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Attenuation of brain stimulation self-starvation: Adaptation effects
Physiology and Behavior, Vol. 9, pp. 831--837. Brain Research Publications Inc., 1972. Printed in U.S.A.
Attenuation of Brain Stimulation Self-starvation: Adaptation Effects R E B E C C A M. S A N T O S A N D A R Y E H R O U T T E N B E R G 1
Cresap Laboratory of Neuroscience and Behavior, Department of Psychology, Northwestern University, Evanston, Illinois 60201
(Received 31 May 1972)
SANTOS, R. M. AND A. ROUTTENBERG. Attenuation of brain stimulation self-starvation: adaptation effects. PHYSIOL. BEHAV. 9 (5) 831-837, 1972.-Rats first trained for 10-15 days to bar-press for lateral hypothalamic brain stimulation while feeding ad lib and then for 17-18 days to maintain weight by eating in a Skinner box under a 22.75-hr food deprivation schedule demonstrate a preference for brain stimulation when food and brain stimulation are simultaneously available. This maladaptive behavior has been called "self-starvation." If the experimental procedure is modified so that in addition to the training in self-stimulation and feeding the animals are given daily a 1.25-hr self-stimulation session and immediately before or after the self-stimulation session a 1.25-hr feeding period for eight days prior to brain stimulation food competition, animals do not meet self-starvation criteria. Moreover when self-starvation has been demonstrated it can be attenuated in a second brain stimulaion food competition preceded by the eight-day alternate feeding and self-stimulation phase of the modified procedure. The attenuating effect of this manipulation of procedure is discussed in terms of adaptation to stress. Adaptation
Self-starvation
Self-stimulation
Hypothalamus
THE I N I T I A L d e m o n s t r a t i o n [9, 12] that rats would forego f o o d essential to survival in o r d e r to obtain hypothalamic stimulation has been confirmed b y subsequent reports [3, 11, 14, 15]. Other studies have shown that rats also prefer brain stimulation to water even when thirsty [5, 6 ] , to caring for pups shortly after giving birth to t h e m [ 13], to heat in a refrigerated e n v i r o n m e n t [ 1 ], to a salt solution after a d r e n a l e c t o m y [ 3 ] , and to an intact s t o m a c h when brain stimulation is accompanied b y intragastric loading of an excessive v o l u m e of nutrient [8 ]. In the case of f o o d deprived rats that chose to self-stimulate rather than eat, R o u t t e n b e r g and Bulloch [ 11] f o u n d that the self-starvation effect could be a t t e n u a t e d by the administration of drugs, namely, c h l o r p r o m a z i n e and pentobarbital. The present experiments determined w h e t h e r it was possible to reduce the self-starvation effect by manipulation o f the training procedure.
Food deprivation
Sprague-Dawley strain weighing b e t w e e n 251 and 370 g at the t i m e of operation.
Apparatus A 9.25 by 8.0 by 7.5-in. Skinner b o x with a 2.5-in. bar protruding f r o m one of its walls was used t h r o u g h o u t the experiment. Lever depression triggered a stimulator which delivered current through an electrode to the brain of the animal. Water intake in the Skinner box was measured by inverted graduated cylinders.
Procedure The animals were implanted with bipolar electrodes aimed at the medial forebrain bundle (MFB) at the level of the lateral h y p o t h a l a m u s as described in the R o u t t e n b e r g and Bulloch study [ 11 ]. A f t e r a p p r o x i m a t e l y one week o f recovery the animals were subjected to the following phases of the procedure. (1) Self-stimulation phase. Animals were tested for self-stimulation as described in the R o u t t e n b e r g and Bulloch [ 11 ] study in a 75-min session for f o u r days. The current was 25 uA rms, 60 Hz sine wave and had a m a x i m u m duration of 0.5 sec. Animals were fed and
EXPERIMENT 1: FAILURE TO DEMONSTRATE SELF-STARVATION METHOD
Animals The animals were 28 naive adult male albino rats o f the
J Rebecca M. Santos was supported by a Northwestern University Graduate Fellowship and the research was supported by MH 17255 to Aryeh Routtenberg to whom reprint requests should be sent. We thank Michael Sloan, Karl Diedrichs, Elaine Bresnahan, and Arthur Anderson, Jr. for their assistance. 831
832
SANTOS AND ROUTTENBERG
watered ad lib except during the testing periods. {2) Feeding phase. The rats were placed under 22.75-hr food deprivation and allowed access to food for 75 min daily. During the feeding sessions Purina food chow pellets were scattered on the floor of the Skinner box and water was a v a i l a b l e in graduated cylinders. Intracranial stimulation was not available at any time of the feeding phase. This phase lasted four days. Daily food and water intake during feeding periods were measured from this phase onward. (3) Alternate feeding and self-stimulation phase. The food deprived animals during this stage remained in the Skinner box 2.5 hr daily. During half this period they were allowed to feed and drink and during the other half, to self-stimulate in the absence of food and water. The two possible sequences of feeding and self-stimulation periods within a day were counterbalanced across the eight days of this phase in an abba paradigm [ 16]. {4) Competition phase. The food deprived animals were then placed in the Skinner box for 75 min daily, during which food and self stimulation were simultaneously available. After three days of the competition phase the animals were sacrificed with an overdose of sodium pentobarbital (Diabutal) and perfused with saline solution and 10% formalin. Brains were sectioned at 35 tz and stained by the Weil and thionin methods for myelinated axons and cell bodies, respectively, to facilitate localization of electrode placements. The criteria for self-starvation were those established by Routtenberg and Lindy [12], namely, a 50% or greater reduction in food intake and a mean 30 g weight loss during the three-day competition phase.
