Physiology & Behavior, Vol. 15, pp. 679--688. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A.
Alterations in Ingestive Behaviors After Bilateral Lesions of the Amygdala in the Rat BLANCHE M. BOX AND G. J. MOGENSON
Department o f Physiology, University o f Western Ontario, London, Canada (Received 12 February 1975) BOX, B. M. AND G. J. MOGENSON. Alterations in ingestive behaviors after bilateral lesions of the amygdala in the rat. PHYSIOL. BEHAV. 15(6) 679-688, 1975. - Bilateral electrolytic lesions in the dorsal aspect of the central amygdaloid nucleus of the rat produced aphagia and adipsia which was similar to but not as severe as or identical to that observed with lesions of the lateral hypothalamus. After tube-feeding to recovery these animals showed permanent deficits in motor coordination as well as in their ability to respond normally to external stimuli. Lesions in the same lateral plane but more ventral or posterior produced none of these changes. An immediate mild, transient hyperphagia was produced by lesioning the ventral posterior aspect of the lateral amygdala, and an impaired neophobic response was observed by lesioning the dorsal aspect of the lateral amgdala. These studies show that it is possible to localize discrete areas in the amygdala which have specific effects on ingestive behaviors. It is suggested that the amygdala modulates on-going hypothalamic activity via the stria terminalis from the dorsomedial area and the ventral amygdalofugal pathway from the ventral and lateral amygdala. Amygdala Food intake Hyperphagia
Limbic system
Feeding
Drinking
DURING the past few years investigators have attempted by means o f lesioning and electrical and chemical stimulation to localize areas within the amygdaloid complex which are related to feeding and drinking. The results are by no means conclusive and a review of the literature reveals that many of the findings appear to be contradictory. Amygdaloid lesions in various species have been reported to produce hyperphagia (dog: [16] ; rat: [18] ; cat: [27] ; monkey: [37]), hyperphagia and hyperdipsia (rat: [7]), hypophagia (rat: [61), aphagia and adipsia (dog: [3] ; rat: [36] ) and no effect (rat: [1,35]). Fonberg has reported that when discrete lesions were made in the dorsomedial amygdala of the dog there was an aphagia [13] but when the lesions destroyed the ventral posterior aspect of the lateral amygdala there was an immediate postoperative hyperphagia [ 14]. The purpose of the present study was to attempt to reproduce Fonberg's canine findings in the rat and to further investigate the extent of the influence of the amygdaloid complex in the regulation of feeding and drinking in the rat.
Aphagia
Adipsia
8 a.m. to 8 p.m. daily. Food and water were available ad lib except during special tests as indicated.
Surgery and Histology Surgery was carried out under sodium pentobarbital anesthesia (Nembutal, 60 mg/kg, IP). Penlong S (penicillin G, 200,000 I.U./ml, Rogar/STB, London, Ontario) was administered prophylactically (0.2 ml per rat). Bilateral electrolytic lesions were placed in various parts of the amygdaloid complex by passing a direct cathodal current ( 1 . 5 - 2 . 0 mA for 2 0 - 3 0 sec) through stainless steel (26 rats) or platinum iridium (34 rats) wire insulated with Formvar except for 0.5 mm at the tip. The exposed skull was levelled between bregma and lambda in the stereotaxic apparatus and lesions were made using bregma as a reference point. In control rats the electrode was allowed to merely puncture the dura. At the conclusion of the experiment, animals were given a lethal dose of Nembutal and perfused with saline followed by 10 percent buffered Formalin. Brains were removed and sectioned at 50 u using the frozen tissue technique. Every fifth section was stained with cresyl violet or thionin to localize the lesions and identidication of the site was facilitated using the stereotaxic atlas of K6nig and Klippel [22].
METHOD
Animals Male Wistar rats weighing 300 + 25 g at the time of surgery were used and were assigned randomly to the various groups. They° were caged individually in an air conditioned room (22 + l°C) which was illuminated from
Diets Before and after surgery animals were fed a synthetic diet which provided 5.66 Cal/g (34 percent carbohydrate,
1This research was supported by grants from the Medical Research Council of Canada and the National Research Council of Canada. 679
680 63 percent fat and 14 percent protein with adequate amounts of vitamins and minerals) except during special test periods as indicated when they received dry Purina rat chow. This relatively high fat diet was chosen in order to avoid spillage and to measure food intake accurately. A previous pilot study had shown that some rats with amygdaloid lesions spilled their diet excessively making accurate food intake measurements difficult. Rats which were adipsic and aphagic for at least 48 hr following surgery were tube-fed according to the method described by Mok et al. [26] until they began to eat the synthetic diet.
