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Tail pinch and handling facilitate feeding behavior in Aplysia
BEHAVIORAL AND NEURAL BIOLOGY 32, 126-132 (1981)
BRIEF REPORT Tail Pinch and Handling Facilitate Feeding Behavior in Aplysia IRVING K U P F E R M A N N AND K L A U D I U S Z R . WEISS
Division of Neurobiology and Behavior, Departments of Anatomy, Physiology, and Psychiatry, College of Physicians and Surgeons and School of Dental and Oral Surgery, Columbia University and The New York State Psychiatric Institute, New York, New York 10032 This study investigated whether nonfood stimuli can facilitate feeding behavior in Aplysia. A tail-pinch stimulus resulted in a reduction of the latency of several indices of feeding behavior evoked by seaweed presented 4 min after the tail pinch. Handling similarly reduced, subsequent feeding latencies. The effect of handling was found to be time dependent. Compared to nonhandled animals, animals tested immediately after handling showed increased latencies to respond to food, whereas animals tested 4 min after handling showed significantly reduced feeding latencies. The findings are consistent with a model in which a variety of stimuli can converge on a food-arousal system, which then exerts divergent actions on several motor systems.
Under appropriate deprivation conditions exposure of animals to appetitive stimuli typically induces an arousal state characterized by enhanced speed and strength of responding. We have been studying the neural and behavioral characteristics of the arousal state produced by food stimuli presented to the marine mollusc Aplysia californica. When food-deprived Aplysia are briefly exposed to a food stimulus, they exhibit a food-arousal response, characterized by an increased speed and strength of biting responses. As well as specific effects on feeding behavior, food stimuli have more general effects and can alter a number of reflexes not directly related to feeding (Fentress, 1973; Kupfermann, 1974; Advokat, 1980). Thus, one can hypothesize an arousal system that is triggered by food stimuli, and which then exerts divergent effects on a variety of reflexes. In higher animals, evidence for a central arousal state includes the observation that specific behaviors can be affected by a variety of nonspecific stimuli that converge on the putative arousal system. For instance, feeding behavior in rats can be facilitated not only by 126 0163-1047/81/050126-07502.00/0 Copyright© 1981 by AcademicPress, Inc. All rights of reproductionin any form reserved.
FEEDING BEHAVIOR IN
Aplysia
127
food stimuli but also by nonfood stimuli such as loud sounds (Kupfermann, 1964) or tail pinch (Antelman, Szechtman, Chin, & Fisher, 1975). We now show that in Aplysia, nonfood stimuli such as tail pinch and handling can facilitate or suppress feeding behavior, and that the direction of the effect is time dependent. These data are consistent with the hypothesis that feeding in Aplysia is under the control of a central arousal state. Animals for these experiments consisted of a pool of 20 A. californica weighing 125-180. They were maintained in individual cages in cooled seawater tanks. The animals were kept on a 12-hr light-dark cycle, and were tested within 1 hr after the onset of the light period. Approximately 3 hr before the onset of the dark period, the animals were given a quarter of a sheet (approximately ½ g) of dried seaweed (Laver brand), and more pieces were periodically added to their cages until the animals ceased to feed. Before every daily test period, each animal was observed to determine if it exhibited any signs of spontaneous arousal, in which case it was not used for that day. Spontaneous arousal was judged to be present when the animal showed either walking behavior or head waving. Both these behaviors are correlated with greatly enhanced feeding responses (Kupfermann, 1974; Preston & Lee, 1973), and since the purpose of these experiments was to determine if nonfood stimuli could enhance feeding, we felt that the elimination of animals that already gave signs of a state of enhanced feeding responses would decrease the variance of the data and simplify their interpretation. Typically 10 to 20% of the animals were not run on a given day because they exhibited signs of arousal. In order to test the latency of feeding behavior, animals were contacted with a piece of seaweed measuring 3 x 3 mm. The seaweed was first touched to the top of the head. As soon as the animal began to lift its head, the seaweed was touched to its lips. Usually, a few seconds after the lips were contacted, the animal lifted its head up fully, and assumed the characteristic head-waving position. Contact with the food was maintained until a biting response occurred. A second experimenter measured the time from the initial touch of food to the top of the head to: (1) contact with lips; (2) assumption of the head-up posture, and (3) occurrence of a biting response. For each experiment, statistical analysis was done on three parameters of feeding behavior: (1) the time from initial food contact (to the top of the head) to assumption of the head up position, a measure of the latency of the appetitive phase of feeding; (2) the time from contact to the lips to the biting response, a measure of the latency of the consummatory phase of feeding; (3) the time from initial food contact to the biting response, a measure that indicates the overall latency to bite and reflects both appetitive and consummatory phases of feeding. Unless otherwise noted, all individual statistical comparisons were done with two-tailed t tests.