RESULTS None of the animals self-starved according to the Routtenberg and Lindy [12] criteria. The analysis of variance, however, revealed a greater tendency for animals with high ( > 5 0 0 bar-presses/15 min) rates of self-stimulation (N=18) to decrease food intake from precompetition to competition. Animals with low (<500 bar presses/15 min) rates of self-stimulation (N=10) showed little change in food intake (Stage x Group F=8.201, d/= 1,26, p< 0.01 ). High self-stimulators showed a 21.2% reduction in food intake, low self-stimulators, 0.7%. Weight measures also showed a greater reduction in high self-stimulators than in low self-stimulators. The mean weight of high self-stimulators decreased from 319.0 g to 304.9 g from precompetition to competition while that of the low self-stimulators changed from 299.9 g to 300.2 g (Stage x Group F=8.958, dr= 1,26, p< 0.01 ). The Stage x Group interaction was also present in self-stimulation measures both for the first 15 min (F=14.664, dr=l,26, p<0.001) and the entire 75 min (F=6.992, df=1,26, p
observed in this experiment were associated with the mamillothalamic tract.
EXPERIMENT 2: SELF-STARVATION AS A FUNCTION OF PRECOMPETITION ADAPTATION PROCEDURE The experimental procedure in Experiment 1 failed to yield self-starvation even when electrode placements appeared to be in the identical regions as those in the Routtenberg and Bulloch study [I 1]. The procedure in Experiment 1 was similar to that of Routtenberg and Bulloch [11] except that in Experiment 1 there was an alternate feeding and self-stimulation phase prior to the competition phase. Thus during the eight days before competition, food and self-stimulation training were presented within the same day in contrast to the Routtenberg and Bulloch [ 11 ] procedure where only one of the two events was present within a day. Experiment 2 investigated the hypothesis that the alternate feeding and self-stimulation training before competition was the crucial attenuating i n f l u e n c e on the brain stimulation self-starvation effect.
METHOD
Animals The animals were 50 naive adult albino rats of the Sprague-Dawley strain weighing between 232 and 300 g at the time of surgery. Only animals whose highest 15 min self-stimulation rate in 12-testing days was greater than 400 were retained for the experiment. Fourteen [14] animals did not meet this criterion. Other animals suffered either electrode loss (N-4) or illness (N-3). A total of 28 animals completed the experiment.
Pro cedure The animals were implanted with electrodes in the MFB at the lateral hypothalamic level as described in Experiment 1. After a week of recovery they were taken through three phases. (1) Self-stimulation phase. Self-stimulation testing was conducted as in Experiment 1 except that the sessions lasted 15 min daily for 12 days. (2) Feeding phase. The animals were placed under 22.75-hr deprivation and allowed to feed on food pellets scattered on the floor of the Skinner box for 75 min daily. Electrodes were connected to the stimulator but current level was set at 0 ~A. This phase lasted until animals stabilized weight. If during any three-day period the weight on the third day was equal to or greater than the weight on the first day, weight stabilization was considered to have occurred. The feeding phase lasted an average of 17 days. (3) Competition phase. This phase was identical to the competition phase of Experiment 1. The above schedule was similar to that used by Routtenberg and Bulloch [ 11 ]. After the completion of the three phases the animals were returned to ad lib feeding to facilitate recovery from food reduction effects of the competition phase. They were then divided randomly between two conditions. In one condition the animals underwent the eight-day alternate feeding and self-stimulation phase described in Experiment
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION
833
TABLE 1 SUMMARY OF FOOD INTAKE, WEIGHT, AND SELF-STIMULATION RATES FOR HIGH AND LOW SELF--STIMULATION, EXPERIMENT 1 Mean daily food intake (in g)
High self-stimulators (N=18) Low self-stimulators (N=I0)
Last three days before competition
Competition
Percent reduction in food intake*
14.6 13.8
11.5 13.7
21.2 0.7
Mean daily weight (in g)
High self-stimulators (N=18) Low self-stimulators (N=10)
Last three days before competition
Competition
Mean weight losst
319.0 299.9
304.9 300.2
14.1 -0.3
Mean daily self-stimulation rates Last three days before competition
High self-stimulators (N=18) Low self-stimulators (N=10)
15 min 831.7 86.4
75 min 3154.8 399.9
Competition 15 min 390.1 41.7
75 min 1825.1 162.2
Percent reduction in food intake = (Mean daily food intake last three days before competition -Mean daily food intake during competition) x 100 Mean daily food intake last three days before competition tMean weight loss= Mean daily weight last three days before competition -Mean daily weight during competition
1 (Modified group, N=14). In the other condition the animals went through the feeding phase of the Routtenberg and Bulloch procedure for eight days (RB or Control group, N=I5). A second three-day competition phase for both the Modified and the RB group immediately followed. At the end of the experiment the animals were sacrificed and their b rains removed for histological examination using procedures described in Experiment 1. RESULTS The self-starvation effect [ 11 ] was replicated in the first competition phase of Experiment 2 and was subsequently attenuated in a second competition phase when preceded by the eight-day alternate feeding and self-stimulation phase. This finding confirmed the view that the eight-day training procedure could attenuate the self-starvation effect. Analysis of variance of food intake revealed a significant reduction in food intake for both groups during both
competition phases. Food intake reduction was less during the second competition phase and this attenuation was greater for the Modified group (Competition x Stage x Group F=5.204, dr=l, 27, p<0.05). Thus the Modified group reduced food intake by 44.3% during Competition 1 and 21.9% during Competition 2. The corresponding values for the RB group were 35.7% and 30.7%. Since interest was focused on self-starvation a separate analysis was carried out only on the data of animals that met self-starvation criteria during Competition 1. A significant Group x Competition interaction (F=8.214, dr=l,7, p<0.05) was obtained in the analysis of percent reduction in food intake of self-starvers. This interaction indicated a greater attenuation in self-starvation during Competition 2 for the Modified group. The attenuating effect of the procedural modification is emphasized in the case of self-starvers as Fig. 1 shows. Weight measures did not yield a significant Competition x Stage x Group interaction (F=2.166, dr=l,27). Self-stimulation scores both for the initial 15 min and the
834
SANTOS AND ROUTTENBERG
