Analysis of behavioral deficits produced by lesions in the dorsal and ventral midbrain tegmentum

Physiology &Behavior,Vol. 25, pp. 829-843. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Analysis of Behavioral Deficits Produced by Lesions in the Dorsal and Ventral Midbrain Tegmentum I SARAH FRYER LEIBOWITZ

AND NORMAN

J. H A M M E R

The Rockefeller University, New York, N Y 10021 AND L U C Y L. B R O W N

Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, N Y 10461 R e c e i v e d 24 F e b r u a r y 1979 LEIBOWITZ, S. F., N. J. HAMMER AND L. L. BROWN. Analysis of behavioral deficits produced by lesions in the dorsal and ventral rnidbrain tegmentum. PHYSIOL. BEHAV. 25(6) 829--843, 1980.--The impact of midbrain lesions on ingestive behavior in the rat was examined under a variety of pharmacological and dietary conditions. Electrolytic lesions in the dorsal midbrain tegmentum, ventrolateral to the central gray, produced moderate to severe (4 to 19 days) aphagia and adipsia in the rat. This effect was followed by complete recovery of ad lib food intake but a sustained suppression of water intake and urine output. Caloric regulation, drug-induced anorexia, drinking in response to intracellular and extracellular dehydration, and feeding as a function of dietary palatability appeared normal. Persistent deficits, however, were observed in the rats' feeding response to glucoprivation, in their consumption of sucrose or saccharin solutions, in post-fast compensatory food intake, and in their ability to maintain a nocturnal pattern of feeding. In contrast to these changes, electrolytic lesions in the ventral midbrain tegmentum produced an increase in food intake and body weight gain. This effect was associated with no other food- or water-related behavioral changes, with the exception of amphetamine- or mazindolinduced anorexia which was attenuated or abolished by the lesion. Amphetamine Catecholamines Circadian rhythm 2-Deoxy-D-glucose Feeding behavior Histamine Hypothalamus Insulin Isoproterenol Sucrose intake

T H E R E are several studies which have shown midbrain lesions to have profound effects on ad lib food and water intake. For example, lesions in the dorsal midbrain tegmentum, ventrolateral to the central gray, result in aphagia and adipsia [18, 32, 44], in contrast to ventral tegmental lesions which produce hyperphagia and obesity [1]. Lesions further ventral, in the region of the substantia nigra, have been shown to cause profound aphagia and adipsia [59]. Detailed analyses of this substantia nigra lesion have also revealed severe but reversible sensorimotor impairments, as well as persistent motivational and regulatory deficits in feeding and drinking [15,36]. Such analyses of the dorsal and ventral midbrain tegmental lesions have not yet been conducted, with the exception of one study [2] which showed the ventral lesion to have little effect on insulin-induced feeding and dietary preferences. Histochemical analysis of these lesions, with respect to their effects on midbraln catecholamine (CA) projections,

Drinking behavior Mazindol Midbrain

indicate that the syndrome of the substantia nigra lesion results from damage to the dopaminergic nigrostriatal pathway originating from the A9 cells of the pars compacta [59]. The hyperphagia produced by ventral midbrain lesions has been attributed to the disruption of ventral noradrenergic or adrenergic fibers of the central tegmental tract [1]. This hypothesis has been questioned, however, and it has been proposed that non-catecholaminergic fibers may be involved [41]. With respect to the dorsal midbrain tegmental lesion, the resulting aphagia and adipsia do not appear to be a consequence of CA fiber damage, since the phenomenon cannot be reproduced by injections of the CA neurotoxin, 6-hydroxydopamine (6-OHDA), into the same area [27]. Studies conducted in this laboratory, in brain-cannulated rats, have demonstrated that lesions in the dorsal and ventral midbrain tegmentum not only affect ad lib food intake but also dramatically alter the animals' responsiveness to hypothalamic CA drug manipulations which modify feeding

~This research was supported by NIH research grants MH 22879, MH 06418, and NS 09649, by an Alfred P. Sloan Fellowship (SFL), and by funds from the Whitehall Foundation (SFL). The authors gratefully acknowledge the excellent technical assistance of Messrs. Kevin Chang and Steven Chin. We thank Smith Kline and French Laboratories and Sandoz, Inc. for their generous supply of drugs.

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/120829-15502.00/0

S30

I~EIB()WlTZ AND HAMMER

127,281. While this evidence links hypothalamic CA receptor systems to midbrain projections and suggests that they may be involved in stimulating or suppressing feeding, the specific nature of the function subserved by these projections remains unclear. The objective of the present study was to conduct a more detailed analysis of the impact of midbrain lesions on the rats' ingestive behavior exhibited under a variety of pharmacological and dietary conditions. METHOD

A trim a Is The subjects were 41 male albino Sprague-Dawley rats weighing 350--400 g at the start of the experiment. They were housed individually in metabolism cages and, except where indicated, they were maintained on a light-dark cycle, with the 12-hr light phase beginning at 6:00 a.m. followed by a 12-hr dark phase. Food intake (corrected for spillage), water intake, and body weight of all rats were recorded daily between 2:00 and 5:00 p.m. Water was made available through calibrated tubes with drinking nozzles, and food was either lab cbow pellets or special diets (see below) placed in small glass dishes. Urine was collected in calibrated tubes, the volume recorded, and osmolality determined using a Fiske osmometer. Neurological examinations and tests with different drugs generally occurred between 9:00 a.m. and 1:00 p.m.

~urgel:v Under pentobarbital anesthesia, the animals were placed in a Kopf stereotaxic instrument. The bilateral lesions were made with stainless steel electrodes (size 00 insect pins), which were insulated with Epoxylite and bared to a 0.5 mm conical tip. The rat's exposed skull was first adjusted so that it was level between bregma and lambda. The electrodes were positioned in the midbrain according to the following coordinates. For the lesions in the dorsal midbrain tegmentum, the electrode tip was aimed 0.5 to 1.0 mm anterior to the interaural line, 1.2 to 1.4 mm lateral to the midline, and 6.8 to 7.0 mm ventral to skull surface. F o r the lesions in the ventral midbrain tegmentum, the coordinates were the same except for the depth reading, which was 7.8 to 8.3 mm below skull surface. After lowering the electrode into the midbrain, the lesion was made using a I mA direct anodal current for durations of 10 to 15 sec and a rectal cathode. Sham-lesioned rats received identical treatment except that no current was passed through the lesion electrode. After the lesion, those rats that failed to exhibit spontaneous eating and drinking were offered highly palatable foods (moist or liquid diets, sucrose solutions, and chocolate cookies) and, if necessary, were given intragastric loads (twice daily) of 50% sweetened condensed milk solution with whole egg until spontaneous eating was restored. Testing Procedure Neurological examination. During the first two weeks after lesion, all animals were examined for sensorimotor impairments on at least three separate occasions. These tests, based on those described by Marshall [35,36], included examination of the rat's orientation toward sensory stimuli (that is, head turning toward odor, visual stimulus, sound, whisker touch, and probes to the face and body); use of limbs (that is, accurate position of limbs during righting, climbing, walking, grasping, and pushing probe from mouth);