128
K U P F E R M A N N AND WEISS
The test stimuli in these experiments consisted of either a tail pinch, or a complex handling stimulus. Tail-pinch stimuli were applied by grasping the tail of the animal between the tips of a broad-tipped forceps (Misdom Frank, Russian forceps, 15 cm, 0.6 cm tip diameter) and squeezing for 5 sec. Sufficient pressure was applied to elicit locomotion, without, however, triggering inking. A crude estimate indicated that approximately 2 kg of pressure was applied to the middle of the forceps. To provide handling stimuli, the animal was lifted from the substrate into the air. It was then turned on its back and held for 5 sec. Finally, the animal was replaced upside down in its own cage or in a clean cage. The animals then spontaneously righted themselves, within 30 sec, and usually locomoted for about a minute. Tail pinch. The effect of tail pinch on feeding was examined since this stimulus provokes rapid locomotion in Aplysia and gives the impression of being a potent general arousing stimulus. Furthermore, tail pinch can facilitate feeding behavior in mammals (Antelman et al., 1975). Pilot experiments indicated that the arousing effect of tail pinch may not be evident until several minutes after the pinch. Therefore in this experiment animals (N = 8) were tail pinched, and were then tested 4 min later for the latency to respond to food. Their latencies were compared to that of nonpinched animals (N = 8) that were tested at the same time of day. Figure 1A shows that, compared to the nonpinched controls, the pinched animals took a significantly shorter time for each of the three measured parameters: time to get into the head up position, time from lip contact to bite, and total time to bite (p < .01 for all cases). For each A 60 OT
TE
FAL
~D uP
BITE
HEADUP C
G 5O
4O
~ 3o
1"
v- 2o I
io 0
c
CP
cP
i L
CL
CL
FIG. 1. Effect of tail pinch and handling (lift) on feeding behavior of Aplysia. (A) Effect of a tail pinch on the time from initial contact with food to the initiation of a biting response (total), on the time to initiate the consumatory response (lip bite), and on the time to initiate appetitive behavior (head up). A tail pinch significantly reduced the latency on all three measures (p < .01). (B) Effect of handling on the latencies o f the same measures of feeding as in A. Handling significantly reduced the total time (p < .01), lip-bite time (p < .01), and the head-up time (p < .05). C, controls; P, pinched; L, lifted. Bar graphs indicate means -+ SE.