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FIG. 1. Mean percent reduction in food intake of the Modified and RB groups during both competition phases, Experiment 2. Standard errors of the mean are Competition 1: Modified group, 6.051, RB group, 3.108; Competition 2: Modified group, 7.834, RB group, 5.322. Data are only for the self-starvinganimals with a greater than 50% reduction in food intake during Competition 1. See text for explanation. entire 75-min session during the competition phase did not indicate any systematic relationship with changes in food intake (Competition x Group F=2.947 and 1.246 for 15-min and 75-min respectively dr=l,27). Table 2 summarizes the food intake, weight, and self-stimulation data for both Modified and RB groups. Additional evidence for the attenuating effect of the modified procedure on self-starvation came from the data of three animals in Experiment 2. These animals were given an additional competition phase. Two of these animals belonged to the RB group and one belonged to the Modified group. After Competition 2, they were given a two-day recovery period during which they were fed ad lib. The two RB animals then had an alternate feeding and self-stimulation phase prior to the third competition, thus undergoing a RB-RB-Modified paradigm through the three competition phases. The animal that belonged to the Modified group had a RB-Modified-RB paradigm, that is, an eight-day feeding phase prior to the third competition. The results for the three animals are summarized in Table 3. The table shows that in the RB-Modified-RB paradigm food intake reduciton was attenuated from Competition 1 to Competition 2 but returned to Competition 1 level in the third c o m p e t i t i o n . On the other hand, in the RB-RB-Modified paradigm, there was a slight attenuation in food intake reduciton during Competition 2 and an even greater one in the third competition. Histological examination indicated that electrode tips fell in or around the MFB extending anteriorly from the lateral hypothalamus and posteriorly to the supramamillary decussation. The electrode placements are plotted in Fig. 2.
g
FIG. 2. Electrode placements of animals in Experiment 2. Circles represent animals in the Modified group; squares, animals in the RB group. Filled circles and squares indicate animals with a greater than 50% reduction in food intake in Competition 1. Frames A to I are Fig. 33b to 40b and 42b in K/Snigand Klippel (1963). Brain sections for three animals were lost during histology.
DISCUSSION Brain stimulation self-starvation is a powerful effect since animals will actually perish on such a regimen [9]. The present experiments demonstrate that it is nevertheless possible to attenuate this self-starvation by manipulating the temporal relationship of the training events prior to brain stimulation-food competition. In one other study attenuation of self-starvation was accomplished by the use of pharmacological agents [11 ] in contrast to the present study wherein attenuation resulted from a simple procedural manipulation. The present findings parallel the attenuation of self-starvation in the activity wheel using adaptation procedures [ 10]. In seeking an explanation of the present results Routtenberg's [ 10] interpretation of attenuation of self-starvation in the activity wheel thus seems relevant. In this study [10] rats introduced simultaneously to a 23-hr food deprivation schedule and a cage containing an activity wheel were unable to maintain weight. Routtenberg
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION
835
TABLE 2 SUMMARY OF FOOD INTAKE, WEIGHT, AND SELF-STIMULATION RATES FOR THE MODIFIED AND RB GROUPS, EXPERIMENT 2 Mean daily food intake (in g) Last three days Precompetition 1
Precompetition 2
15.8 15.1
16.0 16.3
Modified group (N=14) RB group (N=15)
Percent food reduction*
Competition 1
2
8.8 9.7
1
12.5 11.3
2
44.3 35.8
21.9 30.7
Mean daily weight (in g) Last three days
Modified group (N=14) RB group (N=15)
Competition
Mean weight loss~"
Precompetition 1
Precompetition 2
1
2
322.7 319.0
325.6 328.8
305.9 299.9
316.4 308.8
1
2
16.8 19.6
9.2 20.0
Mean daily self-stimulation rates Last three days before competition 1
Modified group (N=14) RB group (N=15)
Competition
2
1
2
15 min
75 min
15 min
75 min
15 min
75 min
15 min
75 min
835.1 728.4
-
1040.4 -
4411.7 -
632.7 500.0
2811.9 2242.4
585.5 647.1
2468.6 2666.8
Note.-Self-stimulation sessions prior to Competition 1 lasted only 15 min for all animals. There were no self-stimulation sessions for the RB group prior to Competition 2. *Percent reduction in food intake= (Mean daily food intake last three days before competition -Mean daily food intake during competition) x 100 Mean daily food intake last three days before competition tMean weight loss = Mean daily weight last three days before competition -Mean daily weight during competition proposed two sources of stress in that experimental situation: (1) novelty stress from the sudden introduction to the activity wheel; and, (2) deprivation stress from the food deprivation. Deprivation stress was concluded to be the more important factor since adaptation to the food deprivation schedule eliminated the self-starvation effect but preadaptation to the activity wheel did not. In brain stimulation self-starvation in the present study the deprivation factor arises from the 22.75-hr food deprivation schedule. The novelty factor might be said to arise from the simultaneous presence of the hunger drive a n d the opportunity to self-stimulate during the competition phase. Adaptation to deprivation stress may be assumed under the RB procedure in as much as the competition phase is introduced only after weight stablization has occurred, that is, after 1 7 - 1 8 days of the feeding phase. The assumption is consistent with the finding that rats take 18 days to adapt to a 23-hr food deprivation schedule [2]. The procedure is thus analogous to Experiment 2 of the Routtenberg [10] study where rats were adapted to the
food deprivation schedule before introducing the activity wheel cage. Where the deprivation factor was thus removed, self-starvation was eliminated in the activity wheel but not in the self-stimulation situation. Therefore it may be concluded that deprivation stress is not as critical to brain stimulation self-starvation as it is to activity wheel self-starvation. The modified experimental procedure in Experiment 1 parallels Experiment 3 of the Routtenberg [10] study in which animals were preadapted to the novelty factor. Those animals were placed in an activity wheel three hr daily prior to introduction to the activity wheel cage and the deprivation schedule. This procedure had no effect on self-starvation. In the present study preadaptation to the novelty factor may have occurred during the daily alternate feeding and self-stimulation phase. In particular this procedure allows the animal to bar-press for brain stimulation under conditions of food deprivation. As a result the stress of the simultaneous presence of food deprivation and availability of brain stimulation during competition may have been reduced whereas under the
836
SANTOS AND ROUTTENBERG TABLE 3 SUMMARY OF RESULTS FOR THREE ANIMALS TAKEN TROUGH THREE COMPETITIONPHASES Animal No.