and mouth movements tin terms of lapping and biting a wooden probe). In addition, lests of irritability were conducted in which the rat's response to a sudden slap against the cage, to capturing and handling, were recorded. Level of activity (number of squares crossed) was measured during a 2-rain test on a 2×3 ft table surface marked off in 6-in, squares. l)rug and hormone manipulations. During weeks 2 to 7 after lesion, several tests of the rat's responses to regulatory challenges and anorexic drugs were conducted. These tests were begun after the animal could maintain its weight on ad lib pellets and water. The compounds examined (see ref. [26[ for review) included: histamine, which causes hypotension (through vasodilation) and hypovolemia (through increased capillary permeability) and consequently stimulates water intake: the dipsogen, isoproterenol, which acts in part through the renin-angiotensin system of the kidney: hypertonic saline, which causes intracellular dehydration and consequently increased water consumption; 2-deoxy-D-glucose, which stimulates food intake by blockade of glycolysis; insulin, which elicits eating consequent to hypoglycemia; and amphetamine and mazindol, which suppress food intake through the release of endogenous catecholamines that act in part on receptors of the perifornical lateral hypothalamus. (1) Histamine. In a 2-hr test. the rats (food- and watersatiated) received two subcutaneous (0.1 cc) injections, the first being sterile physiological saline followed 60 min later by histamine dihydrochloride (Sigma Chemical Co.) dissolved in saline (4 mg/kg). Water intake was measured an hour after each injection, and food was not available during the test. (2) Isoproterenol. A test similar to the histamine test was conducted with l-isoproterenol-d-bitartrate (Winthrop Laboratories, 0.1 mg/kg in 0.1 cc saline), except that water intake measurements were taken at 90-min intervals. (3) Hypertonic saline. Food- and water-satiated rats were given intraperitoneal injections of isotonic saline (3 cc of 0.9% NaC1), followed 60 min later by an equivalent injection of hypertonic (1 M) saline. The amount of water consumed, in the absence of food, was recorded 60 rain after each injection. (4) 2-Deoxy-D-glucose (2-DG). Food (pellets) and water intake measurements were taken 4 hr after intraperitoneal injection (0.5 cc) of 300 and 600 mg/kg of 2-DG (Sigma Chemical Co.) or its saline vehicle. Injections were made on separate days according to a Latin square design. The animals were food- and water-satiated. (5) Insulin. A test similar to the 2-DG test was conducted with regular Iletin insulin (Lilly, 4 and 8 U/kg), except that injections were given subcutaneously in a volume of 0.1 cc. (6) Amphetamine (AMPH). For this test, the rats were placed on an intermittent food-deprivation schedule, whereby they had water but no food for 23 hr prior to the test and then food (pellets) and water ad lib for the next 25 hr. d-Amphetamine sulphate (Smith, Kline and French Laboratories) or its saline vehicle was administered intraperitoneally (0.5 cc) on separate days, and food intake measurements were taken 60 rain after injection. Three doses of AMPH (0.25, 0.5, and 1.0 mg/kg) and the vehicle were tested according to a Latin square sequence, and three dose-response tests were conducted on each rat. (7) Mazindol (MAZ). These tests were the same as those described above for AMPH, except that only two series of dose-response tests were conducted, and the doses tested were 0.75, 1.5, and 4.0 mg/kg.

B E H A V I O R A L D E F I C I T S A N D MIDBRAIN L E S I O N S

Dietary manipulations. During weeks 7 to 15 after lesion, the rats were maintained on food and water ad lib, and daily measurements of food intake, water intake, body weight, and, in some cases, urine output and urine osmolality were taken. During the first few weeks of this test series, the rats were tested (on separate days) on a variety of foods, namely, lab chow pellets, water mash (30% water, 70% lab chow powder), 0.1% and 0.4% quinine mash (0.1% or 0.4% w/v quinine hydrochloride solution mixed with powder in the same proportion as the water mash), and high-fat mash (33% Crisco oil and 67% lab chow powder). Tap water was available at all times. The pellets, water mash, and high-fat mash were available for 7 consecutive days; the quinine diets were offered for 3 consecutive days. The order of presentation of the different diets was randomized according to a Latin square sequence, and one to two days of water mash (to restabilize the rats' feeding behavior) was interspersed between switching from one diet to another. The data collected for the f~rst day on a new diet were not included in the overall mean. A similar sequence of tests was conducted during the next few weeks, when the rats' water was adulterated and lab chow pellets were available. The solutions tested (on separate days) were water, 1.25%, 5%, and 20% w/v sucrose, 0.125% w/v saccharin, and 0.01 and 0.05% w/v quinine. Each solution was available for at least 2 consecutive days, and one to two days of water was interspersed before switching to a new solution. The different solutions were presented according to a Latin square sequence. The final tests of this series involved measuring the rats' circadian rhythm of feeding, drinking, and urine output, and their ability to regulate gram or calorie intake when presented with diets of varying caloric density. The test of circadian rhythm was conducted over a 2-day period while lab chow pellets and tap water were available, and food intake, water intake, and urine output measurements were taken at 12-hr intervals. For the caloric tracking test, a high-fat mash (33% oil, 67% powder) was used, in which the ratio of Crisco oil (9.3 kcal/g) to mineral oil (0.0 kcal/g) mixed with powder (4.3 kcal/g) was varied to achieve diets of 2.9, 3.6, 4.4 and 6.0 kcal/g. Each of these diets was presented separately for 5 consecutive days, and their order of presentation was determined according to a Latin square sequence. Tap water was always available. Deprivation experiments. The final series of tests was conducted between 15 and 22 weeks after lesion, in which the animals' response to food (pellets) and water deprivation was measured. For the first two tests, the animals were deprived of food (water-satiated) or of water (food-satiated) for a 24-hr period, and measurements of food intake and water intake (when available), as well as urine output, were taken during this time of deprivation and compared with measurements taken under ad lib food and water conditions (on days within the same week as the deprivation test). On the day after the 24 hr of deprivation, the animals were given the substance (food or water) of which they had been deprived, and a measurement of consumption was taken after a 2-hr test period. Subsequent to these brief tests, the effects of intermittent or extended food deprivation periods on postfast food intake and body weight were measured. The intermittent deprivation schedule, carried out over a 16-day period, alternated 24 hr of food availability with 24 hr of food deprivation. (Water was available every day.) Subsequent to this test, the animals were given a 3-day fast (with water available) followed by 7 days of food and water ad lib. Each

831 of these deprivation schedules was preceded by a 5-day period during which both food and water were available, and baseline intake scores were obtained.

Histological Analysis At the end of these experiments, between 20 and 24 weeks after lesion, the animals were sacrificed under pentobarbital anesthesia. They were perfused through the heart with isotonic saline followed by a buffered solution of 10% Formalin. The brain was removed, and frozen sections of 50 /z were then cut and alternate sections stained with cresyl violet. In estimating the extent of damage produced by a lesion, the tendency of lesions to contract over time was taken into account. From projections of the stained sections, the area of totally ablated tissue, at the center and caudal aspect of the lesion, was drawn relative to nearby surviving structures. The K r n i g and Klippel atlas [21] was used as a guide, although it should be noted that the angle of cut in the present study was slightly more oblique than the angle used in that atlas. The ring of demyelination that typically surrounds a brain electrocoagulation was included within the area of damage. The broad annulus of pathologic tissue surrounding the central cavity was usually not included in the lesion analysis, since axon and cell damage to that region is generally difficult to interpret, and numerous CA axons in histofluorescence analyses can be seen surviving in that area [27,28].