F E E D I N G B E H A V I O R IN
129
Aptysia
measure, the mean of the nonpinched animals was approximately twice as long as for the pinched animals. Handling. One possible explanation for the food-arousal effects of tail pinch is that the tail-pinch stimulus triggers walking behavior, which in turn increases the contact of the animal with traces of food particles and other arousing chemostimuli that might be adhering to the floor of their home cage. To explore this possibility we wished to test animals in a freshly cleaned cage in which they had not been living. Since this was not possible to do without handling the animals, we investigated whether handling per se could potentiate feeding behavior. Two additional reasons why we studied the effects of handling were that we wished to determine if stimuli other than tail pinch could facilitate feeding and we felt that compared to tail pinch, handling was a somewhat more reproducible stimulus. In the first experiment, animals were tested either 4 rain after handling (lift) or without handling (control). Figure 1B illustrates that handled animals, compared to controls, showed reduced total time to bite, lip to bite, and head up (p's < .01, .01, .05). Figure 2A illustrates the results of a replication of this experiment, in which the tester was blind, that is, was not told which animals had been handled. The results of this experiment were similar to the previous nonblind experiment: handled animals responded with a shorter latency as indicated by all three measures of feeding behavior (p < .01). Having established that handling could serve to facilitate feeding behavior, we could then control for contact with food stimuli in the cage. In B
A UP-BITE 3
70
HEA
UP
TOq
LIP- BtTE
4EADUP
b
c
6O 5O
4o 50 2O LO
C FIG. 2. Effect of handling Blind control. Handled animals m e a s u r e s . (B) Control for food latencies for total (p < .01) and indicate m e a n s _+ SE.
CL
CL
C
CL
CL
on feeding behavior of Aplysia--control procedures. (A) displayed significantly shorter latencies (p < .01) on all three stimulation. Handled animals displayed significantly shorter lip-bite latencies (p < .01), C, controls; L, lifted. Bar graphs
130
KUPFERMANN AND WEISS
this experiment, the handled animals were returned to and tested in a clean cage, instead of their home cage. Figure 2B illustrates that handled animals exhibited reduced total-bite latencies, and lip-bite latencies even when returned to a clean cage (p < .01, for both cases). Thus, it does not appear as if handling facilitates feeding because of contact with food stimuli adhering to the cage. Preliminary experiments suggested that the facilitatory effects of handling and tail pinch are seen only after a delay following the experimental manipulation. The immediate effect appeared to be an inhibition of feeding behavior. To determine whether the effects of handling were time dependent, animals were tested for feeding under three conditions: (1) nonhandled, (2) immediately after handling, and (3) 4 min after handling. The animals in the immediate group were tested as soon as they righted themselves when they were returned to their cage (almost always within 30 sec). One test trial per animal was run each day, for 3 days, and each animal was tested under every condition. The order of conditions was balanced to eliminate possible order effects. One-way, repeated measures analysis of variance indicated a significant main effect of the test conditions for each of the measures of feeding behavior. F ratios for total, lip-bite and head-up measures were 27, 15, and 14, p < .001 in each case, df = 2,22. All individual comparisons within each measure were significant (p < .05 in each case, t tests of correlated means, one tailed). Figure 3 illustrates that compared to latencies of nonhandled animals, feeding latencies were significantly decreased in the handled group that was tested with the 4-min delay; whereas feeding latencies were significantly increased in the handled group that was tested immediately after handling. TOTAL
L]
P - BITE
b
HEAD UP C
ioo
!191 8O
i~i;
m Iit'l
F-
4o
20
!~
4 0 4C0 4C0 Fro. 3. Time d e p e n d e n c e of the effect of handling on feeding behavior. All three measures showed a statistically significant increase oflatencies immediately after handling and a statistically significant decrease of latencies 4 min after handling. Abbreviations: C, controls; O, tested with no delay; 4, tested with a 4-rain delay.