Competition 1
Competition 2
Competition 3
Procedure RB
Mod
RB
3054(Modgroup) Percent reduction in food intake
46.8
5.7
46.1
Weight loss
26.0
23.0
31.1
15-min. SS*
1147.3
584.0
1271.7
75-min. SS*
4554.7
2965.3
4861.7
RB
RB
Mod
Percent reduction in food intake
48.0
40.6
15.8
Weight loss
29.7
27.3
10.3
15-min SS
1279.0
1339.7
1154.0
75-min SS
4883.3
4442.3
3411.3
Percent reduction in food intake
73.3
58.4
43.6
Weight loss
73.0
47.3
27.7
15-min SS
1063.3
1258.3
926.0
75-min SS
4271.7
4841.0
2479.7
3052 (RB group)
3053 (RB group)
*Self-stimulation rate during competition phase only. Routtenberg and Bulloch schedule the presence of both brain stimulation and food deprivation would have been a completely new experience. Thus, self-starvation may have been prevented in this manner under the modified experimental procedure. Adaptation to novelty stress may also explain the observation in Experiment 2 that a second competition under the Routtenberg and Bulloch [1 1] procedure also results in attenuation of self-starvation on the part of the control group may be explained by postulating that the first competition phase provided the adaptation experience. Therefore it is likely that the difference in the effects of the modified and Routtenberg and Bulloch procedures would be greater in the absence of a first competition. However, an evaluation of food intake reduction in high self-stimulators in Experiment 1 with that of animals in
Experiment 2 during Competition 1 as a baseline could not be made due to differences in length of self-stimulation sessions, self-stimulation phase, and feeding phase. The present study has shown that minor changes in the experimental situation can alter an animal's response to brain stimulation without a change in stimulus parameters. These results are reminiscent of the switching of goal objects in stimulus-bound behavior which Valenstein, Cox, and Kakolewski [ 17, 18, 19 ] accomplished by simply removing the initial goal object. Valenstein e t al., attributed the goal switching to the environmental modification of a s i n g l e p l a s t i c hypothalamic system. An alternate explanation was proposed by Wise [20, 21 ] who contended that the emergence of the second reponse was due to a decline in its threshold with repeated stimulation. While it is not inconceivable that behavioral changes effected by
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION manipulation of the experimental situation may be subserved by threshold changes, the present data do not
allow a m o r e specific physiological self-starvation and its attenuation.
837 explanation
of
REFERENCES 1. Carlisle, H. J. and E. Snyder. The interaction of hypothalamic self-stimulation and temperature regulation. Sep. Experientia 26: 1092-1093, 1970. 2. Dufort, R. H. The rat's adjustment to 23-, 47-, and 71-hr food deprivation schedules. Psychol. Rep. 14: 663-669, 1964. 3. Eckert, N. and M. Lewis. Competition between drives for i n t r a c r a n i a l s t i m u l a t i o n and sodium chloride by adrenalectomized NaCl-deprived rats. J. comp. physiol. PsychoL 64: 349-352, 1967. 4. K~nig, J. F. R. and R. A. Klippel. The Rat Brain. A Stereotaxic Atlas of the Forebmin and Lower Parts of the Brain Stem. Baltimore, Maryland: William and Wilkins, 1963. 5. Mogenson, G. J. Effects of drugs on the preference between electrical stimulation of the lateral hypothalamus and water. Psychonortt ScL 17: 13-14, 1969. 6. Morgan, C. W. and G. J. Mogenson. Preference of water-deprived rats for stimulation of the lateral hypothalamus rather than water.Psychonom. Sci. 6: 337-338, 1966. 7. Myers, J. L. Fundamentals of Experimental Design. Boston, Massachusetts: Allyn and Bacon, 1966. 8. Panksepp, J., J. A. Trowill and A. Trehub. An inexpensive electrofistular swivel for negative feedback control of self-stimulation. £ exp. Analysis Behav. 10: 571-579, 1967. 9. Routtenberg, A. Self-starvation caused by "feeding center" self-stimulation.Am. Psychol. 19: 502, 1964. 10. Routtenberg, A. "Self-starvation" of rats living in activity wheels: Adaptation effects. J. comp. physiol. Psyehol. 66: 234-238, 1968. 11. Routtenberg, A. and G. C. BuUoch. "Self-starvation" and rewarding brain stimulation: Differential effects of chlorpromazine and pentobarbital. Learn. Motiv., 2: 83-94, 1971.
