Sensitization in planaria

Physiology and Behavior. Vol. I, pp. 305-308. Pergamon Press Ltd., 1966 Printed in Gruat Britain

Sensitization in Planaria' H. M A C K

B R O W N , R O B E R T E. D U S T M A N

AND EDWARD

C. B E C K

Veterans Administration Hospital and University of Utah, Salt Lake City, Utah (Received 19 D e c e m b e r 1965)

BaowN, H. M., R. E. Dus'rMA~ ANDE. C. B~CK. Sensitization in planaria. PHYSIOL.BEHAV.| (4) 305- 308, 1966.--Planaria were trained under conditions that result in little or no learnin$ in other animals (temporally separated light and shock) and with different levels of conditioned stimulus illuminance. Teraporally separated light and shock resulted in increased responsiveness which resembled altered behavior of planaria trained with simultaneous light and shock. Increased illuminaacc also caused increased responsiveaaess. The data indicated that shock (a) sensitizes planaria to light and (b) caus~ spontaneous division of the animals, resulting in shorter more responsive worms. The combined effects of these variables resulted in i ~ reslmns¢ levels which others have attributed to learning. Planaria

Dugesiatigrina

Learning

Sensitization

SHOAT PLAr~AaJA respond more frequently to a 3 sec light exposure than longer worms [3, 4, 17]. This finding is pertinent to studies that have used light response frequency as a measure of learning [1, 5, 11, 12, 16]. These studies report that successive presentations of paired light and shock result in a gradual increase in the number of responses to light, and that light paired with cathodal shock results in a greater rate of learning than light paired with anodal shock. It is conceivable that these results are the consequence of spontaneous division of the animals which results in shorter more responsive worms and enhanced light sensitivity caused by electrical shock. One way to ascertain the role that electric shock plays in the performance of planaria in learning experiments is to train planaria with light and shock that are temporally separatzd. As shown for a number of different conditioned responses in a variety of other species, a response incremmat that resembles learning often occurs under this condition when the animal is sensiti:axl by the stimuli [2, 7, 9, 13, 14, 18]. The effects of conditioned stimulus illuminance on the responsiveness of the animals is unknown. Since planaria are negatively phototaxic, illuminance could also be an important variable that partially determines planaria responsiveness in conditioning experiments. It was the purpose of the present study to obtain information on the effects of conditioned stimulus illuminance and to describe planaria behavior under experimental conditions that distinguish between learning and altered behavior due to sensitization. METHOD

Apparatus. Training receptacles consisted of two, i l in., clear acrylic semicircular troughs having inside cross-sectional areas of 0.5 cm s. The conditioned stimulus (CS) was light with uniform flux and minimal heat obtained from two tungsten ribbon f i i ~ t lamps operated at a color temperature of

2870°K. llluminance was measured with a Salford electric photometer. The illuminance at the receptacle was varied from 100-500 ft--c, by varying the distance from light source to trough. To control mechanical vibration, RC networks were used to time flash duration, shock duration, and interstimulus interval. The unconditioned stimulus (UCS) was a train of electric shocks obtained from a Grass S4-A stimulator (set t o deliver 100hz. square waves) applied through silver electrodes at the ends of the trough. Pulse duration was 5 msec and stimulus strength was 0.6 mA. Training procedure. Planaria ( Dugesia tigrina) were trained under controlled ambient light (1 ft-c.) and temperature (21°C.) conditions. The training procedure adopted minimized the necessity of touching the worms. This was a necessary precaution since light responsiveness is augmented when planaria are irritated [15]. Animals were trained individually. A five rain acclimatization period was allowed after the worm was placed in the trough. Three training procedures were used. (1) Habituation. One group (N -4) was habituated 8 days, 30 trials/day to a light with illuminance of 1130 ft-c. Habituation trials consisted of a 3 sec exposure to the CS only, A second group (N:=4) was habituated in the same manner except that the CS illuminance was 500 ft-c. (2) Conditioning. Conditioning trials consisted of a 3 sec CS (light) and a i se.c UCS (train of shocks) during the final second of light exposure. One group (N =:4) was trained to a CS illuminance of 100 ft-c,, another (N -8) to 500 ft-c. illuminance. (3) Separated CS-UCS. One group ( N - 5 ) was trained with light (500 ft-c.) and shock temporally separated by several sec (trace or backward conditioning). A 5-15 sec period was allowed to intervene between the cessation of the CS and the onset of the UCS. This variable delay was necessary in order to allow the worm to resume gliding after presentation of the CS. Thus the time between CS and UCS (5-15 sec) varied from worm to worm and trial to trial. Each animal trained with light and shock received 30 trials/day

