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Conditioned aversion to brain-stimulation reward: Effects of electrode placement and prior experience
Brain Research, 170 (1979) 523-531 © Elsevier/North-Holland Biomedical Press
523
C O N D I T I O N E D AVERSION TO B R A I N - S T I M U L A T I O N R E W A R D : EFFECTS OF E L E C T R O D E P L A C E M E N T A N D PRIOR E X P E R I E N C E
ANTHONY G. PHILLIPS and ANGUS C. McDONALD Department of Psychology, University of British Columbia, Vancouver V6T 1 W5 (Canada)
(Accepted November 9th, 1978)
SUMMARY A conditioned aversion to rewarding amygdaloid brain-stimulation was established by injecting rats with toxic doses of 0.15 M LiCI immediately after an initial self-stimulation session. The aversion had extinguished by the third self-stimulation session, 96 h after conditioning. This effect cannot be attributed to general depressant effects of LiC1 on self-stimulation as treatment with LiCI 24 h before the second selfstimulation session was ineffective. Furthermore, this conditioned aversion was locus specific as LiCI injections immediately after the first test session had no disruptive effects on self-stimulation in the substantia nigra. The parallels between conditioned aversion to rewarding brain-stimulation in the amygdala and taste aversion were strengthened by the fact that the novelty of brain-stimulation reward was an important factor in the conditioning effect. These data have important implications for understanding the sensory properties of brain-stimulation reward.
INTRODUCTION Despite the wide-ranging advances in our knowledge of the neural correlates of brain-stimulation-reward (BSR), there is a paucity of information on the sensory properties of this phenomenon a. Positive BSR placements have been found in the olfactory bulb11,1,5 and in the region of the tractus solitarius 1 a structure important for relaying gustatory information 10. These findings, along with the modulation of selfstimulation by olfactory 11 and gustatory stimuli 14, suggest that certain aspects of BSR may be related to sensory processes10,11, 22. At a more general level, rewarding electrical stimulation of the brain has been shown to serve as an effective conditioned stimulus (CS) and discriminative stimulus in both classical 4 and instrumental conditioning paradigmsS, is. While these data confirm the stimulus or 'cue' properties of BSR 6 very few attempts have been made to utilize
524 these 'cue' properties to determine structural and functional relations among different brain areas. Available data indicate a great deal of perceptual similarity between rewarding brain-stimulation at different loci 17. One possible interpretation of these findings is that BSR at two different electrode sites is mediated by a common neuroanatomical substrate 19. A recent experiment has shown that electrical stimulation of the amygdala can be substituted for taste as a CS in a conditioned taste aversion paradigm 13. Stimulation in the neostriatum was ineffective. Afferents from the parabrachial gustatory neurons project to the amygdala 9, raising the possibility that the sensory properties of amygdaloid brain-stimulation may be mediated by this gustatory relay system. The generality of these earlier findings may be limited however by the fact that the brain-stimulation was not controlled by the subject. What remains to be determined is whether animals working for rewarding brain-stimulation in a free operant situation can associate the sensory properties of brain-stimulation with visceral malaise. Such information would provide important new insights into the experiential aspects of intracranial reinforcement. The following experiments were designed to determine whether it is possible to condition an aversion to brain-stimulation reward by pairing it with toxicosis induced by injections of lithium. Stimulation sites included the amygdala and substantia nigra (SN). Previous studies have shown that self-stimulation can be elicited from both of these regions7, zl. The novelty of the CS has been shown to be an important factor in conditioned taste aversion 5,16 and therefore experience with brain-stimulation reward, prior to poisoning, was varied in these experiments. This paradigm appears ideally suited to studying the sensory properties of brain-stimulation reward, as rats developed a conditioned aversion to self-stimulation in the amygdala. METHODS General Procedure Subjects were male Wistar rats weighing between 280-320 g at the time of surgery. The animals were anesthetized with sodium pentobarbital (50 mg/kg), placed in a K o p f stereotaxic apparatus, and small diameter bipolar nichrome electrodes (Plastic Products Co. MS 303, 0.005 in) were chronically implanted using standard procedures. The coordinates for the basolateral nucleus of the amygdala (ABL) were 0.7 mm posterior to bregma, 4.6 m m lateral to the midline, and 8.6 m m ventral to the dorsal surface of the skull. The incisor bar was set at 5.0 m m above the interaural line. A second series of animals were implanted with electrodes aimed at the SN. The coordinates for the SN were 3.0 m m anterior to the stereotaxic zero, 2. l m m lateral to the midline and 2.1 m m dorsal to the stereotaxic zero. The incisor bar was set at 4.2 m m below the interaural line. Following recovery from surgery, each animal was placed in a nose-poke apparatus. This consisted of a black plywood box with a 4.4 cm hole located in one wall 3.0 cm above the floor. When the animal placed its nose into this hole it activated a photoelectric relay and delivered a 0.2 sec train of 60 Hz AC current of varying