Diet Accessibility Test An earlier pilot study had shown that some rats with amygdaloid lesions had difficulty in obtaining synthetic diet if it was placed in a jar which contained a screen baffle to prevent spillage, or in chewing chow pellets if they were placed in a food hopper. In some rats the body weight change was measured when chow was presented in hoppers only vs that when chow was scattered on the cage floor.
BOX AND MOGENSON Results obtained on the various measures were averaged and reported as mean -+ SEM. Significance was determined using the Student's t test. RESULTS
Ad lib Feeding and Drinking: Medial Lesions Eleven rats with lesions in the dorsomedial amygdala in the area of the central amygdaloid nucleus showed aphagia and adipsia accompanied by body weight loss (Fig. 1).
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,
~1-- .
~ .
.
o--o Controls(8) - - Lesioned('', .
.
.
.
.
.
Special Tests o f Ingestive Behavior Four groups of lesioned animals and their controls were subjected to regulatory challenges to determine if their response was similar to control rats. Of the lesioned groups tested, two had lesions in the medial plane (one of these had previously been adipsic and aphagic) and two had lesions in the lateral plane (one of these had previously been hyperphagic).
i
•
Deprivation tests. Animals were tested for prandial drinking by food-depriving overnight and the water consumption during the 18 hr period was measured• They were also water-deprived overnight and food intake measured for the same period. Fluid and food preference tests. Four to 6 weeks after surgery animals were offered a two-bottle choice of tap water and various solutions (0.1 percent saccharin, 5 percent sucrose, and 2 percent NaCI) during a 24 hr period• Food was available ad lib during these tests except for the saccharin; it was presented for the first 6 hr without food available and for the remaining 18 hr with food. Ten weeks after surgery a food preference test similar to that used by Rolls and Rolls [32] was carried out. Animals were food deprived overnight and tested in the morning. Six different foods - sultana raisins, raw cauliflower, cheddar cheese, Canadian bacon, chocolate chip cookies and the familiar rat chow were placed in individual food cups 6 cm in dia. Each cup contained about 5 g of chopped food and the cups were arranged in random order in a Plexiglas cage (30 × 20 x 30 cm) without water available. A stopwatch was started when the rat was placed in the cage and the time when the rat started and stopped eating each type of food was recorded. After 15 min the rat was returned to its home cage and the quantities of each food consumed were determined. Taste aversion test. After 18 hr of water deprivation animals were offered tap water which contained 0.002 percent quinine sulfate on one occasion and 0.005 percent quinine sulfate on another occasion. Fluid consumption was measured during a 6 hr period. This test was carried out 5 weeks after surgery.
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TIME (doys) FIG. 1. Mean daily food intakes, water intakes and body weights for control and lesioned rats for ten days after surgery. Lesions were in the region of the central amygdaloid nucleus. Tube feeding of a liquid diet was begun 48 hr after surgery and continued until rats ate and drank spontaneously. Vertical bars indicate SEM. These rats were tube-fed until they ate more than 5 g of diet per day and drank spontaneously. The recovery period was of relatively short duration ( 5 - 7 days) and once the animals began to eat spontaneously food intake returned to normal levels almost immediately without going through the classical stages observed in animals with lateral hypothalamic lesions. There was consequent resumption of body weight gain. Immediately after recovery these rats began to spill their diet. They were observed to scoop up bite-sized pieces of diet with the forepaws, bring it to the mouth and then drop it again without eating (Fig. 2C). This may have been due to a partial defect in the motor coordination necessary to the complete act of ingestion. The rats soon became greasy looking as illustrated in Fig. 2B and did not groom themselves properly. We also noted that they had a
THE AMYGDALA AND INGESTIVE BEHAVIORS
A
681
[3