a Data in this paper are part of a thesis, "Experimental procedures and state of nucleic acids as factors contributing to "learning" phenomena in planaria", submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the Ph.D. degree in Psychology. 305

BROWN, DUSTMAN AND BECK

306 for a period of 8 days. Animals received an equal number of head directed anodal and cathodal shocks. Two blocks of 15 trials each were administered daily; 20-25 rain intervened between blocks, lntertrial interval was approximately 30 sec. Response criteria. Trials were admirfistered only when the animal was gliding smoothly along the trough (ciliary movement). The criterion for a response to the CS was either a longitudinal contraction or a head turning movement of at least 45 degrees. Head turning movements were approximately I0 times more frequent than contractions, A contraction was the dominant response to the UCS. Qualitative and quantitative differences in the response to the UCS were observed depending on stimulating conditions. If the worm's head was oriented towards the anode, head turning movements (25 per cent) and contractions that proceeded posteriorly were seen. With the head towards the cathode, r e s p o n d s consisted exclusively of contractions that proceeded anteriorly. A response was counted only if it occurred during the initial 2 sec of the CS. Planaria responsiveness was defined as the daily per cent response (number of responses elicited by light as a percentage of the total daily trials). RESULTS

E/]ects of c s i/luminance. Figure I A shows that light responsiveness during habituation was directly related to light intensity. Worms habituated to light with 500 ft..c.

illuminance (solid circles) had a higher daily response level than worms habituated to 100 ft-c. illuminance (open circles). Statistical analysis for these data is shown in Table I. The Duncan's test shows that the mean daily per cent response o f the 100 ft-c. habituation group was 10.2 and the 500 fi-c. group was 23.7. The difference between these means (13,5) was significant (p < 0.05). Effects of temporal relationship of CS-UCS. Figure lB. when compared with Fig. IA. shows that overlapping light and shock enhanced planaria light response frequency. The performance of worms conditioned to 100 ft-c. light and overlapping electrical shock was significantly higher than that of animals habituated to 100 ft-c. light (open circles in A and B, Fig. 1). The solid circles show the same results for animals conditioned and habituated to 500 ft-c. illuminance. Results such as these have suggested to others that learning is responsible for the r ~ p o n s e increment. However, as shown in Fig. IB (triangles). light and shock separated by 5--15 sec yielded similar results. Statistical analysis (Table I. Duncan's test) showed that there was no difference in the performance of animals trained with overlapping light and shock (500 ft..c.) and light and shock temporally separated by sevea'al seconds. Thus electrical shock enhanced light responsiveness independently of the temporal relationship of the CS and UCS. DI~U~k~ION

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DAYS FIG, I, Effect of light intensity and temporal relationship of CS and UCS on the average daily per cent response of whole, mature DIanaria. The top graph shows the average daily per cent response of animals habituated to light with iilurninanoes of 100 ft-c. (open circles) and 500 ft-c. (solid circles). The bottom graph shows the responsiveness of animals conditioned with overlapping light and shock (open circles and solid circle) and animals condi0oned with light and shock temporally separated by 5-15 sec (triangles), CS illuminance for the groups represented by the solid circles and triangles was 500 ft-c,; C S iUurninance for groups represented by the open circles was 100 ft-c.