525 intensity to the chronic electrode assembly. The animals were habituated to the boxes for 30 min after which the baseline rate of responding was established over a second 30 min period. The next day the animals were screened for self-stimulation. Selfstimulation was defined as responding for BSR at a rate that was at lease twice the baseline level and consequently a different criterion was established for each animal. On the first self-stimulation session, current intensities were set at 20/~A for each subject. If an animal failed to reach criterion during the first session, the intensity was increased 5 #A per daily session until the criterion was met. Animals which did not reach criterion after 5 daily sessions were dropped from the experiment. At the completion of the experiment, the animals were asphyxiated with COz, their brains rapidly removed and stored in 1 0 ~ formalin. Brains were frozen, sectioned at 40 # m and sections containing electrode tracts were mounted and stained with Luxol fast blue and counterstained with clesyl violet.
Experiment 1: Effects of LiC1 illness on amygdaloid self-stimulation Subjects meeting the criterion for self-stimulation of the amygdala were assigned to one of 4 treatment groups. Eight animals comprised the ABL-LiC1 group which received an i.p. injection of 0.15 M LiC1 (2 ml/100 g body weight) immediately after the end of the 30 min test during which they reached criterion for self-stimulation. After the injection half of the subjects were returned to the test apparatus and allowed to self-stimulate for another 10 min period. This procedure would provide an immediate indication of the disruptive effects of the injection on ABL self-stimulation. Subsequent self-stimulation sessions were given 48 h and 96 h after the conditioning trial. As a further control for the trauma of the injection, a second group of rats received i.p. injections of sterile NaC1 (0.9 ~). The third group of 6 animals (ABL-LiC1 24 h) was incorporated into the design as a control for the possible depressant effects of LiC1 on self-stimulation. These animals were injected with LiCI 24 h alter the criterion self-stimulation session. Two additional sessions were conducted, again at 48 h intervals after the first self-stimulation session. The fourth group of 8 animals received no-injections and provided a baseline against which to compare the 3 treatment groups. A conditioned aversion to amygdaloid self-stimulation would be demonstrated if a significant reduction in rate was observed only in the ABL-LiC1 group.
Experiment 2: Effect of LiCl-illness on amygdaloid self-stimulation after repeated selfstimulation tests Eight animals were given repeated experience with amygdaloid self-stimulation in five 30 rain test sessions. Each test was conducted at 48 h intervals. On Day 9, following tlae end of the fifth self-stimulation session, each animal was injected with 0.15 M LiC1 as in Experiment 1. Post-poisoning tests were run on days 11 and 13.
Experiment 3: Effect of LiCl-illness on S N self-sthnulation Sixteen rats were tested for self-stimulation in a similar manner to that described in Experiment 1. The fact that SN animals self-stimulate at a higher rate than
526 amygdaloid subjects necessitated a minor procedural change to ensure comparable rates between these two placements. Specifically, the current intensity was adjusted in the first 5 min of the first self-stimulation session to a level that would result in a score of between 200-400 responses in 30 min. RESULTS
Experiment 1: Effect of LiCl-illness on amygdaloid self-stimulation The results of this experiment are shown in Fig. 1. The mean self-stimulation scores for all 4 groups ranged between 250-400 responses/30 min during the preinjection test. Poisoning with LiC1 greatly attenuated the self-stimulation rate in the ABL-LiC1 group on the second test, but the rate had returned to baseline 96 h after the conditioning trial. N o reduction in self-stimulation was observed in the 3 control groups, including those treated with LiCI 24 h after the first self-stimulation session. These data were analyzed by a repeated measure A N O V A which revealed a significant groups effect (F(3,25) ~- 6.86, P < 0.01). Additional A N O V A tests showed that significant group differences only occurred on the second self-stimulation trial and post hoc t-tests confirmed that the ABL-LiCI group had a mean self-stimulation rate that was significantly different from the three control groups (P < 0.05). Control scores were not significantly different from one another.