FIG. 2. A control rat (A) and a rat with central amygdaloid lesions (B) showing the greasy appearance of the lesioned rat. Note that the forepaw is almost bare of hair. Food spillage as shown in (C) by these animals was excessive. slight tremor reminiscent of the Parkinsonian syndrome. When handled by the experimenter they became stiff and rigid although they appeared to be hyperactive when in their cages. There was excessive loss of body hair (see Fig. 2B). One month after surgery, these rats were switched from the high fat diet to chow pellets which were at first presented on the cage floor. As shown in Fig. 3 they continued to gain weight and feeding continued normally. However, when the pellets were later presented in hoppers attached to the cage, it was noted that the lesioned rats lost weight rapidly, suggesting that they were unable to obtain the food. When pellets were again presented on the cage floor they immediately began to regain lost weight. This deficit was noted after three successive test periods several weeks apart. Histological examination of brain sections of this group at the conclusion of the experiment revealed that lesions destroyed the dorsal aspect of the central amygdaloid nucleus and in some cases the far lateral aspect of the internal capsule as well as the stria terminalis (Fig. 4A, B). The stereotaxic coordinates used for these lesions were 1.8 mm posterior to bregma, 3.5 mm lateral and 8.3 mm below the dura. In two other groups the animals with lesions in a similar lateral plane but more ventral (n = 7) or posterior (n = 9) there were no distrubances in ad lib food and water intake immediately following surgery. Figure 4C shows the loca-
tion of lesions in these groups. Ventral lesions destroyed the ventral aspect of the central amygdaloid nucleus as well as most of the basomedial nucleus and part of the medial nucleus. The more posterior lesions destroyed almost the entire aspect of the medial amygdala and in some cases impinged on the crus cerebri and periamygdaloid cortex. It is of interest to note that these animals developed a mild hyperphagia during the second and third week following surgery but it did not persist (Table 1, Group MA). Although their water intake was similar to that of the controls before lesioning, after the surgery they drank slightly but significantly less than the controls for the first 3 weeks.
Ad lib feeding and drinking: lateral lesions Four animals with lesions aimed at the posteroventral aspect of the basolateral amygdaloid nucleus showed a mild hyperphagia the first week after lesioning which was not accompanied by a corresponding hyperdipsia. They ate significantly more than the controls and also more than they had eaten the week prior to the surgery (p
Alterations in Ingestive Behaviors After Bilateral Lesions of the Amygdala in the Rat BLANCHE M. BOX AND G. J. MOGENSON
Department o f Physiology, University o f Western Ontario, London, Canada (Received 12 February 1975) BOX, B. M. AND G. J. MOGENSON. Alterations in ingestive behaviors after bilateral lesions of the amygdala in the rat. PHYSIOL. BEHAV. 15(6) 679-688, 1975. - Bilateral electrolytic lesions in the dorsal aspect of the central amygdaloid nucleus of the rat produced aphagia and adipsia which was similar to but not as severe as or identical to that observed with lesions of the lateral hypothalamus. After tube-feeding to recovery these animals showed permanent deficits in motor coordination as well as in their ability to respond normally to external stimuli. Lesions in the same lateral plane but more ventral or posterior produced none of these changes. An immediate mild, transient hyperphagia was produced by lesioning the ventral posterior aspect of the lateral amygdala, and an impaired neophobic response was observed by lesioning the dorsal aspect of the lateral amgdala. These studies show that it is possible to localize discrete areas in the amygdala which have specific effects on ingestive behaviors. It is suggested that the amygdala modulates on-going hypothalamic activity via the stria terminalis from the dorsomedial area and the ventral amygdalofugal pathway from the ventral and lateral amygdala. Amygdala Food intake Hyperphagia
Limbic system
Feeding
Drinking
DURING the past few years investigators have attempted by means o f lesioning and electrical and chemical stimulation to localize areas within the amygdaloid complex which are related to feeding and drinking. The results are by no means conclusive and a review of the literature reveals that many of the findings appear to be contradictory. Amygdaloid lesions in various species have been reported to produce hyperphagia (dog: [16] ; rat: [18] ; cat: [27] ; monkey: [37]), hyperphagia and hyperdipsia (rat: [7]), hypophagia (rat: [61), aphagia and adipsia (dog: [3] ; rat: [36] ) and no effect (rat: [1,35]). Fonberg has reported that when discrete lesions were made in the dorsomedial amygdala of the dog there was an aphagia [13] but when the lesions destroyed the ventral posterior aspect of the lateral amygdala there was an immediate postoperative hyperphagia [ 14]. The purpose of the present study was to attempt to reproduce Fonberg's canine findings in the rat and to further investigate the extent of the influence of the amygdaloid complex in the regulation of feeding and drinking in the rat.