FEEDING BEHAVIOR IN Aplysia
131
The present results demonstrate that two types of complex nonfood stimuli can facilitate feeding behavior in Aplysia. Tail pinch and handling both result in a decreased latency to exhibit appetitive behavior and the consummatory biting response. Study of the effects of handling revealed that the phase of facilitation seen at 4 min after handling was preceded by a phase of suppression of feeding. Previous findings in Aplysia have indicated that aversive stimuli can facilitate withdrawal responses and inhibit appetitive (feeding) responses, whereas an appetitive stimulus (food) can have the opposite effect (Advokat, 1980; Hawkins & Advokat, 1977; Kupfermann & Pinsker, 1968). The present findings indicate that a mildly aversive stimulus can facilitate as well as inhibit an appetitive response. A variety of behavioral schema can be used to explain the effects of a test stimulus on subsequent feeding behavior. For example, it is possible that test stimuli elicit behavioral responses that can either compete with or facilitate feeding. Our observations, however, indicated that 4 min after tail pinch or handling, the animals were quiescent and were behaviorally indistinguishable from nonstimulated animals. Alternatively, as suggested by Davis, Mpitsos, Pinneo, & Ram (1977) certain test stimuli may inhibit or facilitate subsequent responses, independent of whether or not the test stimulus elicits a response. This explanation of the present results is awkward in view of the fact that the same stimulus could produce opposite behavioral effects as a function of time. Thus, our results indicate that the effects of a given stimulus on subsequent behavior cannot be described simply as facilitatory or inhibitory. In these experiments time was a critical covariable that determined the nature of the effects of handling. We prefer to deal with the present data within the framework of the concept of behavioral state--that is, a set of internal variables that determine the direction and intensity of behavioral responses. Within this framework, the time-dependent effects of handling could be due to either of two factors. First it is possible that handling elicits a high state of arousal that decays with time. The behavioral effects of arousal may be U shaped, as has been suggested for arousal in higher animals (Hebb, 1955). Thus, very high levels of arousal may interfere with behavior, whereas lower levels facilitate behavior. Alternatively, the stimuli provided by a condition such as handling may elicit two central excitatory states, one inhibiting feeding, the other facilitating feeding. The results obtained in the present experiments would be observed if the inhibitory state were initially stronger, but decayed faster than the excitatory state. More definitive answers to this question may be obtained by studying the neural substrates of these behavioral modifications. Previous work on Aplysia indicated that arousal of the feeding system in animals exposed to food stimuli is mediated by a pair of giant serotonergic neurons (Weiss & Kupfermann, 1977). The serotonergic neurons, in turn appear to be con-
132
KUPFERMANN AND WEISS
trolled by a central arousal system, that may be composed of histaminergic and other neurons in the cerebral ganglion (Weiss, Shapiro, Koester, & Kupfermann, 1978). The central arousal system appears to have effects on multiple behaviors including feeding (Weiss & Kupfermann, 1977) as well as cardiovascular responses (Dieringer, Koester, & Weiss, 1978). The current findings suggest that the inputs from a variety of different stimuli can converge on this arousal system, which in turn has divergent effects on several different response systems. This model may be susceptible to direct test by cellular studies of the nervous system of Aplysia.
ACKNOWLEDGMENTS We thank Drs. T. Carew, E. R. Kandel, and J. Koester for comments on an earlier draft, and K. Hilten for the figures. This study was supported in part by Grant NS 12492, Grant 1 P01 GM23540 Scope D and by a Research Scientist Development Award, 1 K02 MH00304.