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Fig. 1. Self-stimulation rates over 3 consecutive test sessions separated by 48 h intervals. All groups had electrodes located in the basolateral nucleus of the amygdala (ABL) and those treated with LiC1 received injections either immediately (ABL-LiCl) or 24 h [ABL-LiCI (24 h)] after the initial selfstimulation session (Data represent mean 4- S.E.M.).
527
Fig. 2. Location of electrode placements for the subjects in the 4 groups in Experiment I. ( e ) ABLLiCI; (m) ABL-NaCI ; (A) ABL-LiC1 (24 h); (V) ABL no injection.
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Fig. 3. Effect of LiC1 on amygdaloio self-stimulation after 5, 30 min test sessions separated by 48 h intervals. (Data represent mean 4- S.E.M.).
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Fig. 4. Self-stimulation rates over 3 consecutive test sessions separated by 48 h intervals. Both groups had electrodes in the SN Group, SN-LiCI received a toxic dose of LiCI immediately after the first self-stimulation session. The second group were injected with NaC1 (Data represent mean zt- S.E.M.).
Histological e x a m i n a t i o n o f the electrode p l a c e m e n t s f r o m the a n i m a l s in each g r o u p confirmed that the electrodes t e r m i n a t e d either within, o r on the edge o f the ABL. The electrode placements for all animals in each g r o u p are shown in Fig. 2.
Experiment 2: Effect of LiCl-illness on amygdaloid self-stimulation after repeated selfstimulation tests The results o f this e x p e r i m e n t are shown in Fig. 3. The m e a n self-stimulation rate h a d stabilized by the fifth test session, after which the animals were p o i s o n e d with LiCl. A slight reduction in self-stimulation rate was observed during the sixth and seventh tests, but this difference did n o t reach statistical significance (F(6,24) = 1.46, P > 0.05).
Experiment 3: Effect of LiCl-illness on SN self-stimulation Self-stimulation scores for b o t h S N g r o u p s r a n g e d between a m e a n o f 200-299 responses/30 min on the first test session (see Fig. 4). These m e a n values increased significantly in the S N - L i C I a n d SN-NaC1 g r o u p s over the two r e m a i n i n g test sessions. N o significant g r o u p differences were observed (F(1,15) = 1.66, P > 0.05).
529
Fig. 5. Location of electrode placements from subjects in group SN-LiC1 (O) and SN-NaCI (m).
Placements of the stimulating electrodes were localized in both the SN pars compacta and pars reticulata. No significant group differences in electrode placements were observed (see Fig. 5). DISCUSSION Rats injected with toxic doses of LiCl following their initial experience with rewarding brain-stimulation at sites in the amygdala, developed a temporary aversion to the brain-stimulation. This conditioned aversion had extinguished by the third test session; 96 h after toxicosis. The effect cannot be attributed to the disruption of a weak rewarding effect by the trauma of i.p. injections because control animals injected with sterile NaC1 failed to develop an aversion. In fact, the trauma asssociated with LiCI toxicity did not have a generalized depressive effect on operant responding, as animals receiving electrical stimulation in the extrapyramidal system were unaffected on the subsequent self-stimulation test. The parallels between the present study and tra-