It was shown in the present study that an increase in planaria light responsivity accompanied a training condition (temporal separation of light and shock) that can be regarded as either backward or trace conditioning. If the pm'adigm is regarded as backward conditioning, the incr~t~gt res. ponsivene~ must be ascribed to sensitization [10]. If the paradigm is regarded as trace conditioning, both m i t i z a t i o n and learning are plausible explanations of the results. Howveer, trace conditioning in other animals is a long and tedious process that only occurs after a greatly extended iwiriod o f training, and rarely does the interval between the C$ and UCS extend beyond 2-3 sec. It is diff~ult to argue that planaria are able to associate temporally separated events se~lral times more rapidly than saY a cat or a dog, espechilly when the light and shock is separated by as much as 15 sec. Rather, modification of planaria behavior under the conditions of our experiments seems more appropriately described as due to the sensitizing effects of the training stimuli. Cornweli [6] also used spaced light and shock to train planaria and found increased light responsiveness. However, he used a massed trial training procedure which may have influenced these results: all training trials were administered consecutively during a single day. Comparable results were obtained in the present study from animals trained at daily intervals, ruliflg out effects due to massed trials. Apparently the ¢e~umn that conditioning (light and shock) results in a higher response level than habituation (light alone) is that planaria are temporarily sensitized by the shock used during a trai~ing trial. When their responsiveness to light is evaluated in a subsequent trial, before sensitization has decayed, inereas¢~ responsiveness is observed. Therefore irritation due to electrical stimulation appears to enhance worm respcmsivity tO light. It might be expected that the more the animal is irritated the higher the likelihood of a response to light. As noted in the pres¢~ study, h~ad~cathodal stimulation resulted in more frequent and vigorous contractions (more irritation) than bead-anodal stimulation. Consistent with this observation is the finding of Barnes and Katzung [i] that h e a d ~

SENSITIZATION IN P L A N A R I A

307 TABLE 1

ANALYSIS OF VARIANCE AND DUNCAN'S TEST OF THE AVERAGEDALLY PER CENT RESPONSEOK PLANARIAHABITUATEDAND CONDITIONEDTO DIFFERENTILLUMINANCES(100 FT-C, AND 500 FT-C.). DATA FROM THESE GROUPS WAS COMPARED WITH THAT OF ANIMALS CONDITIONED TO LIGHT (500 FT-C.) TEMPORALLYSEPARATEDFROM SHOCK BY 5-15 SEC SOURCE Between Subjects A (Groups) Subjects W/Groups Within Subjects B (Days) A × B B × Subjects W/Groups Total

SS

df

MS

F

n

20,421 14,531

4 20

5.105 727

7.03

<0.01

4,142 5,965 25,156 70,215

7 28 140 199

592 213 180

3.29 1.19

<.0.01 _-0.05

3

4

5

13.5"

20.0** 6.5

27.7** 14.2" 7.7

28.3** 14.8" 8.3 0.6

23,7

30.2

3%9

38.5

DUNCAN'S MUt.TIPLE RANGE T~ST 1

1. 2. 3. 4. 5.

100 fl-c. habituation 500 ft-c. habituation Overlapping CS--UCS (100 ft-c.) Temporally separated CS-UCS (500 ft-c.) Overlapping CS-UCS (500 ft-c.)

Mean *p <0.05

10.2

2

**p <0.01

cathodal stimulation results in higher response levels than head-directed anodal stimulation. Electrical shock had another important effect on planaria light responsivity. It was noted in our study that electrical stimulation caused spontaneous division of all the animals in the conditioning groups 3-7 days after training ~as initiated. Previous work has shown higher responsiveness in short worms [3, 4, 17]. Thus random spontaneous shortening of a group of animals during training can contribute to the average daily response increment shown in this and other studies. It is interesting to note in this regard that an experiment conducted over a short period of time (ruling out spontaneous division) reported that conditioned planaria could not be distinguished from unconditioned planaria on the basis o f their light reactivity following training [8]. The observation that the light response frequency of planaria remains essentially the same during daily habituation