12. Routtenberg, A. and J. Lindy. Effects of the availability of rewarding septial and hypothalamic stimulation on barpressing for food under conditions of deprivation. J. comp. physiol. Psychol. 60: 158-161, 1965. 13. Sonderegger, T. B. Intracranial stimulation and maternal behavior. Proc. a. Cony. Am. psychol. Ass. 5: 245-246, 1970. 14. Spies, G. F o o d versus intracranial self-stimulation reinforcement in food-deprived rats. J. comp. physiol. Psychol. 60: 153-157, 1965. 15. Stutz, R. M., R. R. Rossi and A. M. Bowring. Competition between food and rewarding brain shock. Physiol. Behav. 7: 753-575, 1971. 16. Underwood, B. J. Experimental Psychology. New York: Appleton-Century-Crofts, 1966, pp. 33-36. 17. Valenstein, E. S., V. C. Cox and J. Kakolewski. Modification of motivated behavior elicited by electrical stimulation of the hypothalamus. Science 159:1119-1121,1968. 18. Valenstein, E. S., V. C. Cox and J. Kakolewski. Hypothalamic motivational systems: Fixed or plastic neural circuits? Science 163: 1084, 1969. 19. Valenstein, E. S., V. C. Cox and J. Kakolewski. Reexamination of the role of the hypothalamus in motivation. Psychol. Rev. 77: 16-31, 1970. 20. Wise, R. A. Hypothalamic motivational systems: Fixed or plastic neural circuits? Science 162: 377-379, 1968. 21. Wise, R. A. Plasticity of hypothalamic motivational systems. Science 165: 929-930, 1969.
Attenuation of Brain Stimulation Self-starvation: Adaptation Effects R E B E C C A M. S A N T O S A N D A R Y E H R O U T T E N B E R G 1
Cresap Laboratory of Neuroscience and Behavior, Department of Psychology, Northwestern University, Evanston, Illinois 60201
(Received 31 May 1972)
SANTOS, R. M. AND A. ROUTTENBERG. Attenuation of brain stimulation self-starvation: adaptation effects. PHYSIOL. BEHAV. 9 (5) 831-837, 1972.-Rats first trained for 10-15 days to bar-press for lateral hypothalamic brain stimulation while feeding ad lib and then for 17-18 days to maintain weight by eating in a Skinner box under a 22.75-hr food deprivation schedule demonstrate a preference for brain stimulation when food and brain stimulation are simultaneously available. This maladaptive behavior has been called "self-starvation." If the experimental procedure is modified so that in addition to the training in self-stimulation and feeding the animals are given daily a 1.25-hr self-stimulation session and immediately before or after the self-stimulation session a 1.25-hr feeding period for eight days prior to brain stimulation food competition, animals do not meet self-starvation criteria. Moreover when self-starvation has been demonstrated it can be attenuated in a second brain stimulaion food competition preceded by the eight-day alternate feeding and self-stimulation phase of the modified procedure. The attenuating effect of this manipulation of procedure is discussed in terms of adaptation to stress. Adaptation
Self-starvation
Self-stimulation
Hypothalamus
THE I N I T I A L d e m o n s t r a t i o n [9, 12] that rats would forego f o o d essential to survival in o r d e r to obtain hypothalamic stimulation has been confirmed b y subsequent reports [3, 11, 14, 15]. Other studies have shown that rats also prefer brain stimulation to water even when thirsty [5, 6 ] , to caring for pups shortly after giving birth to t h e m [ 13], to heat in a refrigerated e n v i r o n m e n t [ 1 ], to a salt solution after a d r e n a l e c t o m y [ 3 ] , and to an intact s t o m a c h when brain stimulation is accompanied b y intragastric loading of an excessive v o l u m e of nutrient [8 ]. In the case of f o o d deprived rats that chose to self-stimulate rather than eat, R o u t t e n b e r g and Bulloch [ 11] f o u n d that the self-starvation effect could be a t t e n u a t e d by the administration of drugs, namely, c h l o r p r o m a z i n e and pentobarbital. The present experiments determined w h e t h e r it was possible to reduce the self-starvation effect by manipulation o f the training procedure.
Food deprivation
Sprague-Dawley strain weighing b e t w e e n 251 and 370 g at the t i m e of operation.
Apparatus A 9.25 by 8.0 by 7.5-in. Skinner b o x with a 2.5-in. bar protruding f r o m one of its walls was used t h r o u g h o u t the experiment. Lever depression triggered a stimulator which delivered current through an electrode to the brain of the animal. Water intake in the Skinner box was measured by inverted graduated cylinders.
Procedure The animals were implanted with bipolar electrodes aimed at the medial forebrain bundle (MFB) at the level of the lateral h y p o t h a l a m u s as described in the R o u t t e n b e r g and Bulloch study [ 11 ]. A f t e r a p p r o x i m a t e l y one week o f recovery the animals were subjected to the following phases of the procedure. (1) Self-stimulation phase. Animals were tested for self-stimulation as described in the R o u t t e n b e r g and Bulloch [ 11 ] study in a 75-min session for f o u r days. The current was 25 uA rms, 60 Hz sine wave and had a m a x i m u m duration of 0.5 sec. Animals were fed and
EXPERIMENT 1: FAILURE TO DEMONSTRATE SELF-STARVATION METHOD
Animals The animals were 28 naive adult male albino rats o f the
J Rebecca M. Santos was supported by a Northwestern University Graduate Fellowship and the research was supported by MH 17255 to Aryeh Routtenberg to whom reprint requests should be sent. We thank Michael Sloan, Karl Diedrichs, Elaine Bresnahan, and Arthur Anderson, Jr. for their assistance. 831
832
SANTOS AND ROUTTENBERG
watered ad lib except during the testing periods. {2) Feeding phase. The rats were placed under 22.75-hr food deprivation and allowed access to food for 75 min daily. During the feeding sessions Purina food chow pellets were scattered on the floor of the Skinner box and water was a v a i l a b l e in graduated cylinders. Intracranial stimulation was not available at any time of the feeding phase. This phase lasted four days. Daily food and water intake during feeding periods were measured from this phase onward. (3) Alternate feeding and self-stimulation phase. The food deprived animals during this stage remained in the Skinner box 2.5 hr daily. During half this period they were allowed to feed and drink and during the other half, to self-stimulate in the absence of food and water. The two possible sequences of feeding and self-stimulation periods within a day were counterbalanced across the eight days of this phase in an abba paradigm [ 16]. {4) Competition phase. The food deprived animals were then placed in the Skinner box for 75 min daily, during which food and self stimulation were simultaneously available. After three days of the competition phase the animals were sacrificed with an overdose of sodium pentobarbital (Diabutal) and perfused with saline solution and 10% formalin. Brains were sectioned at 35 tz and stained by the Weil and thionin methods for myelinated axons and cell bodies, respectively, to facilitate localization of electrode placements. The criteria for self-starvation were those established by Routtenberg and Lindy [12], namely, a 50% or greater reduction in food intake and a mean 30 g weight loss during the three-day competition phase.