530 ditional experiments on conditioned taste aversion are strengthened by the fact that novelty of the CS was an important determinant ot conditioning. Rats are less likely to show an aversion to bland and familiar tastes after poisoning~, 16. Similarly, animals in Experiment 2 failed to show a significant aversion to amygdaloid self-stimulation when poisoned after several self-stimulation sessions. Evidence for the importance of the temporal contiguity between ABL selfstimulation and LiC1 toxicity in establishing this conditioned aversion comes from the fact that animals injected with LiCI 24 h after the first self-stimulation session did not display a response decrement. These data also argue against any long-term unconditioned effects of LiC1 on the operant response. In this regard, it is important to note that although LiC1 has been shown to attenuate self-stimulation in both the lateral hypothalamus 2 and SN 7, these effects were only observed after 2 or 3 daily injections and disappeared after repeated treatment with this drug. The present findings are particularly important as they extend the CS properties of brain-stimulation to a situation where stimulation can be defined unequivocally as reinforcing because it is directly controlled by the animal's behavior. Lenzer 6 has maintained that an analysis of the sensory or 'cue' properties of reinforcing brainstimulation is essential for a complete understanding of BSR. This proposition now would appear indisputable and can be elaborated upon further in light of the present results. It has been suggested that the internal stimuli produced by BSR can serve best as a CS in a continuous reinforcement situation, as it is assumed that this CS property decays rapidly 6. The successful conditioning of amygdaloid stimulation to the unconditioned stimuli (US) provided by LiCI, may have been facilitated in some animals by permitting them to maintain self-stimulation during the onset of toxicosis. The fact that animals in the ABL-LiCl group which did not receive the postinjection testing, also developed a conditioned aversion would argue against this possibility. Nevertheless, the 'decay' factor is poorly understood and needs to be examined more precisely. In this regard, the conditioned aversion paradigm could provide the necessary information by establishing the maximal CS-US interval in which conditioning will still occur. The present data clearly show that the sensory properties of BSR in the amygdala can serve as a CS, thereby confirming and extending a recent finding la. In the early study, rats receiving non-contingent amygdaloid stimulation while drinking plain water prior to poisoning with LiCl developed a temporary aversion to brainstimulation plus water. Amygdaloid stimulation not paired with LiC1 had no significant effect on subsequent water intake. The property of brain-stimulation which can serve as a CS in these aversion experiments appears to be locus specific. Caudate stimulation could not act as a CS in the earlier bait-shyness study 13 and animals in the present study did not develop an aversion to SN self-stimulation. The sensory properties of amygdaloid stimulation which can be conditioned to visceral discomfort may be related to the fact that this structure receives an input from both the pontine taste area 9 and the anterior olfactory nucleus 20. If this is indeed the case, if should be possible to use localized brain-stimulation in conjunction with the present behavioral testing paradigm to trace the anatomical and functional interactions of those neural structures involved in taste and olfactory perception.
531 ACKNOWLEDGEMENTS This research was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h Co u n ci l an d N a t u r a l Sciences a n d E n g in e e r in g Research C o u n c i l o f C a n a d a . A. G. Phillips is a K i l l a m Senior R e s e a r c h Scholar. T h e authors t h a n k Dr. D. M. Wilkie for helpful suggestions a n d invaluable discussion.
