Food intake and amphetamine anorexia after selective forebrain norepinephrine loss

Brain Research, 82 (1974) 211-240 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

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F O O D I N T A K E A N D A M P H E T A M I N E A N O R E X I A A F T E R SELECTIVE F O R E B R A I N N O R E P I N E P H R I N E LOSS

J. ERIC AHLSKOG* Dartmouth Medical School, Hanover, N.H. 03755 (U.S.A.)

(Accepted July 26th, 1974)

SUMMARY Hyperphagia leading to obesity followed selective electrolytic destruction of the ventral noradrenergic bundle (ventral NAB; the main norepinephrine-containing pathway to the rat hypothalamus). Midbrain injection of 8.0 #g 6-hydroxydopamine (6-OH-DA) near this tract, which destroyed all ascending noradrenergic paths to the forebrain, produced a similar result. Male as well as female rats became hyperphagic. Fluorescence histochemistry revealed nearly complete losses of norepinephrine-containing varicosities in almost all known noradrenergically innervated forebrain areas after 6-OH-DA; forebrain assays indicated that norepinephrine levels were lowered to less than 10 ~ of normal. Dopamine was only slightly reduced by the 6-OH-DA while serotonin was unchanged. Pretreatment of 6-OH-DA-injected animals with 50 mg/kg desmethylimipramine, a specific uptake blocker in norepinephrine neurons, blocked the loss of noradrenergic varicosities and prevented overeating. Animals which were injected with 6-OH-DA near the dorsal NAB, sparing the ventral system, did not become hyperphagic. In additional rats, electrolytic lesions or 6-OH-DA in the vicinity of the ventral NAB antagonized amphetamine-induced anorexia. This suggests that in normal animals, the ventral NAB may serve as a substrate for amphetamine in producing appetite loss. These results indicate that the role o f forebrain norepinephrine in the control of food intake is predominantly inhibitory.

INTRODUCTION Evidence from pharmacological studies suggests that activity at brain cate* Address reprint requests to J. Eric Ahlskog, Department of Psychology, Princeton University, Princeton, N.J. 08540, U.S.A.

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cholamine synapses is associated with loss of appetite in the rat. Amphetamine is a potent hunger suppressant (e.g., refs. 26 and 72) that appears to act indirectly, through endogenous stores of catecholamines z°,z1,a2,3a,3a,73. Involvement of norepinephrine and/or dopamine is attested to by tbe fact that disruption of their synthesis with alpha-methyl-p-tyrosine antagonizes amphetamine-induced anorexia 7,s4. The hypothalamus appears to serve as a substrate for at least part of these effects upon appetite. Lateral hypothalamic lesions prevent drug-induced anorexia ls,52 while cannulation of amphetamine into this area tends to inhibit eating lz,as. Since direct intra-hypothalamic injection of this agent is effective, it appears that the local catecholamine terminals mediate suppression of feeding. The noradrenergic innervation of the hypothalamus is quite profuse while terminals containing other catecholamines are only sparsely concentrated there 7s. According to recent histochemical mapping studies 5,6,78 the norepinephrine-containing varicosities derive almost exclusively from hindbrain cells which project into an ascending pathway. This rostrally directed system divides into two components at anterior pontine levels with the ventral portion (ventral noradrenergic bundle, ventral NAB) providing the main noradrenergic input to the hypothalamus as well as certain other areas of the forebrain. The contribution of the dorsal pathway (dorsal noradrenergic bundle, dorsal NAB) to the hypothalamic innervation is relatively slight 7s although a smaller intermediate bundle apparently projects to restricted regions near the third ventricle s3. This evidence appears to suggest that amphetamine suppresses food intake, at least in part, by facilitation of noradrenergic transmission in the vicinity of the hypothalamus and that this effect is mediated by the ventral NAB. Extending this model to the undrugged state, it would appear likely that the normal activity of this ventral system also results in the inhibition of food intake and contributes to consummatory regulation. If the ventral NAB tends to function as a brake on feeding, then disruption of this pathway should cause an increase in food intake. In addition, similar disruption should attenuate amphetamine-induced anorexia. The following series of experiments were conducted to test these hypotheses. Preliminary results of some of these experi: ments have previously been reported2,3; the present paper details and extends these findings. EXPERIMENT I

6-Hydroxydopamine (6-OH-DA) is a pharmacological agent which is selectively taken up by catecholaminergic neurons and subsequently causes their destruction 9,55,74,75,77. Non-catecholaminergic nerves are for the most part unaffected14,1v,44, v0,79. If the ventral NAB is involved in the inhibition of food intake, then local midbrain injection of 6-OH-DA in the vicinity of this pathway should deplete the hypothalamus and basal forebrain of noradrenergic terminals and lead to hyperphagia. Discrete electrolytic lesions confined to this pathway should also prove effective.