RESULTS None of the animals self-starved according to the Routtenberg and Lindy [12] criteria. The analysis of variance, however, revealed a greater tendency for animals with high ( > 5 0 0 bar-presses/15 min) rates of self-stimulation (N=18) to decrease food intake from precompetition to competition. Animals with low (<500 bar presses/15 min) rates of self-stimulation (N=10) showed little change in food intake (Stage x Group F=8.201, d/= 1,26, p< 0.01 ). High self-stimulators showed a 21.2% reduction in food intake, low self-stimulators, 0.7%. Weight measures also showed a greater reduction in high self-stimulators than in low self-stimulators. The mean weight of high self-stimulators decreased from 319.0 g to 304.9 g from precompetition to competition while that of the low self-stimulators changed from 299.9 g to 300.2 g (Stage x Group F=8.958, dr= 1,26, p< 0.01 ). The Stage x Group interaction was also present in self-stimulation measures both for the first 15 min (F=14.664, dr=l,26, p<0.001) and the entire 75 min (F=6.992, df=1,26, p
observed in this experiment were associated with the mamillothalamic tract.
EXPERIMENT 2: SELF-STARVATION AS A FUNCTION OF PRECOMPETITION ADAPTATION PROCEDURE The experimental procedure in Experiment 1 failed to yield self-starvation even when electrode placements appeared to be in the identical regions as those in the Routtenberg and Bulloch study [I 1]. The procedure in Experiment 1 was similar to that of Routtenberg and Bulloch [11] except that in Experiment 1 there was an alternate feeding and self-stimulation phase prior to the competition phase. Thus during the eight days before competition, food and self-stimulation training were presented within the same day in contrast to the Routtenberg and Bulloch [ 11 ] procedure where only one of the two events was present within a day. Experiment 2 investigated the hypothesis that the alternate feeding and self-stimulation training before competition was the crucial attenuating i n f l u e n c e on the brain stimulation self-starvation effect.
METHOD
Animals The animals were 50 naive adult albino rats of the Sprague-Dawley strain weighing between 232 and 300 g at the time of surgery. Only animals whose highest 15 min self-stimulation rate in 12-testing days was greater than 400 were retained for the experiment. Fourteen [14] animals did not meet this criterion. Other animals suffered either electrode loss (N-4) or illness (N-3). A total of 28 animals completed the experiment.
Pro cedure The animals were implanted with electrodes in the MFB at the lateral hypothalamic level as described in Experiment 1. After a week of recovery they were taken through three phases. (1) Self-stimulation phase. Self-stimulation testing was conducted as in Experiment 1 except that the sessions lasted 15 min daily for 12 days. (2) Feeding phase. The animals were placed under 22.75-hr deprivation and allowed to feed on food pellets scattered on the floor of the Skinner box for 75 min daily. Electrodes were connected to the stimulator but current level was set at 0 ~A. This phase lasted until animals stabilized weight. If during any three-day period the weight on the third day was equal to or greater than the weight on the first day, weight stabilization was considered to have occurred. The feeding phase lasted an average of 17 days. (3) Competition phase. This phase was identical to the competition phase of Experiment 1. The above schedule was similar to that used by Routtenberg and Bulloch [ 11 ]. After the completion of the three phases the animals were returned to ad lib feeding to facilitate recovery from food reduction effects of the competition phase. They were then divided randomly between two conditions. In one condition the animals underwent the eight-day alternate feeding and self-stimulation phase described in Experiment
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION
833
TABLE 1 SUMMARY OF FOOD INTAKE, WEIGHT, AND SELF-STIMULATION RATES FOR HIGH AND LOW SELF--STIMULATION, EXPERIMENT 1 Mean daily food intake (in g)
High self-stimulators (N=18) Low self-stimulators (N=I0)
Last three days before competition
Competition
Percent reduction in food intake*
14.6 13.8
11.5 13.7
21.2 0.7
Mean daily weight (in g)
High self-stimulators (N=18) Low self-stimulators (N=10)
Last three days before competition
Competition
Mean weight losst
319.0 299.9
304.9 300.2
14.1 -0.3
Mean daily self-stimulation rates Last three days before competition
High self-stimulators (N=18) Low self-stimulators (N=10)
15 min 831.7 86.4
75 min 3154.8 399.9
Competition 15 min 390.1 41.7
75 min 1825.1 162.2
Percent reduction in food intake = (Mean daily food intake last three days before competition -Mean daily food intake during competition) x 100 Mean daily food intake last three days before competition tMean weight loss= Mean daily weight last three days before competition -Mean daily weight during competition
1 (Modified group, N=14). In the other condition the animals went through the feeding phase of the Routtenberg and Bulloch procedure for eight days (RB or Control group, N=I5). A second three-day competition phase for both the Modified and the RB group immediately followed. At the end of the experiment the animals were sacrificed and their b rains removed for histological examination using procedures described in Experiment 1. RESULTS The self-starvation effect [ 11 ] was replicated in the first competition phase of Experiment 2 and was subsequently attenuated in a second competition phase when preceded by the eight-day alternate feeding and self-stimulation phase. This finding confirmed the view that the eight-day training procedure could attenuate the self-starvation effect. Analysis of variance of food intake revealed a significant reduction in food intake for both groups during both