REFERENCES 1 Carter, D. A. and Phillips, A. G., Intracranial self-stimulation at sites in the dorsal medulla oblongata, Brain Research, 94 (1975) 155-160. 2 Edelson, A., Gottesfeld, Z., David, S. and Yuwiler, A., Effect of lithium and other alkali metals on brain chemistry and behavior, Psychopharmacol. (Berl.), 45 (1976) 233-237. 3 Hall, R. D., Bloom, F. E. and Olds, J., Neuronal and neurochemical substrates of reinforcement, Neurosci. Res. Prog. Bull., 15 (1977) 133-314. 4 Hupka, R. B., Electrical stimulation of the septum and hypothalamus as conditioned stimuli in the rabbit, Physiol. Behav., 5 (1970) 1355-1363. 5 Kalat, J. W. and Rozin, P., 'Learned safety' as a mechanism in long-delay taste-aversion learning in rats, J. comp. physiol. PsychoL, 83 (1973) 198-207. 6 Lenzer, I. I., Differences between behavior reinforced by electrical stimulation of the brain and conventionally reinforced behavior: an associative analysis, Psychol. Bull., 78 (1972) 103-117. 7 Liebman, J. M. and Segal, D. S., Lithium differentially antagonizes self-stimulation facilitated by morphine and (q-)-amphetamine, Nature (Lend.), 260 (1976) 161-163. 8 Mogenson, G. J., Avoidance responses to rewarding brain stimulation: Replication and extension, J. comp. physiol. Psychol., 58 (1964) 465-467. 9 Norgren, R., Taste pathways to hypothalamus and amygdala, J. comp. NeuroL, 166 (1976) 17-30. 10 Pfaffman, C., The pleasures of sensation, Psychol. Rev., 67 (1960) 253-268. 11 Phillips, A. G., Enhancement and inhibition of olfactory bulb self-stimulation by odours, Physiol. Behav., 5 (1970), 1127-1131. 12 Phillips, A. G. and Fibiger, H. C., Dopaminergic and noradrenergic substrates of positive reinforcement: differential effects of D- and L-amphetamine, Science, 179 (1973) 575-577. 13 Phillips, A. G. and LePiane, F. G., Electrical stimulation of the amygdala as a~conditioned stimulus in a bait-shyness paradigm, Science, 201 (1978) 536-538. 14 Phillips, A. G. and Mogenson, G. J., Effects of taste on self-stimulation and induced drinking, J. comp. physiol. Psychol., 66 (1968) 654-660. 15 Phillips, A. G. and Mogenson, G. J., Self-stimulation of the olfactory bulb, Physiol. Behav., 4 (1969) 195-197. 16 Revusky, S. H. and Bedart, E. W., Association of illness with ingestion of novel foods, Science, 155 (1967) 219-220. 17 Stutz, R. M., Stimulus generalization within the limbic system, J. comp.physiol. Psychol., 65 (1968) 79-82. 18 Stutz, R. M., Butcher, R. E. and Rossi, R. R., Stimulus properties of reinforcing brain shock, Science, 163 (1969) 1081-1082. 19 Stutz, R. M., Rossi, R. R. and Hastings, L., Discriminability of intracranial stimuli: the role of anatomical connectedness, Physiol. Behav., 12 (1974) 69-73. 20 White, L. E., Jr., Olfactory bulb projections of the rat, Anat. Rec., 152 (1965) 465-480. 21 Wurtz, R. H. and Olds, J., Amygdaloid stimulation and operant reinforcement in the rat, J. comp. physiol. Psychol., 56 (1963) 941-949. 22 Young, P. T., Palatability: the hedonic response to foodstuffs. In C. F. Code (Ed.), Handbook o[ Physiology, Section 6: Alimentary Canal. L Control of Food and Water Intake, Amer. Physiol. Soc., Washington, 1967, pp. 353-366.
523
C O N D I T I O N E D AVERSION TO B R A I N - S T I M U L A T I O N R E W A R D : EFFECTS OF E L E C T R O D E P L A C E M E N T A N D PRIOR E X P E R I E N C E
ANTHONY G. PHILLIPS and ANGUS C. McDONALD Department of Psychology, University of British Columbia, Vancouver V6T 1 W5 (Canada)
(Accepted November 9th, 1978)
SUMMARY A conditioned aversion to rewarding amygdaloid brain-stimulation was established by injecting rats with toxic doses of 0.15 M LiCI immediately after an initial self-stimulation session. The aversion had extinguished by the third self-stimulation session, 96 h after conditioning. This effect cannot be attributed to general depressant effects of LiC1 on self-stimulation as treatment with LiCI 24 h before the second selfstimulation session was ineffective. Furthermore, this conditioned aversion was locus specific as LiCI injections immediately after the first test session had no disruptive effects on self-stimulation in the substantia nigra. The parallels between conditioned aversion to rewarding brain-stimulation in the amygdala and taste aversion were strengthened by the fact that the novelty of brain-stimulation reward was an important factor in the conditioning effect. These data have important implications for understanding the sensory properties of brain-stimulation reward.
