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Attenuation of amphetamine anorexia by unilateral nigral striatal lesions
Neurophurmucology,
1975, 14. 827-834.
Pergamon
Press.
Printed
in Gt. Britain.
ATTENUATION OF AMPHETAMINE ANOREXIA UNILATERAL NIGRAL STRIATAL LESIONS
BY
R. J. CAREY Veterans Administration
Hospital and State University of New York, Upstate Medical Center
and E. B. GOODALL Veterans Administration (Accepted
Hospital. Syracuse, New York 4 May 1975)
Summary-Rats with unilateral lateral hypothalamic lesions, which destroyed the nigral striatal bundle, exhibited an attenuation of the food intake suppressive effect of (+)-amphetamine. Rats with unilateral medial hypothalamic lesions, however, did not differ significantly from intact controls in terms of responsivity to the anorexic effect of amphetamine. In a second experiment, unilateral substantia nigra lesions attenuated the anorexic effect of amphetamine to an extent comparable with the unilateral lateral hypothalamic lesion. Both the substantia nigra and unilateral lateral hypothalamic lesion severely depleted dopamine and to a lesser extent norepinephrine on the damaged half of the brain. Thus, unilateral falls in brain catecholamines can reduce the anorexic efficacy of amphetamine.
There have been frequent attempts to relate amphetamine-induced anorexia to stimulation of the hypothalamic satiety region (BROBECK, LARSSON and REYES, 1956; KREBS, BINDRA and CAMPBELL, 1968). Recently, this hypothesis has been given new support by AHLSKOG and HOEBEL (1973) and AHLSKOG (1974) who found that lesions which destroy noradrenergic terminals in the hypothalamus and produce obesity also attenuate the anorexic effect of amphetamine. This report suggested that amphetamine acted on a noradrenergic satiety system. While this is an appealing hypothesis, any account of an effect of amphetamine must consider dopamine as well as norepinephrine. An effect on brain dopamine is particularly appropriate for feeding behaviour, since recent studies (UNGERSTEDT, 1971) indicate that the nigra striatal dopamine system is the neuroanatomica1 substrate for feeding behaviour. To evaluate the possible importance of the nigral striatal bundle in the mediation of amphetamine anorexia, the present study tested the effect of amphetamine on food intake in rats with nigral striatal lesions. A major complication, however, in testing food intake in an animal with a bilateral nigral striatal bundle lesion is the low survival rate and the drastically reduced body weight in surviving animals. To circumvent this difficulty the present study used unilateral lesions which have only a transient disruptive effect on feeding behaviour.
METHODS Experiment
1
Subjects. Eighteen naive, male, Sprague-Dawley rats, 120 days old at the start of experimentation were used. The rats were maintained in a room with controlled temperature (70” f 4°F) humidity (60% k 10%) and light (12 hr light, 12 hr dark). Powdered Purina lab chow was provided in spill-proof containers. Procedure. The rats were divided into three surgical groups of six each. There were two operated groups and one group of intact controls. One lesion group was given unilateral lateral hypothalamic lesions positioned to interrupt the nigral striatal bundle and the other group received lesions medial to the nigral striatal bundle. The medial lesion group was included to allow for a comparison of the effects of hypothalamic 827
R. J.CAREY
8’8
and E. B.
&KHIALL
versus n&al striatal bundle damage. Brain lesions were made with a temperature-controlled radiofrequency lesion maker (Radionics, Inc.). A Kopf stereotaxic instrument was used to position the thermister electrode at the appropriate brain site. The stereotaxic coordinates used were: nigral striatal bundle-l.5 mm posterior to bregma, 8.8 mm below dura, 2.0 mm lateral to the midline; medial hypothalamic-l.5 mm posterior to bregma, 8.8 mm below dura, 1.2 mm lateral to the midline. The incisor bar was fixed l.Omm above the interaural line. Current was adjusted to achieve a temperature at the electrode tip of 60°C which was maintained for 60 sec. The surgery was aseptic and performed with the rats under deep ether anaesthesia. After the operation, each animal was given an intramuscular injection of 200,000 units of procaine penicillin. At the conclusion of the study all animals were decapitated and the brains rapidly removed. The brains were coronally cut at approximately the level of the crossing of the anterior commissure. The rostra1 portion of the brain was used for norepinephrine and dopamine determinations. Prior to preparation for the catecholamine measurements, the rostra1 portion of the brain was cut in half by a sagittal cut through the midline and each of these sections was separately prepared for chemical analysis. This latter dissection of the brain was performed to give separate samples from the intact and lesion sides of the brains of operated animals. The remaining caudal section of brain was placed in 10% formalin for a 5-day fixation period. After this fixation period, 3 mm thick sections containing the lesions were embedded in paraffin. Subsequently, 6 pm thick sections were cut, mounted, and stained with cresyl violet. All sections were examined microscopically to evaluate the lesions. Brain tissue samples were weighed to the nearest 0.1 mg and homogenized in 3.0ml of ice cold 0.4 M percholoric acid. Tissue homogenates were centrifuged at a force of 20,000 y for 10 min at 4°C and supernatant was decanted and stored at - 20°C. These tissue extracts were later assayed simultaneously for norepinephrine and dopamine content according to the procedure of SHELLENBERGER and GORDON (1971). Sample fluorescence was measured in an Aminco-Bowman Spectrophotofluorometer equipped with a xenon lamp. L-Arterenol bitartrate and dopamine HCl (Sigma