no differences between the classical conditioning of forebrainless and sham- ... conditioning, and sensitization procedures suggests that these groups did not.
Journal of Comparative and Physiological Psychology 1969, Vol. 68, No. 2, 193-198
CLASSICAL CONDITIONING, PSEUDOCONDITIONING, AND SENSITIZATION IN "NORMAL" AND FOREBRAINLESS GOLDFISH' J. BRUCE OVERMIER2 AND PAUL F. CURNOW University of Minnesota Forebrain-ablated and sham-operated goldfish received 10 classical conditioning trials a day for 10 days. Both groups showed acquisition when compared to pseudoconditioning and sensitization controls. However, there were no differences between the classical conditioning of forebrainless and shamoperated Ss, suggesting that the failure of forebrainless fish to learn an active instrumental avoidance response is not due to impairment of associative functions. Furthermore, lack of response magnitude differences between forebrainless and sham-operated Sa within the classical-conditioning, pseudoconditioning, and sensitization procedures suggests that these groups did not differ in arousal even following exposure to shock. However, Sa subjected to neither shocks nor forebrain ablation showed more spontaneous activity than Ss subjected to either or both.
Research concerning the function of the forebrain (telencephalon) in fish (teleosts) has typically failed to uncover any easily discernible motor or sensory deficits following forebrain ablation other than the expected loss of olfaction (Healey, 1957). However, some recent investigations into the function of the fish forebrain found that its complete removal resulted in (a) a marked impairment in the acquisition of an instrumental avoidance response, (6) a complete loss of a previously learned avoidance response, but (c) no impairment in escape responding (Hainsworth, Overmier, & Snowden, 1967; Kaplan & Aronson, 1967; Savage, 1968a). That responding during the conditioned stimulus was impaired while responding to the unconditioned stimulus was unimpaired is interpreted to imply that the deficits in avoid-
ance behavior following forebrain ablation were specific to responding to the signal for shock (the conditioned fear signal). Currently popular theories of avoidance hypothesize that avoidance behavior involves (a) the acquisition by the signal (CS) of arousing motivational and cue properties through classical conditioning, then (6) the CS cues the initiation of the instrumental avoidance response, which (c) is then reinforced by reduction of acquired motivation. Forebrain ablation could selectively impair any of these elements. Because neuroanatomical homologies exist between the fish forebrain and the mammalian limbic system (Droogleever Fortuyn, 1961) and because limbic structures have often been implicated in the acquisition of emotional reactions (McCleary & Moore, 1965), a likely hypothesis is that the observed avoidance deficit 1 This research was supported by grants from the National Institute of Health (MH-1304S) and results from an interference with the acthe Graduate School, University of Minnesota, to quisition of motivational proterties (fear) J. Bruce Overmier and from the National Science by the CS. Foundation, National Institute of Child Health Karamyan (1962) has reported that conand Human Development, and the Graduate ditioned responses can be elicited from norSchool, University of Minnesota, to the Center for Research in Human Learning, University of mal and forebrainless fish (carp) with Minnesota. Thanks are due Anna Geyer and Karl equal ease. However, the brevity of this Schwarzkopf for their aid in the conduct of this report and its lack of control comparisons experiment. leave us unsure of this equality. The 2 Requests for reprints should be sent to J. Bruce Overmier, Department of Psychology, Uni- present experiment tests this interferenceversity of Minnesota, Minneapolis, Minnesota with - the - acquisition - of - arousing - motiva 55455. tional-properties-by-the-CS hypothesis by 193
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inquiring whether or not total forebrain ablation interferes with defensive classical motor conditioning in the goldfish. METHOD Subjects Thirty-six 4-6 in. goldfish (Carassius auratus), obtained from local dealers, were divided into six groups matched on the basis of size, The fish were maintained in community tanks until operated upon, after which they were maintained in individual 3-4 gal. compartments of 15-gal. tanks, each being provided with an air stone or box filter. The aquatic medium was aged dechlorinated tap water at room temperature. The fish were fed 3-4 Noyes pellets and a small amount of Tetramin fish food after each daily session.