competition phases. Food intake reduction was less during the second competition phase and this attenuation was greater for the Modified group (Competition x Stage x Group F=5.204, dr=l, 27, p<0.05). Thus the Modified group reduced food intake by 44.3% during Competition 1 and 21.9% during Competition 2. The corresponding values for the RB group were 35.7% and 30.7%. Since interest was focused on self-starvation a separate analysis was carried out only on the data of animals that met self-starvation criteria during Competition 1. A significant Group x Competition interaction (F=8.214, dr=l,7, p<0.05) was obtained in the analysis of percent reduction in food intake of self-starvers. This interaction indicated a greater attenuation in self-starvation during Competition 2 for the Modified group. The attenuating effect of the procedural modification is emphasized in the case of self-starvers as Fig. 1 shows. Weight measures did not yield a significant Competition x Stage x Group interaction (F=2.166, dr=l,27). Self-stimulation scores both for the initial 15 min and the
834
SANTOS AND ROUTTENBERG
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FIG. 1. Mean percent reduction in food intake of the Modified and RB groups during both competition phases, Experiment 2. Standard errors of the mean are Competition 1: Modified group, 6.051, RB group, 3.108; Competition 2: Modified group, 7.834, RB group, 5.322. Data are only for the self-starvinganimals with a greater than 50% reduction in food intake during Competition 1. See text for explanation. entire 75-min session during the competition phase did not indicate any systematic relationship with changes in food intake (Competition x Group F=2.947 and 1.246 for 15-min and 75-min respectively dr=l,27). Table 2 summarizes the food intake, weight, and self-stimulation data for both Modified and RB groups. Additional evidence for the attenuating effect of the modified procedure on self-starvation came from the data of three animals in Experiment 2. These animals were given an additional competition phase. Two of these animals belonged to the RB group and one belonged to the Modified group. After Competition 2, they were given a two-day recovery period during which they were fed ad lib. The two RB animals then had an alternate feeding and self-stimulation phase prior to the third competition, thus undergoing a RB-RB-Modified paradigm through the three competition phases. The animal that belonged to the Modified group had a RB-Modified-RB paradigm, that is, an eight-day feeding phase prior to the third competition. The results for the three animals are summarized in Table 3. The table shows that in the RB-Modified-RB paradigm food intake reduciton was attenuated from Competition 1 to Competition 2 but returned to Competition 1 level in the third c o m p e t i t i o n . On the other hand, in the RB-RB-Modified paradigm, there was a slight attenuation in food intake reduciton during Competition 2 and an even greater one in the third competition. Histological examination indicated that electrode tips fell in or around the MFB extending anteriorly from the lateral hypothalamus and posteriorly to the supramamillary decussation. The electrode placements are plotted in Fig. 2.
g
FIG. 2. Electrode placements of animals in Experiment 2. Circles represent animals in the Modified group; squares, animals in the RB group. Filled circles and squares indicate animals with a greater than 50% reduction in food intake in Competition 1. Frames A to I are Fig. 33b to 40b and 42b in K/Snigand Klippel (1963). Brain sections for three animals were lost during histology.
DISCUSSION Brain stimulation self-starvation is a powerful effect since animals will actually perish on such a regimen [9]. The present experiments demonstrate that it is nevertheless possible to attenuate this self-starvation by manipulating the temporal relationship of the training events prior to brain stimulation-food competition. In one other study attenuation of self-starvation was accomplished by the use of pharmacological agents [11 ] in contrast to the present study wherein attenuation resulted from a simple procedural manipulation. The present findings parallel the attenuation of self-starvation in the activity wheel using adaptation procedures [ 10]. In seeking an explanation of the present results Routtenberg's [ 10] interpretation of attenuation of self-starvation in the activity wheel thus seems relevant. In this study [10] rats introduced simultaneously to a 23-hr food deprivation schedule and a cage containing an activity wheel were unable to maintain weight. Routtenberg
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION
835
TABLE 2 SUMMARY OF FOOD INTAKE, WEIGHT, AND SELF-STIMULATION RATES FOR THE MODIFIED AND RB GROUPS, EXPERIMENT 2 Mean daily food intake (in g) Last three days Precompetition 1
Precompetition 2
15.8 15.1
16.0 16.3
Modified group (N=14) RB group (N=15)
Percent food reduction*
Competition 1
2
8.8 9.7
1
12.5 11.3
2
44.3 35.8
21.9 30.7
Mean daily weight (in g) Last three days
Modified group (N=14) RB group (N=15)
Competition
Mean weight loss~"
Precompetition 1
Precompetition 2
1
2
322.7 319.0
325.6 328.8
305.9 299.9
316.4 308.8
1
2
16.8 19.6
9.2 20.0
Mean daily self-stimulation rates Last three days before competition 1
Modified group (N=14) RB group (N=15)
Competition
2
1
2
15 min
75 min
15 min
75 min
15 min
75 min
15 min
75 min
835.1 728.4
-
1040.4 -
4411.7 -
632.7 500.0
2811.9 2242.4
585.5 647.1
2468.6 2666.8
Note.-Self-stimulation sessions prior to Competition 1 lasted only 15 min for all animals. There were no self-stimulation sessions for the RB group prior to Competition 2. *Percent reduction in food intake= (Mean daily food intake last three days before competition -Mean daily food intake during competition) x 100 Mean daily food intake last three days before competition tMean weight loss = Mean daily weight last three days before competition -Mean daily weight during competition proposed two sources of stress in that experimental situation: (1) novelty stress from the sudden introduction to the activity wheel; and, (2) deprivation stress from the food deprivation. Deprivation stress was concluded to be the more important factor since adaptation to the food deprivation schedule eliminated the self-starvation effect but preadaptation to the activity wheel did not. In brain stimulation self-starvation in the present study the deprivation factor arises from the 22.75-hr food deprivation schedule. The novelty factor might be said to arise from the simultaneous presence of the hunger drive a n d the opportunity to self-stimulate during the competition phase. Adaptation to deprivation stress may be assumed under the RB procedure in as much as the competition phase is introduced only after weight stablization has occurred, that is, after 1 7 - 1 8 days of the feeding phase. The assumption is consistent with the finding that rats take 18 days to adapt to a 23-hr food deprivation schedule [2]. The procedure is thus analogous to Experiment 2 of the Routtenberg [10] study where rats were adapted to the