Statistics The data were analyzed by a single-factor analysis of variance for independent samples (with unequal group size) or a two-factor analysis of variance with repeated measures on one factor. Specific comparisons were made by a Dunnett t-test (for comparisons between the lesion groups and the control group) or by a Newman-Keuls test (for comparisons between all groups) [60]. Some within-group comparisons were made by a Student t-test for dependent samples (twotailed).

RESULTS

Histological Analysis The rats used in this study were separated into 4 groups, namely, one sham group and three lesion groups formed on the basis of lesion placements illustrated in Fig. 1. The dorsal lesion (N= 12) was located just ventrolateral to the central gray. In some animals, the center of the lesion occurred dorsolateral to the red nucleus (Fig. la and c). In other animals, it was located at the level of the raphe nuclei and extended along the dorsal surface of the superior cerebellar peduncles (Fig. lb). The ventral lesion (N=7) encompassed most of the red nucleus, as well as some tissue lateral and just dorsal to the nucleus (Fig. ld and f). The lesion extended ventrally to the dorsal edge of the medial lemniscus. At its most caudal extent (Fig. le), just anterior to the ventral tegmental nucleus, the lesion caused damage primarily along the lateral edge of the superior cerebellar peduncles. The far-ventral lesion (N=8) was found to remain ventral and ventrolateral to the red nucleus and to extend into the medial portion of the medial lemniscus, the substantia nigra pars compacta, and the substantia nigra pars reticulata (Fig. lh and j). There was little or no damage to the ventral tegmental area of Tsai and the mammillary peduncles. At its caudal

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FIG. 1. Illustrations of electrolytic lesions in the dorsal, ventral, and far-ventral midbrain tegmentum. Overlap drawings of the lesions' acellular area are presented at the level of the red nucleus (a, d, g) and the raphe nuclei (b, e, h). All ventral (N=7) and far-ventral (N=8) lesions extended to both levels, as drawn in the figure. The dorsal lesions (N= 12) were somewhat smaller, such that only two lesions with acellular areas at the red nucleus level (a) extended as far posterior as the raphe nuclei. The caudal extent of these two lesions is indicated by broken lines in lb. These lesions overlap the acellular area of the remaining 10 animals with lesion centers at the level of the raphe nuclei (lb) and damage extending approximately 0.5 mm rostral and caudal to this level. Drawings were made from projections of original sections which were cut at a slightly more oblique angle than that used by the atlas of Krnig and Klippel [21]. c, f, i: Cresyl violet-stained sections showing typical lesions at the level of the red nucleus. Abbreviations: CC, crus cerebri; CG, central gray; DR, dorsal raphe nucleus; IP, interpeduncular nucleus; MG, medial geniculate; ML, medial lemniscus; MLF, medial longitudinal fasciculus; nlV, nucleus of trochlear nerve; Pill, principal nucleus of oculomotor nerve; RN, red nucleus: SC, superior colliculus; SCP, superior cerebellar peduncles: SNc, substantia nigra pars compacta: SNr. substantia nigra pars reticulata.

extent, just rostral to the ventral tegmental nucleus (Fig. li), the lesion was located primarily ventrolateral to the superior cerebellar peduncles and along the medial edge of the cerebral peduncles.

Initit~l Measurements Table 1 summarizes the results on food and water intake, body weight, and urine output and osmolality obtained during the first few post-lesion weeks, relative to measurements taken during the week preceding the lesion. The dorsal midbrain lesion ventrolateral to the central gray produced a period of aphagia and adipsia, lasting from 4 to 19 days and resulting in over 100 g (25 to 30%) body weight loss. The ventral and far-ventral midbrain lesions, in contrast, caused only a transient loss of eating and drinking and approximately a 10 to 15% reduction in body weight. Most of these

ventral animals recovered their pre-lesion body weight within 7 days after lesion, whereas the dorsal animals recovered after approximately 20 days. Food intake measurements taken during the first 7 days after weight recovery showed that all groups stabilized at approximately pre-lesion levels except the ventral lesion group, which showed a 17c~: increase in eating. Water intake measurements also normalized, with the exception of the dorsal lesion animals which showed a 27% decrease in consumption. This was associated with a 59% decrease in urine output, a 63% increase in urine osmolality, and a decrease in urine/water ratio (from 0.48 to 0.27, p<0.01). All other groups appeared essentially normal with respect to their urine measurements.

Neurological Examination All tests of orientation toward sensory stimuli, use of

BEHAVIORAL DEFICITS AND MIDBRAIN LESIONS

833

TABLE 1 MEASUREMENTSOF FOODINTAKE,WATERINTAKE,BODYWEIGHT,AND URINE OUTPUT RECORDED ONE WEEK BEFORE AND TWO TO FOURWEEKS AFTERMIDBRAINLESION Midbrain lesion Sham

Dorsal

Ventral

Far-ventral

Days aphagic and adipsic

0

11

3

4

Body weight loss (g)

3

112

60

55

Days to weight recovery

0

9

4

3

Food intake (g/day) Pre-lesion Post-recovery

32 _+ 0.9 31 1.2

30 _+ 28 -+

Water intake (ml/day) Pre-lesion Post-recovery

68 -+ 63 -+

Urine output (ml/day) Pre-lesion Post-recovery

29 --- 4.1 27 --+ 3.2

Urine osmolality (mOsm) Pre-lesion Post-recovery

6.8 5.5

978 -+ 103 1086 -+ 83

1.2 1.1

30 +_ 35 -

0.9 1.8"

29 +_ 0.5 31 -+ 0.6

66 +- 4.3 48 _+ 3.2t

59 -+ 60 -+

3.1 3.8

68 _+ 3.5 62 _+ 2.9

32 +13 -+

28 -+ 24 _+

3.8 2.7

27 - 3.7 23 -+ 4.6

5.1 2.3t

1036 -+ 98 1686 - 131"

1108 _+ 120 1259 _+ 63

1095 --- 88 1241 _+ 76

Given are mean _+ standard error of the mean. Single-factor analyses of variance of the difference scores (pre-lesion minus post-recovery) revealed significant group effects (at least at p<0.05) for each of the above measures. Individual comparisons between the lesion and sham group scores (using Dunnett's t-test) yielded significant differences at p< 0.05 (*) and p<0.01 (t).

limbs, mouth movements, and irritability failed to reveal any reliable differences between the sham, ventral, and farventral lesion animals. With regard to the dorsal lesion animals, specific changes were observed, primarily during the first week after lesion, which clearly distinguished them from the other groups. These animals appeared normal in many respects. They displayed no akinesia or catalepsy and exhibited normal posture and use of their limbs and normal mouth movements (lapping and biting) in response to a wooden probe. They also appeared to show appropriate head-turning in response to a whisker touch, face or body probe, and auditory, olfactory, and visual stimuli. Visually elicited placing also appeared normal. In the open-field test, however, these animals were very hyperactive, crossing 37 _ 6.7 squares in contrast to 10 ± 1.2 squares for the sham animals (p<0.01). Furthermore, the irritability tests revealed an increased reactivity (jumping response) to a sudden noise and in circling movements in response to capture. These results are confirmed by additional observations made of the rats' spontaneous behavior in the home cage. In this situation, they exhibited excessive circling, exploration, sniffing, and jaw, tongue, and teeth movements. While most rats showed no signs of aversion to the food or water (they would exhibit some eating if their mouths were immersed in food), their ability to orient properly to the food dish or water spout was greatly impaired as reflected, for example, by their tendency to walk indiscriminately through dishes of mash and to lose their orientation to the food dish or water spout after exhibiting a little ingestion. (See [32] for detailed description of this behavior.) During the first post-lesion week, this symptom, as well as the circling and exploratory

behavior, greatly subsided. In some animals, this improvement was associated with more effective and prolonged contacts with the food or water (and recovery of normal feeding and drinking within 2 to 3 days), whereas in other animals, the aphagia and adipsia persisted for several more days. The availability of palatable foods (such as sweetened condensed milk or wet mash) greatly helped the recovery process, and water ingestion generally became apparent at approximately the same time as food ingestion.