REFERENCES Advokat, C. (1980). Modulation of defensive reflexes in Aplysia californica by appetitive stimulation. Behavioral and Neural Biology 28, 253-265. Antelman, S. M., Szechtman, H., Chin, P., & Fisher, A. E. (1975). Tail pinch-induced eating, gnawing and licking behavior in rats: Dependence on the nigrostriatal dopamine system. Brain Research 99, 319-337. Davis, W. J., Mpitsos, G. J., Pinneo, J. M., & Ram, J. L. (1977). Modification of the behavioral hierarchy of Pleurobranchea I. Satiation and feeding motivation. Journal of Comparative Physiology 117, 99-125. Dieringer, N., Koester, J., & Weiss, K. R. (1978). Adaptive changes in heart rate of Aplysia californica. Journal of Comparative Physiology 123, 11-21. Fentress, J. C. (1973). Specific and nonspecific factors in the causation of behavior. In P. P. G. Bateson & P. H. Klopfer (Eds.), Perspectives in Ethology, pp. 155-224. New York/London: Plenum. Hawkins, R. D., & Advokat, C. (1977). Effects of behavioral state on the gill-withdrawal reflex in Aplysia californica. Society for Neuroscience Symposia 3, 16-32. Hebb, D. O. (1955). Drives and the CNS (conceptual nervous system). Psychological Review 62, 243-254. Kupfermann, I. (1964). Eating behavior induced by sounds. Nature (London) 201, 324. Kupfermann, I. (1974). Feeding behavior in Aplysia: A simple system for the study of motivation. Behavioral Biology 10, 1-26. Kupfermann, I., & Pinsker, H. (1968). A behavioral modification of the feeding reflex in Aplysia californiea. Communications in Behavioral Biology Part A 2, 13-17. Preston, R. J., & Lee, R. M. (1973). Feeding behavior in Aplysia californica: Role of chemical and tactile stimuli. Journal of Comparative Physiological Psychology 82, 368-381. Weiss, K. R., & Kupfermann, I. (1977). Serotonergic neuronal activity and arousal of feeding in Aplysia ealifornica. Society for Neuroseience Symposia 3, 66-89. Weiss, K. R., Shapiro, E., Koester, J., & Kupfermann, I. (1978). A histaminergic synaptic potential produced by a voltage-dependent apparent decrease of conductance in the metacerebral cell of Aplysia. Society for Neuroscience Abstracts 4, 210.
BRIEF REPORT Tail Pinch and Handling Facilitate Feeding Behavior in Aplysia IRVING K U P F E R M A N N AND K L A U D I U S Z R . WEISS
Division of Neurobiology and Behavior, Departments of Anatomy, Physiology, and Psychiatry, College of Physicians and Surgeons and School of Dental and Oral Surgery, Columbia University and The New York State Psychiatric Institute, New York, New York 10032 This study investigated whether nonfood stimuli can facilitate feeding behavior in Aplysia. A tail-pinch stimulus resulted in a reduction of the latency of several indices of feeding behavior evoked by seaweed presented 4 min after the tail pinch. Handling similarly reduced, subsequent feeding latencies. The effect of handling was found to be time dependent. Compared to nonhandled animals, animals tested immediately after handling showed increased latencies to respond to food, whereas animals tested 4 min after handling showed significantly reduced feeding latencies. The findings are consistent with a model in which a variety of stimuli can converge on a food-arousal system, which then exerts divergent actions on several motor systems.
Under appropriate deprivation conditions exposure of animals to appetitive stimuli typically induces an arousal state characterized by enhanced speed and strength of responding. We have been studying the neural and behavioral characteristics of the arousal state produced by food stimuli presented to the marine mollusc Aplysia californica. When food-deprived Aplysia are briefly exposed to a food stimulus, they exhibit a food-arousal response, characterized by an increased speed and strength of biting responses. As well as specific effects on feeding behavior, food stimuli have more general effects and can alter a number of reflexes not directly related to feeding (Fentress, 1973; Kupfermann, 1974; Advokat, 1980). Thus, one can hypothesize an arousal system that is triggered by food stimuli, and which then exerts divergent effects on a variety of reflexes. In higher animals, evidence for a central arousal state includes the observation that specific behaviors can be affected by a variety of nonspecific stimuli that converge on the putative arousal system. For instance, feeding behavior in rats can be facilitated not only by 126 0163-1047/81/050126-07502.00/0 Copyright© 1981 by AcademicPress, Inc. All rights of reproductionin any form reserved.