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Methods General procedure Subjects were female Sherman rats weighing 250-370 g at the time of surgery. One hundred and twenty-two animals were housed in single cages and randomly assigned to either a ventral NAB-destruction group or to a control group as described below. Following a minimum habituation period of 5 days, food (Purina Laboratory Pellets) and water consumption were measured every 48 h with corrections made for spillage and evaporation; body weight was also monitored. Measurements were taken for a minimum 8-day baseline and for 30 days immediately following the surgical period. The operation was performed between 12 and 36 h after the last preoperative food and weight measurement and no consummatory data were taken for the 48-h period which included surgery. A number of rats were postoperatively monitored for an extra 10 days (40 days, total) but these additional data were not used in any of the comparisons reported here. Weight measurements extending up to 8 months after surgery were periodically taken on a number of hyperphagic animals. Subjects were continuously added to this experiment over approximately a 1.5-year period; control animals were always run simultaneously with the experimentals.

Surgical procedure, electrolytic lesion group Group L Electrolytic lesion (N = 29). The intended lesion site was near the point of maximum separation of dorsal and ventral noradrenergic bundles 7s at the level of the rostral portion of the oculomotor nucleus; brain stereotaxic coordinates were determined from preliminary studies: 1.0 mm anterior to the earbars; 1.5 mm lateral to the midsagittal sinus; and 6.7 mm below the surface of the leveled cortex (AP 1.0, L 1.5, DV 6.7). Bilateral DC cathodal lesions were made (0.5 mA for 20 sec) using a 0.38 ram, 90 ~ platinum-10 ~ iridium electrode; a rectal anode completed the circuit. Surgery was performed under Nembutal ® anesthesia (35 mg/kg) following atropine pretreatment (0.08 mg/rat). Postoperatively, animals were injected with Mikedimide (35 mg/kg) and 60,000 U o f Duracillin before being returned to the home cage.

Surgical procedure, 6-OH-DA groups Animals in the following 4 ventral NAB-destruction groups were injected with 8.0 #g 6-OH-DA-HC1 according to a standard procedure; groups differed only on the basis of injection site or 6-OH-DA concentration, as noted. In all cases, 6-OH-DA was dissolved in a nitrogen infused saline vehicle containing 0.2 mg/ml ascorbic acid. Stereotaxic injections were made using a 27- or 32-gauge delivery needle connected to a Hamilton microsyringe; injection rate -----0.4-0.5 #l/min. The needle was allowed to remain in the brain for at least 4 min to allow diffusion away from the injection site. Surgery was performed under ether anesthesia and 60,000 U of Duracillin were injected immediately after operating. Variations between groups were as follows. Group lI. 6-OH-DA, 8.0 #g/0.8/zl, level o f oculomotor nucleus (N = 22).

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Stereotaxic coordinates were identical to those used for the electrolytic lesion group (AP 1.0, L 1.5, DV 6.7). Group III. 6-OH-DA, 8.0 #g/0.8 #1, level of oculomotor nucleus; injection into lateral edge o f bundle (N =- 11). To vary the locus of damage the injection site was moved to the lateral edge of the ventral NAB; destruction of this noradrenergic pathway should still occur following medial diffusion of 6 - O H - D A (brain coordinates: AP 1.0, L 1.7, DV 6.8). Group IV. 6-OH-DA, 8.0 #g/4.0 #1, level of oculomotor nucleus (N = 10). The dilute concentration of 6 - O H - D A was used to minimize unspecific damageVL Stereotaxic coordinates were the same as those used for the electrolytic lesion group. Group V. 6-OH-DA, 8.0 #g/0.8 #1, level of trochlear nucleus (N = 16). To further vary the locus of unspecific damage, 6-OH-DA was injected into the ventral NAB at a posterior site (AP 0.2, L 1.5, DV 6.9).

Surgical procedure, control groups The following control groups were treated identically to the ventral NABdestruction groups except for variations in surgery, as noted. Group VI. Sham lesion (N = 10). The electrode was lowered to a position 1.0 m m above that used for the electrolytic lesion group but no current was passed. Group VII. 0.8 #1 vehicle only (N ~ 8). Same procedure and coordinates as used for group II except that 6-OH-DA was not added to the vehicle. Group VIII. 4.0 ,ul vehicle only (N = 8). Same procedure as that used for group IV except for the addition of 6 - O H - D A to the vehicle. Group IX. Sham, skull-opened (N = 8). The skull flap was removed and the dura pricked but the brain was not invaded.

