from the number of stimuli required for habituation and from the mean slopes of the habituation process. Although the effect was most consistently found when lesions were nearer the commissure than the posterior telencephalic border, the greatest effects were in individuals with posterior lesions. One fish with a deep, posterior dorsocentral lesion (DC region of Murakami et al., 1983) took 53 trials for habituation of responses to the stimulus to the contralateral eye compared to 7 for those to the ipsilateral eye. Posterior superficial dorsomedial lesions do not have this effect. This is interesting because DC is the probable projection area for fibers from the nucleus prethalamicus, which in turn receives a massive tectofugal projection (Ito et al., 1980). Damage to DC might thus be expected to reduce the telencephalic ability to process visual information. Similar deficits in habituation of responses to the onset of illumination have recently been found in goldfish with posterior DC lesions (Rooney & Laming, 1986a). The roach (R. rutilus), however, responding to a tap

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stimulus seemed to incur greater deficits with posterior DD lesions (Laming & Hornby, 1981). The difference in these previous results had led to speculation that DM lesions may have had more effect on responses to tap stimuli because it is a region of projection for auditory and lateral line telencephalic afferents (Echteler, 1984). The equivalent projection for visual telencephalic afferents is considered to be dorsolateral (area DL) (Murakami et al., 1983). It was for this reason that that large dorsolateral telencephalic lesions were made in the present work, in an attempt to remove most of the final visual projection. The effects of this operation were that there was no significant change in initial response duration to stimuli to the contralateral eye but there was an extended number of trials required for habituation of that response. The slope of the habituation was identical for both sides. Thus habituation itself had not been affected by this lesion. The implication of this interpretation is that habituation of arousal responses to visual stimuli is not mediated by the final visual projection area of the telencephalon. One feature of the analysis of results in this study which is open to criticism is the use of a straight-line slope to describe the habituation process, which is evidently curvilinear. Its use, in the first instance, derived from the high correlations found in previous work between initial response magnitude and trials to a habituation criterion (Laming & Savage, 1980; Rooney & Laming, 1986b). In the second instance, it is necessary to differentiate effects of treatments on the magnitude of the response, and on its habituation. In the present study the mean slope of the habituation was remarkably consistent between control groups and control sides of the brain in all groups with lesions. It seems, therefore, that this empirical approach has been useful, though a more precise description of the form of the habituation curve is anticipated in the future. This may be by tests using individual or group regression lines with added higher order polynomials as suggested by Krauth (1980) and Petrinovichand Widaman (1984). Tests of this nature have not been included in the present work as (i) the results were statistically evident using less powerful techniques, and (ii) it is probable that arousal habituation curves are a composite of several habituation processes. This derives from the observation, from a large number of lesion studies, that posterior telencephalic lesions or ablations only affect the slope of the habituation curve after some four or five trials have elapsed. Thus the initial (steep) slope of the habituation curve for arousal responses may be mediated by lower brain regions, possibly even in the sensory pathways themselves. This study has described how behavioral arousal and its habituation occurs in the teleost H. rufus, a species in which the behavioral response is clearly expressed. In Holocentrus the anterior commissure is important in transmitting information about visual experience relevant to the performance of nonspecific arousal responses and to their habituation. Medial

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lesions, posterior to the commissure, especially those near DC, severely impair habituation of arousal responses. Lesions in the dorsolateral telencephalon, the ultimate site of visual projection, were not shown to have this effect. By study of a variety of species with differing degrees of telencephalic organization, the role of this brain region in arousal and its habituation is slowly being understood.