FEEDING BEHAVIOR IN
Aplysia
127
food stimuli but also by nonfood stimuli such as loud sounds (Kupfermann, 1964) or tail pinch (Antelman, Szechtman, Chin, & Fisher, 1975). We now show that in Aplysia, nonfood stimuli such as tail pinch and handling can facilitate or suppress feeding behavior, and that the direction of the effect is time dependent. These data are consistent with the hypothesis that feeding in Aplysia is under the control of a central arousal state. Animals for these experiments consisted of a pool of 20 A. californica weighing 125-180. They were maintained in individual cages in cooled seawater tanks. The animals were kept on a 12-hr light-dark cycle, and were tested within 1 hr after the onset of the light period. Approximately 3 hr before the onset of the dark period, the animals were given a quarter of a sheet (approximately ½ g) of dried seaweed (Laver brand), and more pieces were periodically added to their cages until the animals ceased to feed. Before every daily test period, each animal was observed to determine if it exhibited any signs of spontaneous arousal, in which case it was not used for that day. Spontaneous arousal was judged to be present when the animal showed either walking behavior or head waving. Both these behaviors are correlated with greatly enhanced feeding responses (Kupfermann, 1974; Preston & Lee, 1973), and since the purpose of these experiments was to determine if nonfood stimuli could enhance feeding, we felt that the elimination of animals that already gave signs of a state of enhanced feeding responses would decrease the variance of the data and simplify their interpretation. Typically 10 to 20% of the animals were not run on a given day because they exhibited signs of arousal. In order to test the latency of feeding behavior, animals were contacted with a piece of seaweed measuring 3 x 3 mm. The seaweed was first touched to the top of the head. As soon as the animal began to lift its head, the seaweed was touched to its lips. Usually, a few seconds after the lips were contacted, the animal lifted its head up fully, and assumed the characteristic head-waving position. Contact with the food was maintained until a biting response occurred. A second experimenter measured the time from the initial touch of food to the top of the head to: (1) contact with lips; (2) assumption of the head-up posture, and (3) occurrence of a biting response. For each experiment, statistical analysis was done on three parameters of feeding behavior: (1) the time from initial food contact (to the top of the head) to assumption of the head up position, a measure of the latency of the appetitive phase of feeding; (2) the time from contact to the lips to the biting response, a measure of the latency of the consummatory phase of feeding; (3) the time from initial food contact to the biting response, a measure that indicates the overall latency to bite and reflects both appetitive and consummatory phases of feeding. Unless otherwise noted, all individual statistical comparisons were done with two-tailed t tests.
128
K U P F E R M A N N AND WEISS
The test stimuli in these experiments consisted of either a tail pinch, or a complex handling stimulus. Tail-pinch stimuli were applied by grasping the tail of the animal between the tips of a broad-tipped forceps (Misdom Frank, Russian forceps, 15 cm, 0.6 cm tip diameter) and squeezing for 5 sec. Sufficient pressure was applied to elicit locomotion, without, however, triggering inking. A crude estimate indicated that approximately 2 kg of pressure was applied to the middle of the forceps. To provide handling stimuli, the animal was lifted from the substrate into the air. It was then turned on its back and held for 5 sec. Finally, the animal was replaced upside down in its own cage or in a clean cage. The animals then spontaneously righted themselves, within 30 sec, and usually locomoted for about a minute. Tail pinch. The effect of tail pinch on feeding was examined since this stimulus provokes rapid locomotion in Aplysia and gives the impression of being a potent general arousing stimulus. Furthermore, tail pinch can facilitate feeding behavior in mammals (Antelman et al., 1975). Pilot experiments indicated that the arousing effect of tail pinch may not be evident until several minutes after the pinch. Therefore in this experiment animals (N = 8) were tail pinched, and were then tested 4 min later for the latency to respond to food. Their latencies were compared to that of nonpinched animals (N = 8) that were tested at the same time of day. Figure 1A shows that, compared to the nonpinched controls, the pinched animals took a significantly shorter time for each of the three measured parameters: time to get into the head up position, time from lip contact to bite, and total time to bite (p < .01 for all cases). For each A 60 OT
TE
FAL
~D uP
BITE
HEADUP C
G 5O
4O
~ 3o
1"
v- 2o I
io 0
c
CP
cP
i L
CL
CL
FIG. 1. Effect of tail pinch and handling (lift) on feeding behavior of Aplysia. (A) Effect of a tail pinch on the time from initial contact with food to the initiation of a biting response (total), on the time to initiate the consumatory response (lip bite), and on the time to initiate appetitive behavior (head up). A tail pinch significantly reduced the latency on all three measures (p < .01). (B) Effect of handling on the latencies o f the same measures of feeding as in A. Handling significantly reduced the total time (p < .01), lip-bite time (p < .01), and the head-up time (p < .05). C, controls; P, pinched; L, lifted. Bar graphs indicate means -+ SE.