Aphagia
Adipsia
8 a.m. to 8 p.m. daily. Food and water were available ad lib except during special tests as indicated.
Surgery and Histology Surgery was carried out under sodium pentobarbital anesthesia (Nembutal, 60 mg/kg, IP). Penlong S (penicillin G, 200,000 I.U./ml, Rogar/STB, London, Ontario) was administered prophylactically (0.2 ml per rat). Bilateral electrolytic lesions were placed in various parts of the amygdaloid complex by passing a direct cathodal current ( 1 . 5 - 2 . 0 mA for 2 0 - 3 0 sec) through stainless steel (26 rats) or platinum iridium (34 rats) wire insulated with Formvar except for 0.5 mm at the tip. The exposed skull was levelled between bregma and lambda in the stereotaxic apparatus and lesions were made using bregma as a reference point. In control rats the electrode was allowed to merely puncture the dura. At the conclusion of the experiment, animals were given a lethal dose of Nembutal and perfused with saline followed by 10 percent buffered Formalin. Brains were removed and sectioned at 50 u using the frozen tissue technique. Every fifth section was stained with cresyl violet or thionin to localize the lesions and identidication of the site was facilitated using the stereotaxic atlas of K6nig and Klippel [22].
METHOD
Animals Male Wistar rats weighing 300 + 25 g at the time of surgery were used and were assigned randomly to the various groups. They° were caged individually in an air conditioned room (22 + l°C) which was illuminated from
Diets Before and after surgery animals were fed a synthetic diet which provided 5.66 Cal/g (34 percent carbohydrate,
1This research was supported by grants from the Medical Research Council of Canada and the National Research Council of Canada. 679
680 63 percent fat and 14 percent protein with adequate amounts of vitamins and minerals) except during special test periods as indicated when they received dry Purina rat chow. This relatively high fat diet was chosen in order to avoid spillage and to measure food intake accurately. A previous pilot study had shown that some rats with amygdaloid lesions spilled their diet excessively making accurate food intake measurements difficult. Rats which were adipsic and aphagic for at least 48 hr following surgery were tube-fed according to the method described by Mok et al. [26] until they began to eat the synthetic diet.
Diet Accessibility Test An earlier pilot study had shown that some rats with amygdaloid lesions had difficulty in obtaining synthetic diet if it was placed in a jar which contained a screen baffle to prevent spillage, or in chewing chow pellets if they were placed in a food hopper. In some rats the body weight change was measured when chow was presented in hoppers only vs that when chow was scattered on the cage floor.
BOX AND MOGENSON Results obtained on the various measures were averaged and reported as mean -+ SEM. Significance was determined using the Student's t test. RESULTS
Ad lib Feeding and Drinking: Medial Lesions Eleven rats with lesions in the dorsomedial amygdala in the area of the central amygdaloid nucleus showed aphagia and adipsia accompanied by body weight loss (Fig. 1).
t i ~ °
I0
\'\ / \° /
o
,
~1-- .
~ .
.
o--o Controls(8) - - Lesioned('', .
.
.
.
.
.
Special Tests o f Ingestive Behavior Four groups of lesioned animals and their controls were subjected to regulatory challenges to determine if their response was similar to control rats. Of the lesioned groups tested, two had lesions in the medial plane (one of these had previously been adipsic and aphagic) and two had lesions in the lateral plane (one of these had previously been hyperphagic).