Chemical Co.) were used as standards. Tissue monoamine content was calculated as pg monoamine base per g of brain tissue, uncorrected for recovery. Estimated recovery of norepinephrine and dopamine was 79 and 82x, respectively. lntuke measurements. All rats were tested for their 24-hr water intake without food available, once preoperatively and twice postoperatively. This intake determination was made because nigral striatal bundle lesions at the level of the lateral hypothalamus characteristically sharply attenuate water intake in the absence of food (WAMPLER,1971). This intake measure, therefore, provided a behavioural indication of the efficacy of the nigral striatal bundle lesion. The assessment of the effect of amphetamine on food intake was started in the fourth postoperative week. Initially all rats were placed on a 1 hr of food per day deprivation schedule, but with water available ad lib. The rats were given one week to acclimate to this deprivation schedule after which the amphetamine tests began. Food and water intake was measured to the nearest 0.1 g and spillage losses were also measured. The effects of three amphetamine dose levels, 0.5, I .O, and 2.0 mg/kg, were tested for their effect on the rat’s 1 hr food intake. The amphetamine doses were given in ascending order and four non-drug days with I hr of food intake intervened between each increment in amphetamine dose level. The amphetamine ((+)amphetamine HCl, K and K Laboratories) was injected intraperitoneally 15 min prior to the presentation of the food. The dose levels of amphetamine were calculated as the salt and were in an equal volume 1 ml/kg dissolved in 0.9% sodium chloride. Experiment
2
Because of the neuroanatomical proximity of the nigral striatal bundle to the hypothalamus, an additional experiment was conducted to evaluate damage to the nigral striatal bundle by a lesion in the substantia nigra which is outside the hypothalamus. In addition. the drug testing was extended to include another anorexic drug, fenfluramine.
Amphetamine
anorexia
and DA neurones
829
Subjects. Fifteen naive, male, Sprague-Dawley rats, 120 days old at the start of the experiment were used. The rats were maintained in individual cages under the same laboratory conditions as the first experiment. Procedure. The rats were divided into three surgical groups of five each. There were two operated groups and one group of intact controls. One operated group received a unilateral substantia nigra lesion and the other group a unilateral nigral striatal bundle lesion at the level of the lateral hypothalamus. The substantia nigra lesion coordinates were: 3.5 mm posterior to bregma, lateral 2.0 mm, depth 9.0 mm with the incisor bar fixed at 3.2 mm above the interaural plane. Otherwise, surgical, histological and catecholamine determinations were the same as in Experiment 1. In this experiment, the effects of 2 dose levels of amphetamine (1.0 and 2.0 mg/kg) and fenfluramine HCl (2.0 and 4.0 mg/kg) on food intake were measured. On the 15th postoperative day rats were placed on the 1 hr of food per day deprivation schedule. After 1 week on this deprivation schedule, the rats were injected with either amphetamine or fenfluramine 15 min prior to presentation of food for 1 hr. Three rats in each group were first injected with amphetamine and the remaining two rats per group were injected with fenfluramine. On the second injection day the drug treatments were reversed. For both drugs, the dose levels were administered in ascending order and between drug treatments there were four non-drug days with food available for 1 hr.
RESULTS
As expected the unilateral nigral striatal bundle lesions produced a I-2 day period of partial aphagia and adipsa. Complete recovery of preoperative intake was achieved by 7-10 days after surgery. The unilateral nigral striatal bundle lesions also had a marked effect on water intake in the absence of food. As shown in Table 1, on both postoperative tests the water intake of the nigral striatal bundle group was considerably reduced and this decrease was statistically significant at the P < 0.01 level. In contrast, the unilateral medial hypothalamic lesions did not significantly decrease intake. Table 2 shows that the nigral striatal bundle lesion also modified the anorexic effect of amphetamine. The unilateral nigral striatal bundle lesions but not the unilateral medial hypothalamic lesion attenuated the anorexic effect of amphetamine and this attenuation was most conspicuous at the 2.0 mg dose levels. The greater intake of the unilateral nigral striatal bundle group at the 2.0 m/kg dose level was statistically significant when compared with either the control or medial hypothalamic groups (P < 0.01). It is also evident from Table 2 that both the unilateral medial hypothalamic and the nigral striatal bundle lesion reduced intake relative to controls under the non-drug condition. To evaluate the anorexic effect of the amphetamine injections relative to non-drug intakes, the percentage of non-drug food intake consumed under each dose level of (+)-amphetamine was calculated and is presented in Figure 1. The close correspondence between the fall in food intake produced by 0.5 mg and 1.0 mg doses in the intact rats and 1.0 and 2.0 mg doses in the nigral striatal bundle group suggests that the nigral striatal bundle lesion produced about a 50% decrease in anorexic efficacy of amphetamine. Statistical evaluation of the percentage decrease in food intake results showed that the attentuation produced by the nigral striatal bundle lesion was highly significantly statistically (F = 16.72, d.f. = 2, 15, P < 0.01). Table
1. Means
and standard
errors
of the preoperative and two 24-hr postoperative in the absence of food Preoperative
Group Unilateral Unilateral Control
lateral hypothalamus medial hypothalamus
NSB: nigral
striatal
bundle.