F E E D I N G B E H A V I O R IN
129
Aptysia
measure, the mean of the nonpinched animals was approximately twice as long as for the pinched animals. Handling. One possible explanation for the food-arousal effects of tail pinch is that the tail-pinch stimulus triggers walking behavior, which in turn increases the contact of the animal with traces of food particles and other arousing chemostimuli that might be adhering to the floor of their home cage. To explore this possibility we wished to test animals in a freshly cleaned cage in which they had not been living. Since this was not possible to do without handling the animals, we investigated whether handling per se could potentiate feeding behavior. Two additional reasons why we studied the effects of handling were that we wished to determine if stimuli other than tail pinch could facilitate feeding and we felt that compared to tail pinch, handling was a somewhat more reproducible stimulus. In the first experiment, animals were tested either 4 rain after handling (lift) or without handling (control). Figure 1B illustrates that handled animals, compared to controls, showed reduced total time to bite, lip to bite, and head up (p's < .01, .01, .05). Figure 2A illustrates the results of a replication of this experiment, in which the tester was blind, that is, was not told which animals had been handled. The results of this experiment were similar to the previous nonblind experiment: handled animals responded with a shorter latency as indicated by all three measures of feeding behavior (p < .01). Having established that handling could serve to facilitate feeding behavior, we could then control for contact with food stimuli in the cage. In B
A UP-BITE 3
70
HEA
UP
TOq
LIP- BtTE
4EADUP
b
c
6O 5O
4o 50 2O LO
C FIG. 2. Effect of handling Blind control. Handled animals m e a s u r e s . (B) Control for food latencies for total (p < .01) and indicate m e a n s _+ SE.
CL
CL
C
CL
CL
on feeding behavior of Aplysia--control procedures. (A) displayed significantly shorter latencies (p < .01) on all three stimulation. Handled animals displayed significantly shorter lip-bite latencies (p < .01), C, controls; L, lifted. Bar graphs
130
KUPFERMANN AND WEISS
this experiment, the handled animals were returned to and tested in a clean cage, instead of their home cage. Figure 2B illustrates that handled animals exhibited reduced total-bite latencies, and lip-bite latencies even when returned to a clean cage (p < .01, for both cases). Thus, it does not appear as if handling facilitates feeding because of contact with food stimuli adhering to the cage. Preliminary experiments suggested that the facilitatory effects of handling and tail pinch are seen only after a delay following the experimental manipulation. The immediate effect appeared to be an inhibition of feeding behavior. To determine whether the effects of handling were time dependent, animals were tested for feeding under three conditions: (1) nonhandled, (2) immediately after handling, and (3) 4 min after handling. The animals in the immediate group were tested as soon as they righted themselves when they were returned to their cage (almost always within 30 sec). One test trial per animal was run each day, for 3 days, and each animal was tested under every condition. The order of conditions was balanced to eliminate possible order effects. One-way, repeated measures analysis of variance indicated a significant main effect of the test conditions for each of the measures of feeding behavior. F ratios for total, lip-bite and head-up measures were 27, 15, and 14, p < .001 in each case, df = 2,22. All individual comparisons within each measure were significant (p < .05 in each case, t tests of correlated means, one tailed). Figure 3 illustrates that compared to latencies of nonhandled animals, feeding latencies were significantly decreased in the handled group that was tested with the 4-min delay; whereas feeding latencies were significantly increased in the handled group that was tested immediately after handling. TOTAL
L]
P - BITE
b
HEAD UP C
ioo
!191 8O
i~i;
m Iit'l
F-
4o
20
!~
4 0 4C0 4C0 Fro. 3. Time d e p e n d e n c e of the effect of handling on feeding behavior. All three measures showed a statistically significant increase oflatencies immediately after handling and a statistically significant decrease of latencies 4 min after handling. Abbreviations: C, controls; O, tested with no delay; 4, tested with a 4-rain delay.