Drug and Hormone Manipulations With regard to the compounds which elicit drinking through their effects on the fluid compartments of the body (cellular and extracellular spaces), all midbrain lesion rats appeared to respond normally (Table 2). A significant increase in water intake over vehicle baseline (at least at p<0.01) was observed in all groups after peripheral injection of histamine, isoproterenol, and hypertonic saline. As shown in this Table, 2-DG and insulin also elicited a reliable drinking response in all groups (food was available during the tests with these compounds), although the dorsal lesion animals showed a significantly reduced response compared with the sham animals. This change reflects the food intake results for 2-DG and insulin, shown in Fig. 2. In the sham and ventral lesion animals, these two drugs, which decrease glucose utilization and blood levels, respectively, were effective in eliciting a reliable eating response (at p<0.001), consistent with findings reported by Ahlskog [2]. In the dorsal lesion animals, in contrast, both drugs failed to produce this re-

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FABLE 2 DRINKING IN RESPONSETO PERIPHERAL DRUG AND HORMONE MANIPUIA I'tONS IN SHAM-OPERATEDAND LESIONED RATS Water intake Imilliliters) Midbrain lesion Dose Histamine Isoproterenol Hypertonic saline 2-DG Insulin

4 mg/kg 0.1 mg/kg 3cc, IM 300mg/kg 600 mg/kg 4 U/kg 8 U/kg

Sham 7.6 6.0 6.6 5.3 4.8 5.4 6.9

÷ 0.9 ± 0.6 ± 1.2 ± 1.6 _+ 1.0 +_ 0.7 ~ 1.2

Dorsal 6.2 4.9 4.3 1.5 2.4 1.8 2.4

~: 1.5 :_~0.8 ~ 1.2 _+ 0.I, ] . ± 1.0 ± 0.7 _+ 0.? ] +

Ventral 8.1 5.8 6.7 3.1 5.8 4.2 6.1

± 1.4 ± 1.0 ± 1.5 ± 1.0 ~. 1.3 ± 0.8 ± 0.9

Far-ventral 7.0 4.8 7.0 4.1 4.0 3.7 4.8

± 1.5 ± 1.2 :~: 1.4 ± 1.8 ~ 1.0 + 0.8 ± 0.7

Given is mean difference between drug and vehicle scores ± standard error of the mean. While 'all groups appeared similar in terms of their response to histamine, isoproterenol, and hypertonic saline, sing!e-factor analyses of variance performed on the 2-DG and insulin scores (averaged across doses) revealed significant group effects (at p <0.05). Specific comparisons (between lesion groups and sham group) were significant at p<0.05 (*) and p<0.01 (+).

sponse, either during the 3-hr test or over a 24-hr interval, despite the fact that these animals were eating normally under ad lib feeding conditions (Table 1) and drinking normally in response to thirst challenges (Table 2). This loss of responsiveness to glucoprivic challenges, previously demonstrated for 2-DG with knife cuts in the area of the present dorsal lesions [4], does not appear to be a direct consequence of the initial aphagia phenomenon, since animals of the ventral lesion group, which also exhibited some aphagia (for 4 to 6 days), ate normally in response to 2-DG and insulin. While these data with glucodynamic hormones distinguish the dorsal lesion group as exhibiting a dramatic postlesion effect, the results obtained with the anorexigenic drugs distinguish the ventral lesion groups in terms of their change in responsiveness. These data are presented in Fig. 3. Statistical analysis of these findings, using a two-factor analysis of variance, showed the groups to be significantly different, both in response to AMPH, F(3,37)=15.01, p<0.001, and MAZ, F(3,37)=6.23, p<0.01. Specific comparisons (via a Newman-Keuls analysis) between the group scores averaged across doses revealed no differences between the dorsal and sham groups. They showed, in contrast, the ventral and far-ventral lesion animals to be significantly less responsive to AMPH and MAZ (at p<0.05) than the sham animals and, furthermore, showed the far-ventral animals to be significantly less sensitive (at p<0.01) to AMPH than the ventral group. This latter difference was not revealed in the case of MAZ, whose anorexic action appeared generally less affected by the lesion than that of AMPH.

Dietao' Manipulations Figure 4 shows the effects of the three midbrain lesions on ad lib food intake, under various dietary conditions, between 7 and 10 weeks after lesion. The dorsal lesion animals, which were aphagic and adipsic during the first 5 to 20 days after lesion, appeared to eat normally on all diets tested. Both these animals and the sham animals responded appropriately

to the dilution of the water mash (increase intake) and the bitter taste of the quinine diets (decrease intake). The fact that both groups ate the same amount in grams but more in calories of the high-fat mash (6.0 kcal/g) compared with the pellets (4.3 kcal/g) reflects the rats' strong preference for the greasy diet. The ventral and far-ventral lesion groups appeared to respond similarly to the diet manipulations, except that all food intake scores were proportionately higher (20 to 35% relative to the sham animals). (These data revealed this effect for the far-ventral animals, although it was not apparent during the first few post-lesion weeks.) The only differential change detected was with the ventral lesion group which, compared with the other groups, showed a significantly greater increase in their consumption of high-fat diet relative to their consumption of the pellet diet 6o<0.01). Body weight measurements tended to reflect these changes in food intake. That is, the sham and dorsal lesion animals showed similar body weight gain, between 2 and 3 g/day on pellets and water mash and approximately 4.5 g/day on high-fat mash. The ventral lesion groups, in contrast, exhibited an increase in their weight gain and, in general, their scores (g/day) were two-fold greater than the sham group scores. (All sham-lesion comparisons were statistically reliable at least at p<0.05.) On the pellet diet, the ventral lesion group (6.6-+ 0.6 g/day) showed a significantly greater (p<0.01) weight gain than the far-ventral group (4.9 _+ 0.2 g/day). These groups appeared similar, however, on the water mash (approximately 4 g/day) and the high-fat mash (approximately 9 g/day). The water intake scores collected during these food manipulation tests essentially confirmed those presented in Table 1. Adulteration of the water revealed further differences between these groups. In these tests, the primary change was observed in the dorsal lesion animals, which showed a large decrement in their consumption of sucrose or saccharin solutions (Fig. 5). When offered dilute sucrose (1.25% and 5%), the sham animals showed a dramatic increase (p<0.001) in their consumption (+ 179% and 305%, respectively) relative to their intake of water. The ventral lesion animals responded similarly, although their scores were not quite as

835

B E H A V I O R A L D E F I C I T S A N D MIDBRAIN L E S I O N S

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Midbrain lesion FIG. 2. Increase in food intake produced by peripheral injection of 2-deoxy-D-glucose (2-DG; shaded bars, 300 mg/kg; open bars, 600 mg/kg) or insulin (shaded bars, 4 U/kg; open bars, 8 U/kg) in shamoperated animals (N= 14) and in animals that received electrolytic lesions in the dorsal (N= 12), ventral (N=7), and far-ventral (N=8) midbrain tegmentum. See Fig. 1 and text for illustrations and description of midbrain lesions. Single-factor analyses of variance performed on these data (averaged across doses) yielded significant group effects (p<0.01) for 2-DG and insulin. These effects were attributed primarily to the dorsal group which exhibited a reliable decrease in responsiveness (*, p<0.01) compared with the sham animals.