INTRODUCTION Despite the wide-ranging advances in our knowledge of the neural correlates of brain-stimulation-reward (BSR), there is a paucity of information on the sensory properties of this phenomenon a. Positive BSR placements have been found in the olfactory bulb11,1,5 and in the region of the tractus solitarius 1 a structure important for relaying gustatory information 10. These findings, along with the modulation of selfstimulation by olfactory 11 and gustatory stimuli 14, suggest that certain aspects of BSR may be related to sensory processes10,11, 22. At a more general level, rewarding electrical stimulation of the brain has been shown to serve as an effective conditioned stimulus (CS) and discriminative stimulus in both classical 4 and instrumental conditioning paradigmsS, is. While these data confirm the stimulus or 'cue' properties of BSR 6 very few attempts have been made to utilize
524 these 'cue' properties to determine structural and functional relations among different brain areas. Available data indicate a great deal of perceptual similarity between rewarding brain-stimulation at different loci 17. One possible interpretation of these findings is that BSR at two different electrode sites is mediated by a common neuroanatomical substrate 19. A recent experiment has shown that electrical stimulation of the amygdala can be substituted for taste as a CS in a conditioned taste aversion paradigm 13. Stimulation in the neostriatum was ineffective. Afferents from the parabrachial gustatory neurons project to the amygdala 9, raising the possibility that the sensory properties of amygdaloid brain-stimulation may be mediated by this gustatory relay system. The generality of these earlier findings may be limited however by the fact that the brain-stimulation was not controlled by the subject. What remains to be determined is whether animals working for rewarding brain-stimulation in a free operant situation can associate the sensory properties of brain-stimulation with visceral malaise. Such information would provide important new insights into the experiential aspects of intracranial reinforcement. The following experiments were designed to determine whether it is possible to condition an aversion to brain-stimulation reward by pairing it with toxicosis induced by injections of lithium. Stimulation sites included the amygdala and substantia nigra (SN). Previous studies have shown that self-stimulation can be elicited from both of these regions7, zl. The novelty of the CS has been shown to be an important factor in conditioned taste aversion 5,16 and therefore experience with brain-stimulation reward, prior to poisoning, was varied in these experiments. This paradigm appears ideally suited to studying the sensory properties of brain-stimulation reward, as rats developed a conditioned aversion to self-stimulation in the amygdala. METHODS General Procedure Subjects were male Wistar rats weighing between 280-320 g at the time of surgery. The animals were anesthetized with sodium pentobarbital (50 mg/kg), placed in a K o p f stereotaxic apparatus, and small diameter bipolar nichrome electrodes (Plastic Products Co. MS 303, 0.005 in) were chronically implanted using standard procedures. The coordinates for the basolateral nucleus of the amygdala (ABL) were 0.7 mm posterior to bregma, 4.6 m m lateral to the midline, and 8.6 m m ventral to the dorsal surface of the skull. The incisor bar was set at 5.0 m m above the interaural line. A second series of animals were implanted with electrodes aimed at the SN. The coordinates for the SN were 3.0 m m anterior to the stereotaxic zero, 2. l m m lateral to the midline and 2.1 m m dorsal to the stereotaxic zero. The incisor bar was set at 4.2 m m below the interaural line. Following recovery from surgery, each animal was placed in a nose-poke apparatus. This consisted of a black plywood box with a 4.4 cm hole located in one wall 3.0 cm above the floor. When the animal placed its nose into this hole it activated a photoelectric relay and delivered a 0.2 sec train of 60 Hz AC current of varying
525 intensity to the chronic electrode assembly. The animals were habituated to the boxes for 30 min after which the baseline rate of responding was established over a second 30 min period. The next day the animals were screened for self-stimulation. Selfstimulation was defined as responding for BSR at a rate that was at lease twice the baseline level and consequently a different criterion was established for each animal. On the first self-stimulation session, current intensities were set at 20/~A for each subject. If an animal failed to reach criterion during the first session, the intensity was increased 5 #A per daily session until the criterion was met. Animals which did not reach criterion after 5 daily sessions were dropped from the experiment. At the completion of the experiment, the animals were asphyxiated with COz, their brains rapidly removed and stored in 1 0 ~ formalin. Brains were frozen, sectioned at 40 # m and sections containing electrode tracts were mounted and stained with Luxol fast blue and counterstained with clesyl violet.
Experiment 1: Effects of LiC1 illness on amygdaloid self-stimulation Subjects meeting the criterion for self-stimulation of the amygdala were assigned to one of 4 treatment groups. Eight animals comprised the ABL-LiC1 group which received an i.p. injection of 0.15 M LiC1 (2 ml/100 g body weight) immediately after the end of the 30 min test during which they reached criterion for self-stimulation. After the injection half of the subjects were returned to the test apparatus and allowed to self-stimulate for another 10 min period. This procedure would provide an immediate indication of the disruptive effects of the injection on ABL self-stimulation. Subsequent self-stimulation sessions were given 48 h and 96 h after the conditioning trial. As a further control for the trauma of the injection, a second group of rats received i.p. injections of sterile NaC1 (0.9 ~). The third group of 6 animals (ABL-LiC1 24 h) was incorporated into the design as a control for the possible depressant effects of LiC1 on self-stimulation. These animals were injected with LiCI 24 h alter the criterion self-stimulation session. Two additional sessions were conducted, again at 48 h intervals after the first self-stimulation session. The fourth group of 8 animals received no-injections and provided a baseline against which to compare the 3 treatment groups. A conditioned aversion to amygdaloid self-stimulation would be demonstrated if a significant reduction in rate was observed only in the ABL-LiC1 group.