trials and that their response frequency during habituation is directly related to conditioned stimulus illuminance adds emphasis to the idea expressed by others [8] that light is not a neutral stimulus for planaria. 1 h i s finding could resolve the discrepancy in t~,e literature in the n u m b e r of training trials required to " c o n d i t i o n " planaria. With high levels of conditioned stimulus illuminance, planaria could be " c o n d i t i o n e d " in approximately 150 trials [16]. However, 300--500 trials were required to " c o n d i t i o n " planaria with a low CS illuminance [5]. In conclusion we would like to suggest that modification of planaria behavior that has previously been ascribed to learning could have resulted from the unrecognized effects of animal length, CS illuminanc¢ and irritation from electrical shock. We do not maintain that planaria are incapable of learning, but do feel that plarmria learning has not been demonstrated in past studies that have not controlled these vaxi ables.

REFERENCES 1. Barnes, C. D. and B. G. Katzung. Stimulus polarity and conditioning in planaria. Science, N. Y. 141: 728-730, 1963. 2. Bemstein, A. L. Temporal factors in the formation of conditioned eyelid reactions in human subjects. J. Genet. Ps),chol. 10: 173-197, 1934. 3. Brown, H. M., R, E. Dustman and E. C. Beck. Experimental procedures that modify light response frequency of regenerated planaria. Physiol. Behav. 245-249, 1966. 4. Brown, H. M. and E. C. Beck. Does learning in planaria survive regeneration? Fed. Prec. 23: 254, 1964 5. Coming, W. C. and E. R. John. Affect of ribonuclease on retention of conditioned response in regenerated planaria, Science, N.Y. 134: 1363-1365, 1961. 6. Cornwell, P. Classical conditioning with mass trials in the planarian. Worm Runner's Dig. 2: 34--39, 1960.

7. Fitzwater, M. K. and M, N. Reisman. Comparison of forward, simultaneous, backward, and pseudo-conditioning. J, Exp. . Psychol. 44:211-214, 1952. 8. Halas, t!. S.. R. L. James and L. A. Stone. Types of responses elicited in planaria by light. J. Comp. PhysioL Psychol. 54: 302-305. 1961. 9. Kappauf, :W. E, and 14. Schlosberg, Conditioned responses in the white rat. Ill. Conditioning as a function of the length of the period of delay, d. Genet. PsychoL 50: I-5, 1959. 10. Kimble, G. A. Revised Hilgard and Marquis' Conditioning and Learning. New York: Appleton-Century-Crofts, 1961, pp. 155-160. II. McConnell, J. V., A. L. Jacobsen and D. P. Kimble. 'Fhe effects el regeneration upon retention of a conditioned response in the planarian. J. Comp. Physiol. Ps),chol. 52: 1-5, 1959.

308 12. McConnell, J. V. Memory transfer throuffh cannibalism in planarians. J. Neuropsychiat. Suppl. 1.3: 42-48, 1962. 13. Mocller, G. The CS-UCS interval in GSR conditioning. J. Exp. Psychol. 48: 162-166, 1954. 14. Sears, R. R. Effect of optic lobe ablation on the visuo-motor behavior of goldfish. J. Comp. Psychol. 14: 233-265, 1934. 15. Sgonina, K. Vergleichende UntersuchunBen uher die Scnsibilisierung und den bedington Reflex. Arch. Tierz. Psychoi. 3: 224-247, 1939.

BROWN, DUSTMAN AND BECK 16. Thompson, R. and J. McConnell. Classica! conditioning in the planarian dugesia dorotocephala. J. Comp. Physiol. Psychoi. 48: 65-68, 1955. 17. VanDeventer, J. M. and S. C. Rather. Variables affecting the frequency of response of planaria to light. J. Comp. Physiol. Psychol. 57:407-411, 1964. 18. Wolfle, Helen M. Conditioning as a function of the interval between the conditioned and the original stimulus. J. Genet. Psychol. 7: 80-103, 1932.