high. The dorsal lesion rats, however, only increased their intake by + 19% (p>0.10) and 119% (p<0.01), respectively, which is considerably less than the scores of the sham as well as the ventral lesion animals. This reduced responsiveness of the dorsal lesion animals to sweet solutions was similarly observed with the saccharin-adulterated water (see study by Schiff [53] for similar results), and to a slight extent with the concentrated (20%) sucrose solution. In a two-bottle test, in which water and 5% sucrose were simultaneously available, these dorsal lesion animals drank between 80 and 90% of their fluid from the sucrose bottle. This result and the out-

FIG. 3. Percent suppression of food intake, relative to vehicle baseline, observed after peripheral injection of amphetamine and mazindol in sham-operated animals (N= 14) and in animals that received electrolytic lesions in the dorsal (N= 12), ventral (N=7), and far-ventral (N=8) midbrain tegmentum. See Fig. 1 and text for illustrations and descriptions of midbrain lesions. Specific comparisons between the group scores revealed a significant decrease in responsiveness (at p<0.01) to AMPH or MAZ for the ventral and farventral lesion animals (compared with sham animals) and a significantly greater loss of responsiveness to AMPH in the far-ventral group compared with the ventral group (p<0.01).

come of the 20% sucrose test suggest that these animals were essentially normal with respect to their gustatory sensitivity. This suggestion is confirmed by the results obtained with the bitter quinine-adulterated water, where all groups showed an appropriate decrease in consumption relative to water intake. At the lower concentration (0.01%), the dorsal lesion animals showed a slightly smaller reduction in intake; however, this difference did not quite reach statistical significance. Analysis of the rats' circadian rhythm of food (pellet) intake, water intake, and urine output is shown in Table 3. The

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FIG. 4. Daily food intake, averaged over 3 to 7-day periods of a particular diet, exhibited by shamoperated animals ( N = 14) and by animals that received electrolytic lesions in the dorsal ( N = 12), ventral ( N = 7 ) , and far-ventral ( N = 8 ) midbrain tegmentum. See Fig. 1 and text for illustrations and descriptions of midbrain lesions. Single-factor analyses of variance performed on each of the five diets yielded significant groups effects (o<0.05) in each case. Specific comparisons (via Dunnett t-test) between scores of the lesion and sham animals revealed a reliable increase in food consumption at p < 0 . 0 5 * and p < 0 . 0 1 ** for the ventral and far-ventral lesion animals on each diet tested.

TABLE

3

CIRCADIAN RHYTHM OF FOOD INTAKE, WATER INTAKE, AND URINE OUTPUT IN SHAM-OPERATED AND MIDBRAIN-LESIONED RATS Food intake

Sham Dorsal lesion Ventral lesion Far-ventral lesion

Water intake

Urine output

24 hr

Light

24 hr

Light

24 hr

Light

(g)

period (%)

(ml)

period (%)

(ml)

period (%)

30+__2.8 30 _+ 4.3 36 _+ 2.3+ 33 _+ 1.8

17-* 2.3 31 -* 2.6t 15 --- 3,2 13 __- 4.6

8-+ 2.3 12 -+ 3.2 8 -+ 2.6 9 + 3.0

33_+ 4.8 19 -+ 4.5* 28 _+ 3.8 25 _+ 3.5

75_+ 43 _ 68 -+ 62 +--

8.5 3.2+ 9.9 10.6

11 25 14 17

-+ 2.1 -+ 2.6t +_ 3.1 -+ 2.8

Given are 24-hr scores (g or ml) and ratio of light/24-hr scores × 100 (%). Single-factor analyses of variance performed on the 24-hr and the light period (%) scores revealed significant F values (at least at p < 0 . 0 5 ) in all cases except for the water intake, light period scores. Dunnett t-test yielded significant differences at p < 0 . 0 5 * and p < 0 . 0 1 t between individual lesion groups and the sham scores.

BEHAVIORAL DEFICITS AND MIDBRAIN LESIONS

837

200 180 160 E 140 .=

120

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I00 g

Sham Dorsal Ventral Far-ventral

80 60 40 20 Water

1.25% Sucrose

5% Sucrose

20% Sucrose

0.125% Saccharin

0.01% Quinine

0.05% Quinine

FIG. 5. Dally fluid intake, averaged over 2 to 3-day periods of a particular drinking solution, exhibited by sham-operated animals (N= 14) and by animals that received electrolytic lesions in the dorsal (N= 12), ventral (N=7), and far-ventral (N=8) midbrain tegmentum. See Fig. 1 and text for illustrations and descriptions of midbraln lesions. In the case of the sucrose and saccharin solutions, single-factor analyses of variance revealed significant group differences (at p<0.05), and the specific comparisons demonstrated a reliable decrease in fluid consumption at p<0.05 * and p<0.01 ** for the dorsal lesion animals compared with the sham animals. All groups appeared similar when maintained on the quinine-adulterated solutions.

24-hr measurements confirmed the findings (see Table 1) that the dorsal lesion caused a reduction in water intake (43%) and urine output (42%), and the ventral lesion caused a significant increase in food intake (+20%). The nocturnal rhythm normally exhibited by rats, where 85 to 90% of their feeding, drinking, and urination may occur at night, was observed in the sham and ventral lesion rats, consistent with results on food intake reported by Ahlskog [3]. The dorsal lesion animals, however, revealed a disturbance of this pattern, such that a significantly greater proportion of their total food intake (31%, p<0.01 compared with sham rats) and urine output (25%, p<0.01) occurred during the day. This adjustment in circadian rhythm did not cause any differential changes in urine/water ratio relative to the other three groups. All groups exhibited a ratio of 0.40 to 0.45 over the 24-hr measurement period and a higher ratio of 0.60 to 0.80 during the day. The effects of varying caloric density of the animals' diet can be seen in Table 4. All groups showed evidence of caloric tracking, as they decreased their total gram intake with increase in caloric density, F(3,111)=32.67, p<0.001. No differences between the groups were detected, indicating

that the lesions had no apparent effect on the accuracy of caloric regulation. It should be noted, however, that the rats' adjustment of gram intake was not sufficient to maintain calorie intake at a constant level. That is, as the proportion of Crisco oil (9.3 kcal/g) to mineral oil (0.0 kcal/g), and consequently caloric density, was increased, the total calorie intake also increased, F(3,111)=21.79, p<0.001. This error of caloric regulation has been previously reported and attributed to the rats' preference for calorically denser diets, as well as to a palatability preference [10].