FEEDING BEHAVIOR IN Aplysia
131
The present results demonstrate that two types of complex nonfood stimuli can facilitate feeding behavior in Aplysia. Tail pinch and handling both result in a decreased latency to exhibit appetitive behavior and the consummatory biting response. Study of the effects of handling revealed that the phase of facilitation seen at 4 min after handling was preceded by a phase of suppression of feeding. Previous findings in Aplysia have indicated that aversive stimuli can facilitate withdrawal responses and inhibit appetitive (feeding) responses, whereas an appetitive stimulus (food) can have the opposite effect (Advokat, 1980; Hawkins & Advokat, 1977; Kupfermann & Pinsker, 1968). The present findings indicate that a mildly aversive stimulus can facilitate as well as inhibit an appetitive response. A variety of behavioral schema can be used to explain the effects of a test stimulus on subsequent feeding behavior. For example, it is possible that test stimuli elicit behavioral responses that can either compete with or facilitate feeding. Our observations, however, indicated that 4 min after tail pinch or handling, the animals were quiescent and were behaviorally indistinguishable from nonstimulated animals. Alternatively, as suggested by Davis, Mpitsos, Pinneo, & Ram (1977) certain test stimuli may inhibit or facilitate subsequent responses, independent of whether or not the test stimulus elicits a response. This explanation of the present results is awkward in view of the fact that the same stimulus could produce opposite behavioral effects as a function of time. Thus, our results indicate that the effects of a given stimulus on subsequent behavior cannot be described simply as facilitatory or inhibitory. In these experiments time was a critical covariable that determined the nature of the effects of handling. We prefer to deal with the present data within the framework of the concept of behavioral state--that is, a set of internal variables that determine the direction and intensity of behavioral responses. Within this framework, the time-dependent effects of handling could be due to either of two factors. First it is possible that handling elicits a high state of arousal that decays with time. The behavioral effects of arousal may be U shaped, as has been suggested for arousal in higher animals (Hebb, 1955). Thus, very high levels of arousal may interfere with behavior, whereas lower levels facilitate behavior. Alternatively, the stimuli provided by a condition such as handling may elicit two central excitatory states, one inhibiting feeding, the other facilitating feeding. The results obtained in the present experiments would be observed if the inhibitory state were initially stronger, but decayed faster than the excitatory state. More definitive answers to this question may be obtained by studying the neural substrates of these behavioral modifications. Previous work on Aplysia indicated that arousal of the feeding system in animals exposed to food stimuli is mediated by a pair of giant serotonergic neurons (Weiss & Kupfermann, 1977). The serotonergic neurons, in turn appear to be con-
132
KUPFERMANN AND WEISS
trolled by a central arousal system, that may be composed of histaminergic and other neurons in the cerebral ganglion (Weiss, Shapiro, Koester, & Kupfermann, 1978). The central arousal system appears to have effects on multiple behaviors including feeding (Weiss & Kupfermann, 1977) as well as cardiovascular responses (Dieringer, Koester, & Weiss, 1978). The current findings suggest that the inputs from a variety of different stimuli can converge on this arousal system, which in turn has divergent effects on several different response systems. This model may be susceptible to direct test by cellular studies of the nervous system of Aplysia.
ACKNOWLEDGMENTS We thank Drs. T. Carew, E. R. Kandel, and J. Koester for comments on an earlier draft, and K. Hilten for the figures. This study was supported in part by Grant NS 12492, Grant 1 P01 GM23540 Scope D and by a Research Scientist Development Award, 1 K02 MH00304.
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