(NSB)
246 i: 2.87 22.58 i: 2.83 21.27 i_ I.11
water
intakes
in grams
Postoperative 22.56 * 1.45 8.28 * 1.97 17.63 k 3.0
19.93 * 2.53 7.47 k 1.90 16.15 i 2.87
Group
Table
2. Means
and standard
errors
for 1 hr food intakes
ND
in grams
Amphetamine (0.5 mg/kg) ND
of the operated and control groups preceding non-drug (ND) test day
under
each
the immediately
(2.0 mg/kg)
ND
with
(1Q w/k)
level compared
Amphetamine
dose
Amphetamine
amphetamine
R
>
z
Amphetamine
a
Control
l
Unrlateral
0 Untlateral
anorexia
medlal lateral
831
and DA neurones
hypottdamlc hypothalamx
lesion lesion
loo r
( + )-Amphetamme.
mdkg
Fig. I. Mean percentage of baseline food intake consumed under 3 dose levels of (+)-amphetamine for the control and two brain lesion groups. Vertical bars denote standard errors of the mean.
Fig. 2. Representative photomicrographs of cresyl violet stained sections through unii, ateral medial hypothalamic lesion (A) and a unilateral lateral hypothalamic bundle) lesion (B).
a repres entative (nigral striatal
R. J. CAREY and E. B. GOODALL
832 Table
3. Means
and
standard damaged
errors of dopamine and norepinephrine concentrations halves of forebrain sections for each group in Experiment
Downine (m/g) Group Unilateral Unilateral Control
lateral hypothalamus media1 hypothalamus
(NSB)
in intact
Norepinephrine Lesion
Lesion
Intact
0,316 k @12 2-61 * 0.21 2.66 f 0.16
2.73 i 0.08 2.81 + 0.16 2-76 k 0.22
and
brain-
I
0.16 f 0.01 0.27 * 0.03 0.37 & 0.01
(pg/g) Intact o-36 + Q.01 0.37 * 0.03 0.39 f 0.01
A representative brain section through each of the lesions is shown in Figure 2. The nigral striatal bundle lesions were located at the extreme lateral border of the hypothalamus and were well situated to interrupt the nigra striatal projections. The medial hypothalamic lesions were comparable in terms of cavitation but were situated more medially and appeared to primarily damage the medial forebrain bundle and fornix. The catecholamine determinations shown in Table 3 confirmed the efficacy of the lesions in destroying the nigral striatal bundle. Dopamine was greatly reduced in the lesioned half of the brain for the nigral striatal bundle group. To a lesser extent norepinephrine was also decreased on the side of the nigral striatal bundle lesion. The medial hypothalamic lesion, as expected, only moderately lowered norepinephrine levels but not dopamine levels on the lesioned side of the brain. The decreases in dopamine and norepinephrine produced by the nigral striatal bundle lesions were highly significant statistically for dopamine (I; = 58.76, d.f. = 2, 15, P < 0.01) and for norepinephrine (F = 24.20, d.f. = 2, 15, P < 0.01). Both dopamine and norepinephrine levels were not significantly different among groups (P < O-25) for the intact sides of the brain. Table 4 shows that the nigral striatal bundle lesions at the level of the hypothalamus and substantia nigra had comparable effects. Both lesions attenuated the anorexic effect of amphetamine. At the 2 mg of dose of amphetamine both the nigral striatal bundle and substantia nigra groups had a higher food intake than controls (P < 0.01). In contrast, at both the 2.0 and 4.0 mg/kg dose levels of fenfluramine, both operated groups consumed less food than the controls (P < 0.01). It should also be observed, however, that the non-drug intakes of the nigral striatal bundle and substantia nigra groups were consistently less than the controls. Both lesions also greatly reduced dopamine and to a much lesser degree norepinephrine levels on the lesion sides of the brain as shown in Table 5. The nigral striatal bundle lesions were comparable in size and location to those of Experiment 1. The substantia nigra lesions damaged both the para compacta and pars reticularis of the substantia nigra. DISCUSSION
The results of the present study indicate that unilateral decreases in brain catecholamines can attenuate amphetamine-induced anorexia. Since the catecholamine depletion Table
4. Means and standard errors for 1 hr food intakes in grams of operated (+)-Amphetamine and fenfluramine treatments and the preceding Non-drug
Contlol Unilateral Umlateral
Table
substantia ncga lateral hypothalamus
5. Means
and standard
II.92 k 0.57 8 36 k O-61 8.64 k 0 X6
(2.0 w/kg)
5-5X k 002 6,3X + 0.78 5-68 * I.3
0 26 f 0, 3.42 f I-01 2.68 f 0.6
I5
Dopamine Lesion lateral hypothalamus substantia nigra
12.36 i 0 52 Y-16 * o-45 X.86 & 0,86
x 04 i o-93 448 * 0 59 3.74 _+ II.45
errors of dopamine and norepinephrine concentrations halves of forebrain for each group in Experiment 2
Group Unilateral Unilateral Control
ND
(1 0 w&W
(NSB)
0.51 * 0.13 063 f 0.17 2.72 k 0.06
(pg/g) Intact 2.48 + o-1 1 261 * 0.14 2.54 k 0.12
for
Fenfluramme (4.0 mg’kg) (20 wikgt
Amphetamine ND
Group
and control groups (ND) test days
in intact
Norepinephrine Lesion o-15 * 0.02 o-27 * 0.02 0.36 & 0.03
i 52 & 0.52 0 x4 * 0.27 I58 & 038
and lesioned
&g/g) Intact 0.33 * 0.04 0.34 * 0.03 0.38 * 0.02
833
Amphetamineanorexia and DA neurones