Deprivation Experiments The results of the first two tests, in which the rats received a single 24-hr period of food or water deprivation (approximately 15 weeks after lesion), revealed no differences between the groups in terms of the effects of deprivation on their food and water intake during the deprivation period. When deprived of food, all groups decreased their water intake by 50 to 60% (p<0.001), and when deprived of water, they decreased their food intake by 25 to 35% (p<0.001). The 2-hr tests which followed the 24-hr depriva-

83S

I.EIBOWITZ AND HAMMER ]'ABLE INTAKE

(g) OF

HIGH-FAT

DIETS

4

VARYING

IN CALORIC

DENSITY

Caloric density (kcal/g) 2.9 Sham Dorsal lesion Ventral lesion Far-ventral lesion

38 41 56 54

+ 2.1 _+ 4.9 _4-5.2 ± 6.1

4.4

3.6 33 34 51 49

+ 3.3 + 2.2 4- 3.8 ± 4.9

29 ± 30 + 41 ± 44 ±

6.0

2.2 5.2 5.3 4.9

25 _+ 2.1 28 _+ 1.9 38 + 6.1 37 ÷ 3.2

Given are mean daily food intake (g) _+ standard error of the mean. Twofactor analysis of variance revealed a significant F value for the caloric density measure (F(3,11)=32.67, p<0.001) but insignificant values for the group effect and the group x treatments interaction.

~'- ~

tion periods also failed to reveal any reliable differences between the groups. After food deprivation, all groups ate (in 2 hr) between 46 and 54% of their normal daily food intake, and after water deprivation, all groups drank (in 2 hr) between 27 and 33% of their normal daily water intake. Thus, it appears that the midbrain lesions had little impact on the animals' food and water intake responses to a single 24-hr period of deprivation. With respect to urine output, however, the results revealed a clear effect in the dorsal group that distinguished it from the other groups. Whereas the sham and ventral lesion animals conserved their urine under both the water deprivation (50 to 65% decrease in output, p <0.001) and food deprivation (20 to 25% decrease in output, p<0.05) conditions, the dorsal lesion animals showed no significant decrease in urine output during water deprivation (15 _+ 5.0 ml under ad lib conditions and 12 _+ 2.7 ml under deprivation, p>0.10), and actually increased their output, from 18 _+ 3.0 and 26 _+ 4.5 ml (+ 44%, p<0.01) during food deprivation. The percentage of urine output to water intake also increased (from 51% under ad lib conditions to 145% under food deprivation, p<0.001), to a significantly greater extent (p<0.01) than the sham or ventral lesion animals. While these tests failed to show any differential effects of a single day of food deprivation on subsequent (2-hr) food intake, further tests with repeated alternate days of food deprivation revealed a marked deficit in the dorsal lesion animals' compensatory feeding response on days of ad lib food. As shown in Fig. 6, the sham and ventral lesion animals, on alternate days of food availability, ate between 25% (tests 1-3) and 50% (tests 4-8) more than they normally do under non-deprivation conditions. This pattern is clearly different from that of the dorsal lesion animals, which showed a tendency toward compensation during the first 3 test days (20 and 25% increase) but then reduced their food intake during the subsequent 5 days such that they ate even less (by 10%) than they did under ad lib food conditions. A two-factor analysis of variance performed on these data (averaged across days 1-4 and 5--8) revealed a significant group effect, F(3,37)=9.42, p<0.01, and group × test days interaction, F(3,37)=7.65, p<0.01. Individual comparisons showed no difference between the sham and ventral lesion animals but showed the scores of the dorsal animals, on days 4 to 8, to be significantly lower (at p<0.01) than those of the other groups. The body weight scores tended to reflect these food intake changes. The sham animals increased their

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weight by 1.8% and the ventral and far-ventral lesion animals by 4 to 5% (indicative of the hyperphagia exhibited by these animals even under intermittent deprivation). The dorsal lesion animals, in contrast, showed a small but significant decrease in body weight ( - 2 . 6 % , p<0.05 compared with sham rats), reflecting their deficit in compensatory feeding. The effects of a 3-day fast on these animals' food compensatory responses and associated body weight changes confirm the results obtained on the intermittent deprivation schedule. During the fast itself, all groups exhibited approximately a 13% loss of body weight. During the subsequent 7-day period of ad lib food and water, the sham animals exhibited a 13% increase in their daily food intake and re-

B E H A V I O R A L DEFICITS AND MIDBRAIN LESIONS covered 98% of their lost body weight. The ventral and farventral animals exhibited a similar change. However, the dorsal animals showed a dramatically reduced post-fast compensatory response (a 2.5% increase in daily food intake) and body weight recovery (only 75%). These scores were significantly less (at p<0.01) than those of the other three groups.

DISCUSSION

Electrolytic lesions in the dorsal midbrain tegmentum, ventrolateral to the central gray, were found to produce moderate to severe (4 to 19 days) aphagia and adipsia in the rat. This effect, originally described by Gold [18], Lyon et al. [32], and Parker and Feldman [44], was followed by complete recovery of ad lib food intake but a sustained suppression of water intake and urine output. It was also associated with persistent deficits in the animals' feeding response to glucoprivation, in consumption of sucrose or saccharin solutions, in post-fast compensatory food intake, and in the ability to maintain a nocturnal pattern of feeding. In contrast to these changes, electrolytic lesions in the ventral midbrain tegmentum produced an increase in food intake and body weight gain. This effect, originally described by Ahlskog [1], was found in the present study to be associated with no other food- or water-related behavioral changes, with the exception of AMPH- or MAZ-induced anorexia, which was attenuated or abolished by the lesion. The anatomical basis for the aphagia and adipsia produced by the dorsal lesion is unknown. The neurological tests indicated that the rats were active and essentially normal on basic motor capacities. Furthermore, their orientation to isolated sensory stimuli in the test situation (that is, under activating and restricted conditions) appeared normal. Observations of spontaneous behavior in the home cage, however, revealed abnormal behavior with respect to the rats' orientation to the food dish and water spout (the only objects within the cage). It is not clear whether this behavior reflects a deficit in localization and utilization of sensory information or whether it simply reflects a hyperactive arousal. Lyon et al. [32] suggested that these animals may have sensory inattention, and it is possible that this deficit, along with the hyperactivity, hyperexploration, excessive circling and sniffing also observed, may be attributed to the destruction of multiple, long, ascending and descending pathways that provide the basis for normal sensorimotor integration [56]. While these functional deficits may have contributed to the decrease in food or water intake, it is not clear that they were the primary cause of the deficit, since recovery of spontaneous eating sometimes occurred several days after apparent recovery of coordinated eating activity (such as that exhibited when fresh food was first introduced into the cage), and a specific sensorimotor deficit in head orientation to stimuli (after superior colliculus lesions) has been shown to occur without a deficit in food intake [35]. Extensive damage specifically to visceral and gustatory afferents undoubtedly occurred with this lesion [40], which, in view of the importance of orogastric sensations in food intake regulation, may account for some of the feeding deficit. However, lesions in the dorsolateral pons, which also presumably severed these fibers, have been found to produce little change in body weight and, by inference, food intake (Norgren, personal communication). Finally, the possibility remains that the initial impairment in feeding