Apparatus The experimental chamber wag a one-gallon tank 4M> X 9 X 9 in. filled one half full of the medium. Freshly aerated and filtered medium was circulated through the tank continuously. Fish body movements were measured by means of a paddle device similar to that described by Horner, Longo, and Bitterman (1960). A clear Plexiglas paddle, 7 in. long by Vz in. high and located 2 inches above the tank bottom, was attached by a 7-in. rod directly to the exposed crystal of a phonograph cartridge clamped to the side of the tank. The fish were prevented from directly contacting the paddle lever by a 9 X 9 in. clear Plexiglas grid located % in. from the tank wall. Phonocartridge potentials proportional to paddle movements were recorded continuously using a Meterite recorder. Six phonocartridges were used during the course of the experiment; the output of
each was amplified so as to provide maximum pen deflection when recording the UR of a single large fish maintained for apparatus testing. The CS was the onset of a 40-w. red light bulb located 3 in. from the tank wall and was of 10.6sec. duration. A mirror covered the tank wall opposite from the CS light. The US was a .6-sec. train of 18-msec.-on, 82-msec.-off 30-v. dc shocks supplied by a Grass square-wave stimulator. The US was coterminous with the CS and was applied across aluminum plates covering the ends of the tank.
Operations and Verification of the Ablations All fish underwent cranial operations. One half had the forebrains removed while one half had sham operations during which superficial fatty tissue was removed but the brain left undisturbed. The procedures for the operations and verification of the ablations has been described previously by Hainsworth et al. (1967). Essentially, all forebrain tissue of the experimental fish was removed while no brain tissue of the sham-operate control fish was damaged. Figure 1 presents the minimum and maximum ablations observed in each ablation group and a sham-operated brain from a respective control group for comparison.
Procedure Pairs of groups, one forebrainless (F) and one sham-operated control (S), received one of each of the following "conditioning" treatments: (a) classical conditioning (C) in which the CS and US were presented in a paired manner for 10 trials per day using a 10-sec. CS-US onset interval and an intertrial interval (ITI) of 90-150 sec. (M = 120 sec.), (b) pseudoconditioning (P) in which
SENSITIZATION CLASSICAL PSEUDOCONDITIONING CONDITIONING
SHAM
MAXIMUM
MINIMUM FIG. 1. A representative sham-operated brain (top), maximum forebrain ablation (middle), and minimum forebrain ablation (bottom) for each treatment: classical conditioning (left), pseudoconditioning (center), and sensitization (right).
CONDITIONING IN FOREBRAINLESS GOLDFISH the 10.6-sec. CS and the ,6-sec. US were each presented alone 10 times per day in a random order using ITI of 45-75 sec. (M = 60 sec.), and (c) sensitization (S) in which only the 10.6-sec. CS was presented 10 times per day using an ITI of 90-150 sec. (M = 120 sec.). There were 10 such conditioning days. Immediately prior to the first 10 conditioning trials, 5 CS-only adaptation trials were administered. The fish were run in sets of 4-8 at a time over a period of 8 mo. Assignment of fish to the experimental groups within each set was not balanced. However, the experimental groups were not run sequentially, and equal numbers of forebrainless and sham-operated fish within a treatment were run simultaneously. The 10 days of conditioning were not consecutive, a maximum of 2 nonconsecutive days being skipped.
RESULTS Maximum pen deflections in millimeters were measured within each of two 10-sec. intervals: (a) The interval 20-10 sec. prior to CS onset provided the pre-CS spontaneous activity base line, and (b) the interval running concurrent with the CS provided the activity response to the cue. Response magnitude was defined as a magnitude of deflection during the CS minus magnitude of deflection during the pre-CS base-line period. A conditioned response (CR) was defined as being a trial during which response magnitude exceeded 2 mm. Mean daily spontaneous activity, mean daily response magnitude, and mean daily percentage CRs were subjected to analyses of variance. Due to apparatus failures, data was missing on 63 of the 3,600 conditioning trials. Ten of these occurred on Day 2 for a single S of the forebrainless classical-conditioning group and 10 occurred on Day 9 for a single .50; Operation X Procedure: F = 1.99, df = 2/28, p > .10). Critical difference comparisons of the six group means revealed that the classical-conditioning groups differed significantly from all other groups (t > 4.15, df = 28,p< .001)3 but not from each other (t = .60, df = 28, p>.50). Mean daily percentage CRs for all groups is presented in Figure 3. As would be expected based upon the response magnitude data, the classical-conditioning procedure produced significant acquisition of CRs (Procedure: F = 31.176, df = 2/28, p < .01) and the sham classical-conditioning and the forebrainless classical-conditioning groups did not differ from each other (t = .76, df = 28, p > .20). Surprisingly, both the forebrainless-pseudoconditioning and forebrainless-sensitization groups also showed a small increase in percentage CRs over days, while their re3
All t tests and their degrees of freedom are based upon the analyses of variance. The p values are based upon two-tailed tests.