REFERENCES Aronson, L. R. (1963). The central nervous system of sharks and bony fishes with special reference to sensory and integrative mechanisms. In P. W. Gilbert (Ed.), Sharks and survival (pp. 165-241). Boston: Heath. Ebbesson, S. O. E. (1970). On the organization of central visual pathways in vertebrates. Brain Behavior and Evolution, 3, 178-194. Ebbesson, S. O. E. (1980). A visual thalamo telencephalic pathway in a teleost fish (Holocentrus rufus). Cell and Tissue Research, 213, 505-508. Echteler, S. M. (1984). Organisation of central auditory pathways in a teleost fish (Cyprinus carpio). Journal of Comparative Physiology, 156A, 267-280. Herrick, C. J. (1948). The brain of the tiger salamander (Ambystoma tigrinum). Chicago: Univ. of Chicago Press. Ingle, D. (1965). Interocular transfer in goldfish: Color easier than pattern. Science 149, 1000-1002. Ingle, D., & Campbell, A. (1977). Interocular transfer of visual discriminations in goldfish after selective commissure lesions. Journal of Comparative and Physiological Psychology, 91, (2) 327-335. Ito, H., Morita, Y., Sakamoto, N., & Ueda, S. (1980). Possibility of telencephalic visual projection in teleosts, Holocentridae. Brain Research, 197, 219-222. Krauth, J. (1980). Nonparametric analysis of response curves. Journal of Neuroscience Methods, 2, 239-259. 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, (1981). The physiological basis of alert behaviour in fish. In P. R. Laming (Ed.), Brain mechanisms of behaviour in lower vertebrates (pp. 203-224). Cambridge, England, Cambridge Univ. Press. Laming, P. R., & Ebbesson, S. O. E. (1984). Behavioural studies on the Slippery Dick, (Halichoeres bivittatus). Experientia, 40, 767-769. Laming, P. R., McKee, M. (1981). Deficits in habituation of cardiac arousal responses incurred by telencephalic ablation in goldfish (Carassius auratus) and their relation to other telencephalic functions. Journal of Comparative and Physiological Psychology, 95(3) 460-467. Laming, P. R., & Ennis, P. (1982). Habituation of fright and arousal responses in the teleosts Carassius auratus and Rutilus rutilus. Journal of Comparative and Physiological Psychology, 96(3), 460-466. Laming, P. R., & Savage, G. E. (1978). Flow changes in visceral blood vessels of the chubb (Leuciscus cephalus) during behavioural arousal. Comparative Biochemistry and Physiology, A 59(3), 291-293. Laming, P. R., & Savage, G. E. (1980). Physiological changes observed in the goldfish (Carassius auratus) during behavioural arousal and fright. Behavioural and Neural Biology, 29, 255-275. Laming, P. R., & Hornby, P. (1981). The effect of unilateral telencephalic lesions on behavioral arousal and its habituation in the roach, Rutilus rutilus. Behavioural and Neural Biology, 33, 59-65.

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Mark, R. F. (1966). The tectal commissure and interocular transfer of pattern discrimination in cichlid fish. Experimental Neurology, 16, 215-255. Mark, R. F., Peer, O., & Steiner, J. (1973). Integrative functions in the midbrain commissures in fish. Experimental Neurology, 39, 140-156. Marino-Neto, J., & Sabbatini, R. M. E. (1983). Discrete telencephalic lesions accelerate the habituation rate of behavioral arousal responses in Siamese fighting fish (Betta splendens). Brazilian Journal of Medical Biological Research, 16, 271-278. Murakami, T., Morita, Y., & Ito, H. (1983). Extrinsic and intrinsic fibre connections of the telencephalon in a teleost, Sebasticus marmoratus. Journal of Comparative Neurology, 216, 115-131. Parker, R. E. (1973). Introductory statistics for biology. London: Arnold. Petrinovich, L., & Widaman, K. F. (1984). An evaluation of statistical strategies to analyse repeated-measures data. In H. V. S. Peeke & L. Petrinovich (Eds.), Habituation, sensitisation and behavior (pp. 155-201). New York/London: Academic Press. Rooney, D., & Laming, P. R. (1984). Effects of olfactory bulb ablation on cardiac and ventilatory arousal responses and their habituation in goldfish (Carassius auratus). Behavioural and Neural Biology, 42, 120-126. Rooney, D. J., & Laming, P. R. (1986a). Localization of telencephalic regions concerned with habituation of cardiac and ventilatory responses associated with arousal in the goldfish Carassius auratus. Behavioural Neuroscience, 100(1), 45-50. Rooney, D. J., & Laming, P. R. (1986b). Cardiac and ventilatory arousal responses and their habituation in goldfish: Effects of intensity of eliciting stimulus. Physiology and Behaviour, 37, 11-14. Russell, E. M. (1967). Changes in the behaviour of Lebistes reticulatus upon repeated shadow stimulus. Animal Behaviour, 15, 574-585. Savage, G. E. (1969). Telencephalic lesions and avoidance behaviour in the goldfish (Carassius auratus). Animal Behaviour, 17, 362-373. Savage, G. E. (1971). Behavioural effects of electrical stimulation of the telencephalon of the goldfish, Carassius auratus. Animal Behaviour, 19, 661-668. Schroeder, D. M., Vanegas, H., & Ebbesson, S. O. E. (1980). The cytoarchitecture of the optic tectum of the squirrel fish, Holocentrus. Journal of Comparative Neurology, 191, 337-351. Vanegas, H., & Ebbesson, S. O. E. (1976). Telencephalic projections in two teleost species. Journal of Comparative Neurology, 165, 181-196. Vanegas, H., & Ito, H. (1983). Morphological aspects of the teleostean visual system: A review. Brain Research Reviews, 6, 117-137. Vivien, M. L., & Peryrot-Clausade, M. (1974). A comparative study of the feeding behaviour of three coral-reef fishes (Holocentridae) with special reference to the polychaetes of the reef cryptofauna as prey. Proceedings of the Second International Symposium on Coral Reefs, 1, 179-192. Yeo, C. H., & Savage, G. E. (1975). The tectal commissure and interocular transfer of a shape discrimination in the goldfish. Experimental Neurology, 49, 291-298.