839 may, in part, be secondary to the impairment in water ingestion. The available evidence suggests that the aphagia and adipsia observed after dorsal midbrain electrolytic lesions cannot be attributed to damage of the various ascending CA fibers. Injections of 6-OHDA into the dorsal tegmentum, just ventrolateral to the central gray, fail to reproduce these effects [27]. Instead, 6-OHDA there causes a small but significant reduction of food intake (10 to 2(F~), associated with a tendency toward increased water ingestion. This evidence distinguishes these effects of dorsal midbrain lesions from similar but more prolonged and perhaps extensive deficits observed after electrolytic and 6-OHDA lesions in the lateral hypothalamus and along the nigrostriatal dopamine projection [15, 36, 57]. The dorsal midbrain 6-OHDA actually produced an increase in water intake, which is particularly interesting in light of the above-normal excretion of urine detected after dorsal midbrain electrolytic lesions, under conditions of water deprivation (see above). Both of these effects, namely, increased water intake and urine excretion, have also been observed after lesions in the area of the locus coeruleus A6 noradrenergic cells [42,46]. The dorsal midbrain lesion is located 1 to 2 mm rostrai to the locus coeruleus and would be expected to damage some locus fibers as they follow a rostroventral course through the brainstem [27]. It is possible, therefore, that the similar effects of these two brainstem lesions on fluid balance may be attributed to the destruction of a common locus projection. This suggestion is consistent with the evidence that noradrenergic stimulation of the PVN and SON has a potent suppressive effect on water consumption and urine excretion [26] and that locus coeruleus lesions [43], as well as the dorsal midbrain lesion [27], cause a decrease in norepinephrine content or CA fluorescence of these hypothalamic nuclei. This proposed pontine-hypothalamic noradrenergic projection, for regulation of water intake and urine excretion, must remain speculative, however, until more systematic and direct analyses, with specific neurotoxins, histochemical procedures, and functional tests, are conducted on the noradrenergic locus cells and their ascending locus fibers (see ref. [50]). With regard to the hyperphagia and increased weight gain induced by ventral midbrain lesions, this has been attributed to damage sustained by noradrenergic fibers of the central tegmental tract, that is, the ventral noradrenergic bundle [ 1]. The present findings replicate this phenomenon with lesions which are expected to damage these ventral CA fibers. The present study, however, does not evaluate critically the possible contribution of non-catecholaminergic neurons to the feeding change [41]. Greater insight into the neural mechanisms and behavioral functions disrupted by these lesions may be obtained from the additional studies in which a variety of pharmacological, hormone, dietary, and experimental manipulations were performed on the lesioned animals. All groups appeared to respond normally on several of the tests conducted. These included the tests with histamine, isoproterenol, and hypertonic saline, which examined the rats' drinking behavior in response to intracellular and extracellular dehydration; the test in which the caloric density of the diet was varied to assess the rats' accuracy of caloric regulation; and the single 24-hr tests of food- or water-deprivation which examined the rats' ability to regulate independently food and water intake and to work toward replenishing their fluid and energy stores during a 2-hr period after deprivation.

840 A lourth set of tests, in which a variety of tbods were used (pellets, water mash, quinine mash, and high-fat mash) failed to reveal any differential responsiveness of the animals to dietary palatability, with the exception of the ventral lesion group which appeared to overeat to a significantly greater extent on the high-fat mash (+ 54%) compared with the other diets (+ 20 to _+ 30%). This result may in part explain the finding of Ahlskog [2] that ventral lesion animals, when switched from a diet of low caloric density (pellets) to one of high caloric density (high-fat mash), are slower than normal in readjusting their intake to isocaloric levels. While the above tests yielded essentially negative results, additional experiments have strongly distinguished the dorsal lesion animals as showing persisting deficits in food intake regulation under specific circumstances. These deficits were: Ca) a loss of eating in response to glucoprivation, (b) a failure to increase eating in compensation for food deprivation, (c) a decrease of sucrose consumption, and (d) a decreased ability to sustain a nocturnal pattern of eating. The significance of these deficits and their relationship to each other will need to be investigated in future studies. However, it is interesting that a similar constellation of functional disorders has been discovered for the intact hamster [48,54], which can adjust its food intake in response to certain ongoing challenges, such as lactation, exposure to cold, and dietary dilution (similar to dorsal lesion rats), but is somewhat unique amongst mammals in being unable to detect and/or compensate for the effects of food deprivation. Silverman and Zucker likened the nocturnal eating rhythm to a cycle of food deprivation and compensation. These authors found that hamsters, which failed to increase their nutrient intake in response to food deprivation and glucoprivation (induced by 2-DG), also did not engage in this daily cycle and lacked the ability to do so when experimental conditions demanded it. Thus, it appears that the feeding system of the hamster is unresponsive to some signal of energy depletion, and Silverman and Zucker proposed that the hoarding and perhaps hibernation which hamsters engage in may have reduced their need to compensate for periods of food deprivation. The system in rats designed to perform this function may be precisely that which has been damaged by the dorsal lesion. Several lesion stuides in the rat have linked brain CA systems to the regulation of eating response to 2-DG and insulin, as well as to the ingestion of glucose [7~ 11, 13, 36, 57]. While the specific CA projections involved in this function have not yet been identified, the present results, in light of additional published evidence, lead us to propose that norepinephrine (NE) systems that course through the dorsal midbrain tegmentum and project to the medial hypothalamus may contribute to these phenomena, as well as to post-fast compensatory feeding and, perhaps, to the nocturnal pattern of feeding. The dorsal lesion, which in the present study was found to disrupt each of these functions, has invariably been found in a separate study to damage specifically the dorsal NE fibers of the central tegmental tract, which join the medial tegmental radiations and innervate the paraventricular and periventricular nuclei of the hypothalamus [27]. This lesion, furthermore, was found to abolish the eating response induced by presynaptic release of endogenous NE in this brain area, while at the same time potentiate the eating response evoked by postsynaptic receptor stimulation, that is, by exogenous NE. The possibility that damage to this midbrainmedial hypothalamic N E feeding system may to some extent account for the functional deficits observed in the present investigation (i.e., loss of eating to glucoprivation or in corn-

LEIBOWITZ AND HAMMER pensation for rood deprivation, decreased sugar consumption, and altered circadian rhythm) is supported by several additional studies which have demonstrated that: la) 6-OHDA injected into the same area as that affected by the dorsal midbrain lesion is found in preliminary studies (Leibowitz, unpublished results) to attenuate or abolish eating to 2-DG and decrease preference for diets containing sugar. (Further analyses of these animals' circadian rhythm or of their ability to compensate for food deprivation have not yet been conducted.): (b) 2-DG and insulin stimulate the turnover and release of NE in the hypothalamus [37,49]: (c) NE and 2-DG, when injected directly into the rat lateral ventricles, produce a similar sequence and time course of ingestive responses, consisting of initial drinking followed by eating [5]. As demonstrated with N E [26], these responses induced by 2-DG are selectively antagonized by c~-adrenergic blockade [5]; (d) eating induced by peripheral 2-DG is blocked by peripheral injection of s-methyl tyrosine and ventricular injection of 6-OHDA or the c~-adrenergic blocker phentolamine [38]; (e) 2-DG and insulin produce a specific preference for sugar in animals [22, 47, 55] and in humans [58]. Norepinephrine injected into the hypothalamic paraventricular nucleus of the rat also causes an increased preference for carbohydrate [26], as do the tricyclic antidepressants which are believed to act through their stimulating effect on endogenous NE [31[ and are similarly associated with carbohydrate craving in humans [39,45]: if) genetically obese Zucker rats, which are shown to have reduced NE levels specifically within the hypothalamic paraventricular nucleus [12], are also found to exhibit a selective decrement in sweet preference [17]; (g) surgical disruption of the vagus, which relays sensory and visceral information between the periphery and the brain, causes a decrement in sucrose intake [16], while abolishing eating elicited by hypothalamic NE injection [52] and peripheral 2-DG injection [6]; and (h) the anterior hypothalamic area shows a circadian rhythm of endogenous NE levels [331 and NE-elicited feeding may vary as a function of the day-night cycle [34]. (However, see [25].) This evidence notwithstanding, the precise nature of the signal to which the noradrenergic neurons may respond to elicit feeding still remains unclear. While these neurons may serve a compensatory function and become activated by a homeostatic imbalance associated with food deprivation or, specifically, glucoprivation, they may also serve an anticipatory function in preparing the organism for future periods of deprivation. What appears clear from the present study is that NE systems to medial hypothalamus apparently damaged by the dorsal midbrain lesion are not essential for regulation of feeding under conditions of continuous food availability. Further studies with histochemical analyses and selective CA lesions (with 6-OHDA), as well as with more sensitive behavioral tests, will need to be performed to verify the involvement of the midbrain-hypothalamic NE pathway and to define more precisely the nature of the function subserved by this system. In contrast to the dorsal lesion animals, all tests conducted on the ventral and far-ventral lesion animals revealed only one deficit in feeding regulation besides the hyperphagia discussed earlier. This deficit was an attenuation of the animals' anorexic response to peripheral administration of AMPH or MAZ. The dorsal lesion animals responded normally to the feeding suppressive effect of these drugs. The ventral lesion animals, in contrast, showed a 10 to 40% decrease in responsiveness to both drugs (depending on dose),