was predominantly a reduction in dopamine, a significant role for this catecholamine in the mediation of amphetamine anorexia is suggested. In addition to the greater fall in dopamine compared with norepinephrine, the importance of the dopamine depletion was further suggested by the fact that the ineffective medial hypothalamic lesion in Experiment 1 and the effective substantia nigra lesion in the second experiment differed in dopamine but not norepinephrine depletion. The results of the present study, however, contrast with the report of AHLSKOC;and HOEBEL(1973) which implicated norepinephrine in the mediation of amphetamine anorexia. These differences could be reconciled if it is assumed that amphetamine anorexia can be attenuated by a depletion of either dopamine or norepinephrine providing the depletion is of sufficient magnitude. It should be noted, however, that in their study AHLSKOG and HOEBEL (1973) found amphetamine attenuation in animals with lesions positioned to greatly reduce brain norepinephrine but brain catecholamine depletions were not reported for these particular animals. Another significant facet of the present study is that the amphetamine attenuation was produced by a unilateral lesion. One possible way to interpret this finding is to consider that the depletion in total brain catecholamines is the important variable regardless of whether it is bilateral or unilateral. Considering the results of the experiments, it is possible to relate the total brain catecholamine depletion of nearly 50% to the approximately 50% reduction in efficacy of amphetamine. While this is an appealing relationship, the recent report of CREESEand IVERSEN(1975) indicates the need for an alternative explanation. In their study of the motor stimulatory effects of amphetamine, CREESEand IVERSEN (1975) did not observe a graded reduction in amphetamine effect as catecholamine levels decreased. Rather, they found that catecholamines depletion only modified the effect of amphetamine if the depletion was nearly complete. In terms of the present study it might be argued, then, that the attenuation of amphetamine occurred because the unilateral catecholamine depletion was of sufficient magnitude to block the effect of amphetamine on the lesioned side of the brain, thereby limiting the effect of amphetamine to the intact of half the brain. Resolution of this important issue, however, requires additional experimental determinations such as the amphetamine distribution to the intact and injured halfs of the brain. Finally, it is also of interest that the effects of the unilateral lesions in the second experiment closely replicated the findings obtained by FIBIGER, ZIS and MC GEER (1974) using bilateral lateral hypothalamic lesions. In the study of FIBIGER et al. (1974) and in the present study, attenuation of amphetamine anorexia was observed as was an enhancement of fenfluramine-induced anorexia. As suggested by FIBIGER et al. (1974), the fenfluramine results suggest that catecholaminergic systems may antagonize the anorexic effect of fenfluramine. The reduced baseline intake of the brain damaged rats in the present experiment, however, cautions against this interesting possibility. Regardless of this consideration, the dissociative effects of the lesions in the present experiment on the effects of fenfluramine and amphetamine are consistent with biochemical evidence (COSTA, GROPPETTI and REVUELTA, 1971; SOUTHGATE, MAYER, BOXALL and WILSON, 1971) which implicates serotonergic rather than catecholaminergic systems in the mediations of fenfluramine effects. AcknowledqwwntA.
H. Robbin
Company
generously
supplied
the fenfluramine
HCl.
REFERENCES AHLSKOG, J. E. (1974). Food intake and amphetamine anorexia after selective forebrain norepinephrine loss. Brain Rex 82: 211-240. AHLSKOG, J. E. and HOEBEL, B. G. (1973). Overeating and obesity from damage to a noradrenergic system in the brain. Science, N.Y 182:166169. BROBECK, J. R., LARSSON, S. and REYES, E. (1956). A study of the electrical activity of the hypothalamic feeding mechanism. J. Physiol. Land. 132: 358-364. COSTA, E., GROPPETTI, A. and REVUELTA, A. (1971). Action of fenfluramine on monoamine stores of rat tissues. Br. J. Pharmac. 41: 51-64. CREESE, I. and IVERSEN, S. D. (1975). The pharmacological and anatomical substrates of the amphetamine response in the rat. Brain Rex 83: 419-436.
834
R. J. CAREY and E. B. GOODALL
FIBIGER, H. C., Zrs, A. P. and MC GEER, E. G. (1974). Feeding and drinking deficits after h-hydroxydopamine administration in the rat: similarities to the lateral hypothalamic syndrome. Brain Res. 65: 135-148. KREBS, H., BINDRA, D. and CAMPBELL, J. F. (1968). Effects of amphetamine on neuronal activity in the hypothalamus. Physiol. Behav. 4: 685-691. SHELLENBERGER,M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas. Analyt. Biochrm. 39: 356-372. SOUTHGATE, P. J., MAYER, S. R., BOXALL, E. and WILSOX, A. E. (1971). Some 5-hydroxytryptamine-like actions of fenfluramine: a comparison with (+) amphetamine and diethylpopsion. J. Pharm. Pharnmc. 23: 60@605. UNGERSTEDT, U. (1971). Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta. physiol. stand., Suppl. 367: 95-122. WAMPLER, R. S. (1971). Regulatory deficits in rats following unilateral lesions of the lateral hypothalamus. J. camp. physiol. Psychol. 75: 19@l99.