food deprivation schedule before introducing the activity wheel cage. Where the deprivation factor was thus removed, self-starvation was eliminated in the activity wheel but not in the self-stimulation situation. Therefore it may be concluded that deprivation stress is not as critical to brain stimulation self-starvation as it is to activity wheel self-starvation. The modified experimental procedure in Experiment 1 parallels Experiment 3 of the Routtenberg [10] study in which animals were preadapted to the novelty factor. Those animals were placed in an activity wheel three hr daily prior to introduction to the activity wheel cage and the deprivation schedule. This procedure had no effect on self-starvation. In the present study preadaptation to the novelty factor may have occurred during the daily alternate feeding and self-stimulation phase. In particular this procedure allows the animal to bar-press for brain stimulation under conditions of food deprivation. As a result the stress of the simultaneous presence of food deprivation and availability of brain stimulation during competition may have been reduced whereas under the
836
SANTOS AND ROUTTENBERG TABLE 3 SUMMARY OF RESULTS FOR THREE ANIMALS TAKEN TROUGH THREE COMPETITIONPHASES Animal No.
Competition 1
Competition 2
Competition 3
Procedure RB
Mod
RB
3054(Modgroup) Percent reduction in food intake
46.8
5.7
46.1
Weight loss
26.0
23.0
31.1
15-min. SS*
1147.3
584.0
1271.7
75-min. SS*
4554.7
2965.3
4861.7
RB
RB
Mod
Percent reduction in food intake
48.0
40.6
15.8
Weight loss
29.7
27.3
10.3
15-min SS
1279.0
1339.7
1154.0
75-min SS
4883.3
4442.3
3411.3
Percent reduction in food intake
73.3
58.4
43.6
Weight loss
73.0
47.3
27.7
15-min SS
1063.3
1258.3
926.0
75-min SS
4271.7
4841.0
2479.7
3052 (RB group)
3053 (RB group)
*Self-stimulation rate during competition phase only. Routtenberg and Bulloch schedule the presence of both brain stimulation and food deprivation would have been a completely new experience. Thus, self-starvation may have been prevented in this manner under the modified experimental procedure. Adaptation to novelty stress may also explain the observation in Experiment 2 that a second competition under the Routtenberg and Bulloch [1 1] procedure also results in attenuation of self-starvation on the part of the control group may be explained by postulating that the first competition phase provided the adaptation experience. Therefore it is likely that the difference in the effects of the modified and Routtenberg and Bulloch procedures would be greater in the absence of a first competition. However, an evaluation of food intake reduction in high self-stimulators in Experiment 1 with that of animals in
Experiment 2 during Competition 1 as a baseline could not be made due to differences in length of self-stimulation sessions, self-stimulation phase, and feeding phase. The present study has shown that minor changes in the experimental situation can alter an animal's response to brain stimulation without a change in stimulus parameters. These results are reminiscent of the switching of goal objects in stimulus-bound behavior which Valenstein, Cox, and Kakolewski [ 17, 18, 19 ] accomplished by simply removing the initial goal object. Valenstein e t al., attributed the goal switching to the environmental modification of a s i n g l e p l a s t i c hypothalamic system. An alternate explanation was proposed by Wise [20, 21 ] who contended that the emergence of the second reponse was due to a decline in its threshold with repeated stimulation. While it is not inconceivable that behavioral changes effected by
ATTENUATION OF BRAIN STIMULATION SELF-STARVATION manipulation of the experimental situation may be subserved by threshold changes, the present data do not
allow a m o r e specific physiological self-starvation and its attenuation.
837 explanation
of
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12. Routtenberg, A. and J. Lindy. Effects of the availability of rewarding septial and hypothalamic stimulation on barpressing for food under conditions of deprivation. J. comp. physiol. Psychol. 60: 158-161, 1965. 13. Sonderegger, T. B. Intracranial stimulation and maternal behavior. Proc. a. Cony. Am. psychol. Ass. 5: 245-246, 1970. 14. Spies, G. F o o d versus intracranial self-stimulation reinforcement in food-deprived rats. J. comp. physiol. Psychol. 60: 153-157, 1965. 15. Stutz, R. M., R. R. Rossi and A. M. Bowring. Competition between food and rewarding brain shock. Physiol. Behav. 7: 753-575, 1971. 16. Underwood, B. J. Experimental Psychology. New York: Appleton-Century-Crofts, 1966, pp. 33-36. 17. Valenstein, E. S., V. C. Cox and J. Kakolewski. Modification of motivated behavior elicited by electrical stimulation of the hypothalamus. Science 159:1119-1121,1968. 18. Valenstein, E. S., V. C. Cox and J. Kakolewski. Hypothalamic motivational systems: Fixed or plastic neural circuits? Science 163: 1084, 1969. 19. Valenstein, E. S., V. C. Cox and J. Kakolewski. Reexamination of the role of the hypothalamus in motivation. Psychol. Rev. 77: 16-31, 1970. 20. Wise, R. A. Hypothalamic motivational systems: Fixed or plastic neural circuits? Science 162: 377-379, 1968. 21. Wise, R. A. Plasticity of hypothalamic motivational systems. Science 165: 929-930, 1969.