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5
10
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DAYS
FIG. 3. Mean daily percentage CRs (response magnitude > 2 mm.) of the sham (S) and forebrainless (F) fish for each treatment on all conditioning days. (Note that if the CR criterion were response magnitude > 0 mm., the expected percentage CRs in the absence of learning would be 50%.)
spective sham-operated control groups did not (Operation: F = 4.076, dj = 1/28, p < .05; Operation X Procedure X Days: F — 4.44, df = 18/268, p < .01). Within the pseudoconditioning procedure, the increase was enough to overcome the initial depression, and percentage CRs of the forebrain-ablated group eventually surpassed that of the sham-operated group (Days: F = 11.084, df = 9/268, p < .01; Days X Operation: F = 2.06, df = 9/268, p < .05; Days X Procedures: F = 4.44, df = 18/268, p < .01). Within the sensitization procedure, the initial depression was much larger and percentage CRs for the forebrain-ablated group remained below that of its sham-operated control throughout the 10 days of conditioning. Critical difference pairwise comparisons of group means of percentage CRs revealed that the two classical-conditioning groups were significantly different from all other groups (t > 2.80, df = 28, p < .01) and did not differ from each other (t = .76, df = 28, p > .20). In addition, the percentage CRs of sham-sensitization group was significantly greater than that of the forebrainless-sensitization group (t = 3.03, df = 28, p < .01). No other comparisons reached accepted significance levels (all ps > .10). That one control procedure should result in significantly more CRs than another is problematical. However, that the "CRs" of the sham-sensitization group were nonassociative is suggested by
the observation that their frequency did not increase with "training" (see Figure 3). These latter differences between control groups appear to be related to the lower level of spontaneous unconditioned activity of the shocked and/or forebrain-ablated Ss both prior to and during the CS. Mean daily spontaneous activity and the activity during the CS (cued activity) of the sensitization groups is presented in Figure 4 along with indications of the spontaneous activity of all other groups. Spontaneous activity of forebrain-ablated groups tended to be somewhat less than that of their respective sham-operated control groups within all three experimental procedures (Operations: F = 16.03, df = 1/28, p < .01) and tended to increase over days for all groups (Days: F = 5.45, df = 9/268, p < .01). However, the sham-sensitization group showed distinctly more spontaneous activity than all other groups including the forebrainless-sensitization group (Procedure: F = 16.30, df = 2/28, p < .01; Procedure X Operation: F = 3.35, df = 2/28, p < .05; and t > 3.40, df = 28, p < .01 for all relevant comparisons). The high level of activity of the sham-sensitization group prior to CS onset clearly continues throughout the CS period as well,
FIG. 4. Mean daily spontaneous activity (left) and activity during the CS (right) of the forebrainless (F) and sham-operated (S) sensitization (S) groups. (The shaded area in the left panel indicates the range of the mean daily spontaneous activity of the four classical-conditioning and pseudoconditioning groups.)
CONDITIONING IN FOEEBRAINLESS GOLDFISH
and is greater than the CS activity of the forebrainless-sensitization group (t — 4.21, df = 28, p < .01). Because the activity level of the sham-sensitization subjects did not increase over days (see Figure 4), the spontaneous and cued activity differences observed between the sensitization groups cannot be attributed to differences in response learning or to differences in acquired motivation. DISCUSSION No deficits in classical conditioning were observed following forebrain ablation. The conditioning of the forebrainless goldfish was equal to that of the sham-operated controls. The hypothesis that deficits observed in the avoidance behavior of forebrainless fish are due to impairment of the acquisition associative relations between the CS and US is not supported by these results. Other attempts to account for the deficit in avoidance behavior have suggested that memory decay is much more rapid in the forebrainless fish than in normal fish (Savage, 1968b). We feel that attempts to attribute the avoidance deficit to memory decay and interference with short- to longterm memory transfer will have difficulty in accounting for the data. This is because the acquisition of a classically conditioned response must also be dependent upon short- and long-term memory mechanisms. Alternatively, Kaplan and Aronson (1967) have hypothesized that the teleost forebrain exercises a general nonspecific energizing influence over lower brain centers and that it is "concerned with facilitating the acquisition and performance of conditioned responses by contributing to the general arousal level of the organism [p. 447]." The present data are difficult to interpret with respect to this hypothesis. The sham and forebrainless Ss within each of the classical-conditioning, pseudoconditioning, and sensitization groups did not differ from each other in response magnitude. Therefore, if response magnitude is a valid index of arousal level, their hypothesis also is not supported by the present
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experiment. Indeed, the forebrainless Ss tended, if anything, to condition slightly better than the controls as measured by percentage CRs. However, one could reasonably argue that response magnitude is only an index of stimulus specific arousal (or arousability), and that while the Ss do not differ in stimulus specific arousal, they may well differ in general arousal perhaps as indexed by spontaneous activity. The sham and forebrainless Ss within the classical-conditioning and pseudoconditioning procedures, wherein shocks were presented, did not differ with respect to spontaneous activity. However, the sham and forebrainless