1975, 14. 827-834.
Pergamon
Press.
Printed
in Gt. Britain.
ATTENUATION OF AMPHETAMINE ANOREXIA UNILATERAL NIGRAL STRIATAL LESIONS
BY
R. J. CAREY Veterans Administration
Hospital and State University of New York, Upstate Medical Center
and E. B. GOODALL Veterans Administration (Accepted
Hospital. Syracuse, New York 4 May 1975)
Summary-Rats with unilateral lateral hypothalamic lesions, which destroyed the nigral striatal bundle, exhibited an attenuation of the food intake suppressive effect of (+)-amphetamine. Rats with unilateral medial hypothalamic lesions, however, did not differ significantly from intact controls in terms of responsivity to the anorexic effect of amphetamine. In a second experiment, unilateral substantia nigra lesions attenuated the anorexic effect of amphetamine to an extent comparable with the unilateral lateral hypothalamic lesion. Both the substantia nigra and unilateral lateral hypothalamic lesion severely depleted dopamine and to a lesser extent norepinephrine on the damaged half of the brain. Thus, unilateral falls in brain catecholamines can reduce the anorexic efficacy of amphetamine.
There have been frequent attempts to relate amphetamine-induced anorexia to stimulation of the hypothalamic satiety region (BROBECK, LARSSON and REYES, 1956; KREBS, BINDRA and CAMPBELL, 1968). Recently, this hypothesis has been given new support by AHLSKOG and HOEBEL (1973) and AHLSKOG (1974) who found that lesions which destroy noradrenergic terminals in the hypothalamus and produce obesity also attenuate the anorexic effect of amphetamine. This report suggested that amphetamine acted on a noradrenergic satiety system. While this is an appealing hypothesis, any account of an effect of amphetamine must consider dopamine as well as norepinephrine. An effect on brain dopamine is particularly appropriate for feeding behaviour, since recent studies (UNGERSTEDT, 1971) indicate that the nigra striatal dopamine system is the neuroanatomica1 substrate for feeding behaviour. To evaluate the possible importance of the nigral striatal bundle in the mediation of amphetamine anorexia, the present study tested the effect of amphetamine on food intake in rats with nigral striatal lesions. A major complication, however, in testing food intake in an animal with a bilateral nigral striatal bundle lesion is the low survival rate and the drastically reduced body weight in surviving animals. To circumvent this difficulty the present study used unilateral lesions which have only a transient disruptive effect on feeding behaviour.
METHODS Experiment
1
Subjects. Eighteen naive, male, Sprague-Dawley rats, 120 days old at the start of experimentation were used. The rats were maintained in a room with controlled temperature (70” f 4°F) humidity (60% k 10%) and light (12 hr light, 12 hr dark). Powdered Purina lab chow was provided in spill-proof containers. Procedure. The rats were divided into three surgical groups of six each. There were two operated groups and one group of intact controls. One lesion group was given unilateral lateral hypothalamic lesions positioned to interrupt the nigral striatal bundle and the other group received lesions medial to the nigral striatal bundle. The medial lesion group was included to allow for a comparison of the effects of hypothalamic 827
R. J.CAREY
8’8
and E. B.
&KHIALL
versus n&al striatal bundle damage. Brain lesions were made with a temperature-controlled radiofrequency lesion maker (Radionics, Inc.). A Kopf stereotaxic instrument was used to position the thermister electrode at the appropriate brain site. The stereotaxic coordinates used were: nigral striatal bundle-l.5 mm posterior to bregma, 8.8 mm below dura, 2.0 mm lateral to the midline; medial hypothalamic-l.5 mm posterior to bregma, 8.8 mm below dura, 1.2 mm lateral to the midline. The incisor bar was fixed l.Omm above the interaural line. Current was adjusted to achieve a temperature at the electrode tip of 60°C which was maintained for 60 sec. The surgery was aseptic and performed with the rats under deep ether anaesthesia. After the operation, each animal was given an intramuscular injection of 200,000 units of procaine penicillin. At the conclusion of the study all animals were decapitated and the brains rapidly removed. The brains were coronally cut at approximately the level of the crossing of the anterior commissure. The rostra1 portion of the brain was used for norepinephrine and dopamine determinations. Prior to preparation for the catecholamine measurements, the rostra1 portion of the brain was cut in half by a sagittal cut through the midline and each of these sections was separately prepared for chemical analysis. This latter dissection of the brain was performed to give separate samples from the intact and lesion sides of the brains of operated animals. The remaining caudal section of brain was placed in 10% formalin for a 5-day fixation period. After this fixation period, 3 mm thick sections containing the lesions were embedded in paraffin. Subsequently, 6 pm thick sections were cut, mounted, and stained with cresyl violet. All sections were examined microscopically to evaluate the lesions. Brain tissue samples were weighed to the nearest 0.1 mg and homogenized in 3.0ml of ice cold 0.4 M percholoric acid. Tissue homogenates were centrifuged at a force of 20,000 y for 10 min at 4°C and supernatant was decanted and stored at - 20°C. These tissue extracts were later assayed simultaneously for norepinephrine and dopamine content according to the procedure of SHELLENBERGER and GORDON (1971). Sample fluorescence was measured in an Aminco-Bowman Spectrophotofluorometer equipped with a xenon lamp. L-Arterenol bitartrate and dopamine HCl (Sigma Chemical Co.) were used as standards. Tissue monoamine content was calculated