i
•
Deprivation tests. Animals were tested for prandial drinking by food-depriving overnight and the water consumption during the 18 hr period was measured• They were also water-deprived overnight and food intake measured for the same period. Fluid and food preference tests. Four to 6 weeks after surgery animals were offered a two-bottle choice of tap water and various solutions (0.1 percent saccharin, 5 percent sucrose, and 2 percent NaCI) during a 24 hr period• Food was available ad lib during these tests except for the saccharin; it was presented for the first 6 hr without food available and for the remaining 18 hr with food. Ten weeks after surgery a food preference test similar to that used by Rolls and Rolls [32] was carried out. Animals were food deprived overnight and tested in the morning. Six different foods - sultana raisins, raw cauliflower, cheddar cheese, Canadian bacon, chocolate chip cookies and the familiar rat chow were placed in individual food cups 6 cm in dia. Each cup contained about 5 g of chopped food and the cups were arranged in random order in a Plexiglas cage (30 × 20 x 30 cm) without water available. A stopwatch was started when the rat was placed in the cage and the time when the rat started and stopped eating each type of food was recorded. After 15 min the rat was returned to its home cage and the quantities of each food consumed were determined. Taste aversion test. After 18 hr of water deprivation animals were offered tap water which contained 0.002 percent quinine sulfate on one occasion and 0.005 percent quinine sulfate on another occasion. Fluid consumption was measured during a 6 hr period. This test was carried out 5 weeks after surgery.
|
|
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w
I ~urgery
4,
Procedure
!
!
i
|
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I.,""
~ 20.
t o:1"
240
TIME (doys) FIG. 1. Mean daily food intakes, water intakes and body weights for control and lesioned rats for ten days after surgery. Lesions were in the region of the central amygdaloid nucleus. Tube feeding of a liquid diet was begun 48 hr after surgery and continued until rats ate and drank spontaneously. Vertical bars indicate SEM. These rats were tube-fed until they ate more than 5 g of diet per day and drank spontaneously. The recovery period was of relatively short duration ( 5 - 7 days) and once the animals began to eat spontaneously food intake returned to normal levels almost immediately without going through the classical stages observed in animals with lateral hypothalamic lesions. There was consequent resumption of body weight gain. Immediately after recovery these rats began to spill their diet. They were observed to scoop up bite-sized pieces of diet with the forepaws, bring it to the mouth and then drop it again without eating (Fig. 2C). This may have been due to a partial defect in the motor coordination necessary to the complete act of ingestion. The rats soon became greasy looking as illustrated in Fig. 2B and did not groom themselves properly. We also noted that they had a
THE AMYGDALA AND INGESTIVE BEHAVIORS
A
681
[3
FIG. 2. A control rat (A) and a rat with central amygdaloid lesions (B) showing the greasy appearance of the lesioned rat. Note that the forepaw is almost bare of hair. Food spillage as shown in (C) by these animals was excessive. slight tremor reminiscent of the Parkinsonian syndrome. When handled by the experimenter they became stiff and rigid although they appeared to be hyperactive when in their cages. There was excessive loss of body hair (see Fig. 2B). One month after surgery, these rats were switched from the high fat diet to chow pellets which were at first presented on the cage floor. As shown in Fig. 3 they continued to gain weight and feeding continued normally. However, when the pellets were later presented in hoppers attached to the cage, it was noted that the lesioned rats lost weight rapidly, suggesting that they were unable to obtain the food. When pellets were again presented on the cage floor they immediately began to regain lost weight. This deficit was noted after three successive test periods several weeks apart. Histological examination of brain sections of this group at the conclusion of the experiment revealed that lesions destroyed the dorsal aspect of the central amygdaloid nucleus and in some cases the far lateral aspect of the internal capsule as well as the stria terminalis (Fig. 4A, B). The stereotaxic coordinates used for these lesions were 1.8 mm posterior to bregma, 3.5 mm lateral and 8.3 mm below the dura. In two other groups the animals with lesions in a similar lateral plane but more ventral (n = 7) or posterior (n = 9) there were no distrubances in ad lib food and water intake immediately following surgery. Figure 4C shows the loca-
tion of lesions in these groups. Ventral lesions destroyed the ventral aspect of the central amygdaloid nucleus as well as most of the basomedial nucleus and part of the medial nucleus. The more posterior lesions destroyed almost the entire aspect of the medial amygdala and in some cases impinged on the crus cerebri and periamygdaloid cortex. It is of interest to note that these animals developed a mild hyperphagia during the second and third week following surgery but it did not persist (Table 1, Group MA). Although their water intake was similar to that of the controls before lesioning, after the surgery they drank slightly but significantly less than the controls for the first 3 weeks.
Ad lib feeding and drinking: lateral lesions Four animals with lesions aimed at the posteroventral aspect of the basolateral amygdaloid nucleus showed a mild hyperphagia the first week after lesioning which was not accompanied by a corresponding hyperdipsia. They ate significantly more than the controls and also more than they had eaten the week prior to the surgery (p