Experiment 2: Effect of LiCl-illness on amygdaloid self-stimulation after repeated selfstimulation tests Eight animals were given repeated experience with amygdaloid self-stimulation in five 30 rain test sessions. Each test was conducted at 48 h intervals. On Day 9, following tlae end of the fifth self-stimulation session, each animal was injected with 0.15 M LiC1 as in Experiment 1. Post-poisoning tests were run on days 11 and 13.
Experiment 3: Effect of LiCl-illness on S N self-sthnulation Sixteen rats were tested for self-stimulation in a similar manner to that described in Experiment 1. The fact that SN animals self-stimulate at a higher rate than
526 amygdaloid subjects necessitated a minor procedural change to ensure comparable rates between these two placements. Specifically, the current intensity was adjusted in the first 5 min of the first self-stimulation session to a level that would result in a score of between 200-400 responses in 30 min. RESULTS
Experiment 1: Effect of LiCl-illness on amygdaloid self-stimulation The results of this experiment are shown in Fig. 1. The mean self-stimulation scores for all 4 groups ranged between 250-400 responses/30 min during the preinjection test. Poisoning with LiC1 greatly attenuated the self-stimulation rate in the ABL-LiC1 group on the second test, but the rate had returned to baseline 96 h after the conditioning trial. N o reduction in self-stimulation was observed in the 3 control groups, including those treated with LiCI 24 h after the first self-stimulation session. These data were analyzed by a repeated measure A N O V A which revealed a significant groups effect (F(3,25) ~- 6.86, P < 0.01). Additional A N O V A tests showed that significant group differences only occurred on the second self-stimulation trial and post hoc t-tests confirmed that the ABL-LiCI group had a mean self-stimulation rate that was significantly different from the three control groups (P < 0.05). Control scores were not significantly different from one another.
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Fig. 1. Self-stimulation rates over 3 consecutive test sessions separated by 48 h intervals. All groups had electrodes located in the basolateral nucleus of the amygdala (ABL) and those treated with LiC1 received injections either immediately (ABL-LiCl) or 24 h [ABL-LiCI (24 h)] after the initial selfstimulation session (Data represent mean 4- S.E.M.).
527
Fig. 2. Location of electrode placements for the subjects in the 4 groups in Experiment I. ( e ) ABLLiCI; (m) ABL-NaCI ; (A) ABL-LiC1 (24 h); (V) ABL no injection.
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Fig. 3. Effect of LiC1 on amygdaloio self-stimulation after 5, 30 min test sessions separated by 48 h intervals. (Data represent mean 4- S.E.M.).
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Fig. 4. Self-stimulation rates over 3 consecutive test sessions separated by 48 h intervals. Both groups had electrodes in the SN Group, SN-LiCI received a toxic dose of LiCI immediately after the first self-stimulation session. The second group were injected with NaC1 (Data represent mean zt- S.E.M.).
Histological e x a m i n a t i o n o f the electrode p l a c e m e n t s f r o m the a n i m a l s in each g r o u p confirmed that the electrodes t e r m i n a t e d either within, o r on the edge o f the ABL. The electrode placements for all animals in each g r o u p are shown in Fig. 2.
Experiment 2: Effect of LiCl-illness on amygdaloid self-stimulation after repeated selfstimulation tests The results o f this e x p e r i m e n t are shown in Fig. 3. The m e a n self-stimulation rate h a d stabilized by the fifth test session, after which the animals were p o i s o n e d with LiCl. A slight reduction in self-stimulation rate was observed during the sixth and seventh tests, but this difference did n o t reach statistical significance (F(6,24) = 1.46, P > 0.05).
Experiment 3: Effect of LiCl-illness on SN self-stimulation Self-stimulation scores for b o t h S N g r o u p s r a n g e d between a m e a n o f 200-299 responses/30 min on the first test session (see Fig. 4). These m e a n values increased significantly in the S N - L i C I a n d SN-NaC1 g r o u p s over the two r e m a i n i n g test sessions. N o significant g r o u p differences were observed (F(1,15) = 1.66, P > 0.05).