as pg monoamine base per g of brain tissue, uncorrected for recovery. Estimated recovery of norepinephrine and dopamine was 79 and 82x, respectively. lntuke measurements. All rats were tested for their 24-hr water intake without food available, once preoperatively and twice postoperatively. This intake determination was made because nigral striatal bundle lesions at the level of the lateral hypothalamus characteristically sharply attenuate water intake in the absence of food (WAMPLER,1971). This intake measure, therefore, provided a behavioural indication of the efficacy of the nigral striatal bundle lesion. The assessment of the effect of amphetamine on food intake was started in the fourth postoperative week. Initially all rats were placed on a 1 hr of food per day deprivation schedule, but with water available ad lib. The rats were given one week to acclimate to this deprivation schedule after which the amphetamine tests began. Food and water intake was measured to the nearest 0.1 g and spillage losses were also measured. The effects of three amphetamine dose levels, 0.5, I .O, and 2.0 mg/kg, were tested for their effect on the rat’s 1 hr food intake. The amphetamine doses were given in ascending order and four non-drug days with I hr of food intake intervened between each increment in amphetamine dose level. The amphetamine ((+)amphetamine HCl, K and K Laboratories) was injected intraperitoneally 15 min prior to the presentation of the food. The dose levels of amphetamine were calculated as the salt and were in an equal volume 1 ml/kg dissolved in 0.9% sodium chloride. Experiment
2
Because of the neuroanatomical proximity of the nigral striatal bundle to the hypothalamus, an additional experiment was conducted to evaluate damage to the nigral striatal bundle by a lesion in the substantia nigra which is outside the hypothalamus. In addition. the drug testing was extended to include another anorexic drug, fenfluramine.
Amphetamine
anorexia
and DA neurones
829
Subjects. Fifteen naive, male, Sprague-Dawley rats, 120 days old at the start of the experiment were used. The rats were maintained in individual cages under the same laboratory conditions as the first experiment. Procedure. The rats were divided into three surgical groups of five each. There were two operated groups and one group of intact controls. One operated group received a unilateral substantia nigra lesion and the other group a unilateral nigral striatal bundle lesion at the level of the lateral hypothalamus. The substantia nigra lesion coordinates were: 3.5 mm posterior to bregma, lateral 2.0 mm, depth 9.0 mm with the incisor bar fixed at 3.2 mm above the interaural plane. Otherwise, surgical, histological and catecholamine determinations were the same as in Experiment 1. In this experiment, the effects of 2 dose levels of amphetamine (1.0 and 2.0 mg/kg) and fenfluramine HCl (2.0 and 4.0 mg/kg) on food intake were measured. On the 15th postoperative day rats were placed on the 1 hr of food per day deprivation schedule. After 1 week on this deprivation schedule, the rats were injected with either amphetamine or fenfluramine 15 min prior to presentation of food for 1 hr. Three rats in each group were first injected with amphetamine and the remaining two rats per group were injected with fenfluramine. On the second injection day the drug treatments were reversed. For both drugs, the dose levels were administered in ascending order and between drug treatments there were four non-drug days with food available for 1 hr.
RESULTS
As expected the unilateral nigral striatal bundle lesions produced a I-2 day period of partial aphagia and adipsa. Complete recovery of preoperative intake was achieved by 7-10 days after surgery. The unilateral nigral striatal bundle lesions also had a marked effect on water intake in the absence of food. As shown in Table 1, on both postoperative tests the water intake of the nigral striatal bundle group was considerably reduced and this decrease was statistically significant at the P < 0.01 level. In contrast, the unilateral medial hypothalamic lesions did not significantly decrease intake. Table 2 shows that the nigral striatal bundle lesion also modified the anorexic effect of amphetamine. The unilateral nigral striatal bundle lesions but not the unilateral medial hypothalamic lesion attenuated the anorexic effect of amphetamine and this attenuation was most conspicuous at the 2.0 mg dose levels. The greater intake of the unilateral nigral striatal bundle group at the 2.0 m/kg dose level was statistically significant when compared with either the control or medial hypothalamic groups (P < 0.01). It is also evident from Table 2 that both the unilateral medial hypothalamic and the nigral striatal bundle lesion reduced intake relative to controls under the non-drug condition. To evaluate the anorexic effect of the amphetamine injections relative to non-drug intakes, the percentage of non-drug food intake consumed under each dose level of (+)-amphetamine was calculated and is presented in Figure 1. The close correspondence between the fall in food intake produced by 0.5 mg and 1.0 mg doses in the intact rats and 1.0 and 2.0 mg doses in the nigral striatal bundle group suggests that the nigral striatal bundle lesion produced about a 50% decrease in anorexic efficacy of amphetamine. Statistical evaluation of the percentage decrease in food intake results showed that the attentuation produced by the nigral striatal bundle lesion was highly significantly statistically (F = 16.72, d.f. = 2, 15, P < 0.01). Table
1. Means
and standard
errors
of the preoperative and two 24-hr postoperative in the absence of food Preoperative
Group Unilateral Unilateral Control
lateral hypothalamus medial hypothalamus
NSB: nigral
striatal
bundle.