BEHAVIORAL DEFICITS AND MIDBRAIN LESIONS and the far-ventral lesion animals reduced their responsiveness by 50 to 100% in the case of AMPH and 25 to 40% in the case of MAZ. Pharmacological studies of these drugs' anorexigenic action have clearly indicated the involvement of brain CA (dopamine [DA], NE, and perhaps epinephrine [EPI]) in the mediation of this phenomenon [17]. One site of action appears to be the perifornical region of the lateral hypothalamus, where these drugs as well as the CA agonists are effective in suppressing food intake [23, 24, 29, 30]. In a study similar to the present one [28], except that AMPH and MAZ were injected directly into this hypothalamic area of braincannulated rats, very similar results were obtained; that is, the dorsal midbrain lesions produced no change, the ventral midbrain lesions caused a partial loss of AMPH and MAZ anorexia, and the far-ventral midbrain lesions produced a significantly greater effect with AMPH (up to 100% loss of response) and only a partial reduction of MAZ anorexia. In that study, the animals' brains were analyzed with fluorescence histochemistry, and the results indicated that both the ventral and far-ventral lesions (electrolytic and 6-OHDA) caused a dramatic reduction of perifornical CA varicosities. Furthermore, analyses at the level of and caudal to the lesion demonstrated that, for the ventral lesion, the ventral NE (or EPI) fibers of the central tegmental tract were most consistently and extensively damaged, and for the far-ventral lesion, the ventral DA A8 cells and the DA A9 cells of the substantia nigra compacta were the primary target. The dorsal lesion, which had no effect on these ventral CA projections and instead damaged dorsal NE fibers of the central tegmental tract, also had no effect on amphetamine or mazindol anorexia and on perifornical CA varicosities. While the present lesions (Fig. 1) were not analyzed with fluorescence histochemistry, they appeared quite similar to those of the central injection study [28], with possibly more damage occurring to the ventral A8 cells in the case of the ventral lesion and to the ventral adrenergic fibers in the case of the far-ventral lesion. The similarity of results obtained after peripheral and central drug administration argues in favor of the hypothesis that peripherally injected AMPH and MAZ act in part through perifornical hypothalamic CA mechanisms to suppress feeding. Ahlskog [1] originally suggested the involvement of the ventral adrenergic fibers in this phenomenon and, consistent with the present study, he and, subsequently, Samanin et al. [51] demonstrated a partial (50%) reduction in AMPH's anorexic action after lesions in the paths of these fibers. In the Samanin study, the anorexic effect of MAZ was found to be almost abolished by this ventral lesion, which contrasts with the partial attenuation observed in the present experiment. Since histological results were not presented in these other studies, however, comparisons between the specific lesions could not be made, although descriptions of the lesions indicated that they fell immediately caudal to our ventral lesions, that is, behind the DA cell bodies. A particularly significant finding of the present study was that the far-ventral lesions, aimed at the DA A8 and medial A9 cell bodies, were significantly more effective than the ventral lesions, aimed at the ventral adrenergic fibers, in

841 attenuating the anorexic effect of AMPH. While electrolytic [9] and 6-OHDA [15] lesions in the vicinity of the substantia nigra A9 cells have previously been found to interfere with AMPH's action, both lesions (as in the present study) were very likely associated with damage to other CA projections which course just dorsal to this cell group. Thus, it is difficult to determine whether the nigrostriatal system, a major source of extra-hypothalamic CA, is specifically involved in CA-induced anorexia. Evidence against this possibility is provided by the finding that 6-OHDA injections into the caudate nucleus have no effect on AMPH's anorexigenic action [51], and that injections of CA stimulants (including AMPH) directly into the caudate produce no change in baseline food intake [23,29]. These central mapping studies suggest, instead, that h y p o t h a l a m i c DA receptors, specifically in the lateral perifornical area, are involved in feeding inhibition, and the present study indicates that the ventral midbrain may be a source of the cell bodies which project to the hypothalamus and mediate this function. Kizer et al. [20] have revealed a decrease of hypothalamic DA after ventral midbrain lesions in the area of the A8 and A9 cell bodies. It remains to be established, however, that this neurochemical change reflects a direct neuronal link between the midbrain and hypothalamus, as opposed to an indirect or secondary consequence of damage to particular midbrain cells that have impact upon the activity of hypothalamic CA neurons [8]. Further anatomical studies are clearly needed to establish the existence of a midbrain-hypothalamic DA projection. The possibility that such a pathway exists, specifically innervating the perifornical lateral hypothalamus, is supported by the autoradiographic study of Fallon and Moore [14], which shows that cells of the ventral midbrain, in the area of the DA A8 and A9 cell groups, terminate in the perifornical area. Further support comes from the results of lesions in the vicinity of these DA cells, which are found to potentiate the anorexic effect of DA receptor stimulation in the perifornical hypothalamus while causing a marked reduction of CA varicosities in that area and a total loss of AMPH anorexia [28]. From these findings, it is concluded that AMPH anorexia is mediated via at least two CA projections which originate in or course through the ventral midbrain tegmentum and project to the hypothalamus. They are the ventral adrenergic or noradrenergic fibers of the central tegmental tract and the dopaminergic fibers of the A8 or A9 cell groups. The CA projections in the dorsal midbrain tegmentum appear not to be involved. With regard to MAZ, the evidence showing only a partial attenuation of its action with ventral or farventral lesions indicates that this anorexigenic agent may act in part through these midbrain-hypothalamic projections but that other brain systems may play an important role. Pharmacological studies have demonstrated that MAZ, like AMPH, stimulates the release of brain CA. A number of investigations, however, have revealed several differences in the pharmacological profiles of these two drugs [17,30], as did the study of Samanin et al. [51] which showed intrastriatal 6-OHDA injection to have no effect on AMPH but to attenuate MAZ's anorexic action by 40%. The present study further differentiated MAZ and AMPH with respect to their neuroanatomical substrate.

REFERENCES 1. Ahlskog, J. E. Food intake and amphetamine anorexia after selective forebrain norepinephrine loss. Brain Res. 82:211-240, 1974.

2. Ahlskog, J. E. Feeding response to regulatory challenges after 6-hydroxydopamine injection into the brain noradrenergic pathways. Physiol. Behav. 17: 407-411, 1976.

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f,EIBOWI'I'Z A N D H A M M E R

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