529
Fig. 5. Location of electrode placements from subjects in group SN-LiC1 (O) and SN-NaCI (m).
Placements of the stimulating electrodes were localized in both the SN pars compacta and pars reticulata. No significant group differences in electrode placements were observed (see Fig. 5). DISCUSSION Rats injected with toxic doses of LiCl following their initial experience with rewarding brain-stimulation at sites in the amygdala, developed a temporary aversion to the brain-stimulation. This conditioned aversion had extinguished by the third test session; 96 h after toxicosis. The effect cannot be attributed to the disruption of a weak rewarding effect by the trauma of i.p. injections because control animals injected with sterile NaC1 failed to develop an aversion. In fact, the trauma asssociated with LiCI toxicity did not have a generalized depressive effect on operant responding, as animals receiving electrical stimulation in the extrapyramidal system were unaffected on the subsequent self-stimulation test. The parallels between the present study and tra-
530 ditional experiments on conditioned taste aversion are strengthened by the fact that novelty of the CS was an important determinant ot conditioning. Rats are less likely to show an aversion to bland and familiar tastes after poisoning~, 16. Similarly, animals in Experiment 2 failed to show a significant aversion to amygdaloid self-stimulation when poisoned after several self-stimulation sessions. Evidence for the importance of the temporal contiguity between ABL selfstimulation and LiC1 toxicity in establishing this conditioned aversion comes from the fact that animals injected with LiCI 24 h after the first self-stimulation session did not display a response decrement. These data also argue against any long-term unconditioned effects of LiC1 on the operant response. In this regard, it is important to note that although LiC1 has been shown to attenuate self-stimulation in both the lateral hypothalamus 2 and SN 7, these effects were only observed after 2 or 3 daily injections and disappeared after repeated treatment with this drug. The present findings are particularly important as they extend the CS properties of brain-stimulation to a situation where stimulation can be defined unequivocally as reinforcing because it is directly controlled by the animal's behavior. Lenzer 6 has maintained that an analysis of the sensory or 'cue' properties of reinforcing brainstimulation is essential for a complete understanding of BSR. This proposition now would appear indisputable and can be elaborated upon further in light of the present results. It has been suggested that the internal stimuli produced by BSR can serve best as a CS in a continuous reinforcement situation, as it is assumed that this CS property decays rapidly 6. The successful conditioning of amygdaloid stimulation to the unconditioned stimuli (US) provided by LiCI, may have been facilitated in some animals by permitting them to maintain self-stimulation during the onset of toxicosis. The fact that animals in the ABL-LiCl group which did not receive the postinjection testing, also developed a conditioned aversion would argue against this possibility. Nevertheless, the 'decay' factor is poorly understood and needs to be examined more precisely. In this regard, the conditioned aversion paradigm could provide the necessary information by establishing the maximal CS-US interval in which conditioning will still occur. The present data clearly show that the sensory properties of BSR in the amygdala can serve as a CS, thereby confirming and extending a recent finding la. In the early study, rats receiving non-contingent amygdaloid stimulation while drinking plain water prior to poisoning with LiCl developed a temporary aversion to brainstimulation plus water. Amygdaloid stimulation not paired with LiC1 had no significant effect on subsequent water intake. The property of brain-stimulation which can serve as a CS in these aversion experiments appears to be locus specific. Caudate stimulation could not act as a CS in the earlier bait-shyness study 13 and animals in the present study did not develop an aversion to SN self-stimulation. The sensory properties of amygdaloid stimulation which can be conditioned to visceral discomfort may be related to the fact that this structure receives an input from both the pontine taste area 9 and the anterior olfactory nucleus 20. If this is indeed the case, if should be possible to use localized brain-stimulation in conjunction with the present behavioral testing paradigm to trace the anatomical and functional interactions of those neural structures involved in taste and olfactory perception.
531 ACKNOWLEDGEMENTS This research was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h Co u n ci l an d N a t u r a l Sciences a n d E n g in e e r in g Research C o u n c i l o f C a n a d a . A. G. Phillips is a K i l l a m Senior R e s e a r c h Scholar. T h e authors t h a n k Dr. D. M. Wilkie for helpful suggestions a n d invaluable discussion.
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