(NSB)
246 i: 2.87 22.58 i: 2.83 21.27 i_ I.11
water
intakes
in grams
Postoperative 22.56 * 1.45 8.28 * 1.97 17.63 k 3.0
19.93 * 2.53 7.47 k 1.90 16.15 i 2.87
Group
Table
2. Means
and standard
errors
for 1 hr food intakes
ND
in grams
Amphetamine (0.5 mg/kg) ND
of the operated and control groups preceding non-drug (ND) test day
under
each
the immediately
(2.0 mg/kg)
ND
with
(1Q w/k)
level compared
Amphetamine
dose
Amphetamine
amphetamine
R
>
z
Amphetamine
a
Control
l
Unrlateral
0 Untlateral
anorexia
medlal lateral
831
and DA neurones
hypottdamlc hypothalamx
lesion lesion
loo r
( + )-Amphetamme.
mdkg
Fig. I. Mean percentage of baseline food intake consumed under 3 dose levels of (+)-amphetamine for the control and two brain lesion groups. Vertical bars denote standard errors of the mean.
Fig. 2. Representative photomicrographs of cresyl violet stained sections through unii, ateral medial hypothalamic lesion (A) and a unilateral lateral hypothalamic bundle) lesion (B).
a repres entative (nigral striatal
R. J. CAREY and E. B. GOODALL
832 Table
3. Means
and
standard damaged
errors of dopamine and norepinephrine concentrations halves of forebrain sections for each group in Experiment
Downine (m/g) Group Unilateral Unilateral Control
lateral hypothalamus media1 hypothalamus
(NSB)
in intact
Norepinephrine Lesion
Lesion
Intact
0,316 k @12 2-61 * 0.21 2.66 f 0.16
2.73 i 0.08 2.81 + 0.16 2-76 k 0.22
and
brain-
I
0.16 f 0.01 0.27 * 0.03 0.37 & 0.01
(pg/g) Intact o-36 + Q.01 0.37 * 0.03 0.39 f 0.01
A representative brain section through each of the lesions is shown in Figure 2. The nigral striatal bundle lesions were located at the extreme lateral border of the hypothalamus and were well situated to interrupt the nigra striatal projections. The medial hypothalamic lesions were comparable in terms of cavitation but were situated more medially and appeared to primarily damage the medial forebrain bundle and fornix. The catecholamine determinations shown in Table 3 confirmed the efficacy of the lesions in destroying the nigral striatal bundle. Dopamine was greatly reduced in the lesioned half of the brain for the nigral striatal bundle group. To a lesser extent norepinephrine was also decreased on the side of the nigral striatal bundle lesion. The medial hypothalamic lesion, as expected, only moderately lowered norepinephrine levels but not dopamine levels on the lesioned side of the brain. The decreases in dopamine and norepinephrine produced by the nigral striatal bundle lesions were highly significant statistically for dopamine (I; = 58.76, d.f. = 2, 15, P < 0.01) and for norepinephrine (F = 24.20, d.f. = 2, 15, P < 0.01). Both dopamine and norepinephrine levels were not significantly different among groups (P < O-25) for the intact sides of the brain. Table 4 shows that the nigral striatal bundle lesions at the level of the hypothalamus and substantia nigra had comparable effects. Both lesions attenuated the anorexic effect of amphetamine. At the 2 mg of dose of amphetamine both the nigral striatal bundle and substantia nigra groups had a higher food intake than controls (P < 0.01). In contrast, at both the 2.0 and 4.0 mg/kg dose levels of fenfluramine, both operated groups consumed less food than the controls (P < 0.01). It should also be observed, however, that the non-drug intakes of the nigral striatal bundle and substantia nigra groups were consistently less than the controls. Both lesions also greatly reduced dopamine and to a much lesser degree norepinephrine levels on the lesion sides of the brain as shown in Table 5. The nigral striatal bundle lesions were comparable in size and location to those of Experiment 1. The substantia nigra lesions damaged both the para compacta and pars reticularis of the substantia nigra. DISCUSSION
The results of the present study indicate that unilateral decreases in brain catecholamines can attenuate amphetamine-induced anorexia. Since the catecholamine depletion Table
4. Means and standard errors for 1 hr food intakes in grams of operated (+)-Amphetamine and fenfluramine treatments and the preceding Non-drug
Contlol Unilateral Umlateral
Table
substantia ncga lateral hypothalamus
5. Means
and standard
II.92 k 0.57 8 36 k O-61 8.64 k 0 X6
(2.0 w/kg)
5-5X k 002 6,3X + 0.78 5-68 * I.3
0 26 f 0, 3.42 f I-01 2.68 f 0.6
I5
Dopamine Lesion lateral hypothalamus substantia nigra
12.36 i 0 52 Y-16 * o-45 X.86 & 0,86
x 04 i o-93 448 * 0 59 3.74 _+ II.45
errors of dopamine and norepinephrine concentrations halves of forebrain for each group in Experiment 2
Group Unilateral Unilateral Control
ND
(1 0 w&W
(NSB)
0.51 * 0.13 063 f 0.17 2.72 k 0.06
(pg/g) Intact 2.48 + o-1 1 261 * 0.14 2.54 k 0.12
for
Fenfluramme (4.0 mg’kg) (20 wikgt
Amphetamine ND
Group
and control groups (ND) test days
in intact
Norepinephrine Lesion o-15 * 0.02 o-27 * 0.02 0.36 & 0.03
i 52 & 0.52 0 x4 * 0.27 I58 & 038
and lesioned
&g/g) Intact 0.33 * 0.04 0.34 * 0.03 0.38 * 0.02
833
Amphetamineanorexia and DA neurones
was predominantly a reduction in dopamine, a significant role for this catecholamine in the mediation of amphetamine anorexia is suggested. In addition to the greater fall in dopamine compared with norepinephrine, the importance of the dopamine depletion was further suggested by the fact that the ineffective medial hypothalamic lesion in Experiment 1 and the effective substantia nigra lesion in the second experiment differed in dopamine but not norepinephrine depletion. The results of the present study, however, contrast with the report of AHLSKOC;and HOEBEL(1973) which implicated norepinephrine in the mediation of amphetamine anorexia. These differences could be reconciled if it is assumed that amphetamine anorexia can be attenuated by a depletion of either dopamine or norepinephrine providing the depletion is of sufficient magnitude. It should be noted, however, that in their study AHLSKOG and HOEBEL (1973) found amphetamine attenuation in animals with lesions positioned to greatly reduce brain norepinephrine but brain catecholamine depletions were not reported for these particular animals. Another significant facet of the present study is that the amphetamine attenuation was produced by a unilateral lesion. One possible way to interpret this finding is to consider that the depletion in total brain catecholamines is the important variable regardless of whether it is bilateral or unilateral. Considering the results of the experiments, it is possible to relate the total brain catecholamine depletion of nearly 50% to the approximately 50% reduction in efficacy of amphetamine. While this is an appealing relationship, the recent report of CREESEand IVERSEN(1975) indicates the need for an alternative explanation. In their study of the motor stimulatory effects of amphetamine, CREESEand IVERSEN (1975) did not observe a graded reduction in amphetamine effect as catecholamine levels decreased. Rather, they found that catecholamines depletion only modified the effect of amphetamine if the depletion was nearly complete. In terms of the present study it might be argued, then, that the attenuation of amphetamine occurred because the unilateral catecholamine depletion was of sufficient magnitude to block the effect of amphetamine on the lesioned side of the brain, thereby limiting the effect of amphetamine to the intact of half the brain. Resolution of this important issue, however, requires additional experimental determinations such as the amphetamine distribution to the intact and injured halfs of the brain. Finally, it is also of interest that the effects of the unilateral lesions in the second experiment closely replicated the findings obtained by FIBIGER, ZIS and MC GEER (1974) using bilateral lateral hypothalamic lesions. In the study of FIBIGER et al. (1974) and in the present study, attenuation of amphetamine anorexia was observed as was an enhancement of fenfluramine-induced anorexia. As suggested by FIBIGER et al. (1974), the fenfluramine results suggest that catecholaminergic systems may antagonize the anorexic effect of fenfluramine. The reduced baseline intake of the brain damaged rats in the present experiment, however, cautions against this interesting possibility. Regardless of this consideration, the dissociative effects of the lesions in the present experiment on the effects of fenfluramine and amphetamine are consistent with biochemical evidence (COSTA, GROPPETTI and REVUELTA, 1971; SOUTHGATE, MAYER, BOXALL and WILSON, 1971) which implicates serotonergic rather than catecholaminergic systems in the mediations of fenfluramine effects. AcknowledqwwntA.
H. Robbin
Company
generously
supplied
the fenfluramine
HCl.
REFERENCES AHLSKOG, J. E. (1974). Food intake and amphetamine anorexia after selective forebrain norepinephrine loss. Brain Rex 82: 211-240. AHLSKOG, J. E. and HOEBEL, B. G. (1973). Overeating and obesity from damage to a noradrenergic system in the brain. Science, N.Y 182:166169. BROBECK, J. R., LARSSON, S. and REYES, E. (1956). A study of the electrical activity of the hypothalamic feeding mechanism. J. Physiol. Land. 132: 358-364. COSTA, E., GROPPETTI, A. and REVUELTA, A. (1971). Action of fenfluramine on monoamine stores of rat tissues. Br. J. Pharmac. 41: 51-64. CREESE, I. and IVERSEN, S. D. (1975). The pharmacological and anatomical substrates of the amphetamine response in the rat. Brain Rex 83: 419-436.
834
R. J. CAREY and E. B. GOODALL
FIBIGER, H. C., Zrs, A. P. and MC GEER, E. G. (1974). Feeding and drinking deficits after h-hydroxydopamine administration in the rat: similarities to the lateral hypothalamic syndrome. Brain Res. 65: 135-148. KREBS, H., BINDRA, D. and CAMPBELL, J. F. (1968). Effects of amphetamine on neuronal activity in the hypothalamus. Physiol. Behav. 4: 685-691. SHELLENBERGER,M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas. Analyt. Biochrm. 39: 356-372. SOUTHGATE, P. J., MAYER, S. R., BOXALL, E. and WILSOX, A. E. (1971). Some 5-hydroxytryptamine-like actions of fenfluramine: a comparison with (+) amphetamine and diethylpopsion. J. Pharm. Pharnmc. 23: 60@605. UNGERSTEDT, U. (1971). Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta. physiol. stand., Suppl. 367: 95-122. WAMPLER, R. S. (1971). Regulatory deficits in rats following unilateral lesions of the lateral hypothalamus. J. camp. physiol. Psychol. 75: 19@l99.