Histochemical procedure The effects of surgical manipulations were analyzed using the Falck-Hillarp histochemical technique for monoamines zS,2s,31. Representative animals from all groups were taken in the following numbers: group I - - 8, II - - 5, III - - 5, IV - - 3, V - - 3, VI - - 2, VII - - 2, V I I I - - 2, I X - - 2. These rats were decapitated following mild ether anesthesia 1-6 months after surgery. Portions ofneocortex from the dissected brain were taken for smears 62 while the remaining forebrain and midbrain were quickfrozen in isopentane cooled with liquid nitrogen and then freeze-dried. The freezedried brains were subsequently reacted with formaldehyde gas (80 °C, 1.5 h) generated from paraformaldehyde stored at 60 ~o relative humidity, vacuum-embedded in paraffin and stored at - - 2 0 °C until analyzed. Cortical smears were processed according to Olson and Ungerstedt 62 and were used as an index of dorsal NAB destruction 6,7s. Smears and 10-/~m brain sections were examined using a Zeiss microscope equipped with an HBO-200 W/4 super pressure mercury light source (Osram), one BG-38 and two BG-12 exciter filters and a Zeiss '47' barrier filter.

Forebrain assay procedure Forebrain norepinephrine, dopamine and serotonin were neurochemically assayed in eight 6-OH-DA-injected animals (group If) and 8 shams (groups VII and

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IX); procedures were modified from Chang 22, Maickel et a l ) 4 and Haubrich and Denzer 41. Histological procedure Brains from animals not used for histochemical or neurochemical analysis were stained with cresyl violet and studied with a light microscope. Since visible brain damage is maximum 1-7 days postoperatively85, histology was similarly performed on 12 additional rats sacrificed 6 days after surgery. The 12 animals had been evenly divided into 3 groups which received one of the following treatments according to the procedure described above: electrolytic lesion, 6-OH-DA (8.0 #g/0.8 #1), or 6-OH-DA (8.0 #g/4.0 #1). Four other lesioned and two 6-OH-DA-injected rats (8.0 #g/0.8/~1) sacrificed immediately after surgery were also examined. Results Behavioral results Electrolytic lesions or injections of 6-OH-DA in the vicinity of the ventral NAB did result in hyperphagia with average group increments ranging from 24.6 to 31.5 ~ (Table I). Eighty-seven of the 88 animals in the ventral NAB-destruction conditions increased their mean food intake following surgery. Rats in the control groups hardly changed from their preoperative baselines and these animals were significantly different from the ventral NAB-destruction rats (P < 0.001; Table I). Latency of onset of hyperphagia varied slightly among the 6-OH-DA-injected animals. Both large weight gains and large weight losses occurred during the 24 h immediately after surgery (food intake was not recorded during this interval). Hypophagia, extending into the second to third postoperative day was noted in some animals. However, in all cases, the consummatory increments which were to appear were evident by the fourth to fifth postoperative day. Among the electrolytically lesioned animals the t, yperphagia appeared much more quickly. During the first day after surgery large weight gains were not uncommon; 8 of 29 animals gained at least 10 g during this period. The hyperphagia was usually obvious by the end of the first postoperative measurement period which included the second and third day after surgery. The initial, transient, postsurgical hypophagia, observed in some 6-OH-DAinjected rats, was noted in only one electrolytically lesioned animal. In all ventral NAB-destruction groups, once an animal began to overeat, the hyperphagia continued for the duration of the 30-day postsurgical measurement period (Fig. 1). As one might expect, the increases in food consumption led to greater than normal weight gains among the ventral NAB-damaged rats (Fig. 1). In contrast, the rate of body weight gain among the control animals tended to decline over time and the mean postoperative rate was less than the baseline in these groups. The ventral NAB-damaged rats were statistically different from the sham animals as indicated in Table I (P < 0.001). Body weight of a number of the most hyperphagic rats was periodically recorded for up to 8 months after surgery. Among these animals, weight asymptotes were not reached until approximately 4-6 months postoperatively and generally

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Fig. 1. Individual feeding and body weight records from rats having undergone destruction of the ventral NAB (Experiment 1); arrow indicates time of surgery. Top two records from group II, (6-OH-DA injection, level of oculomotor nucleus); middle two: group V, (6-OH-DA injection, level of trochlear nucleus); bottom two: group I (electrolytic lesion).

occurred within a 500-600-g range o f body weight. However, two animals which were still gaining weight at the times of their death (7 and 14 months, postsurgically), each weighed approximately 900 g. A normal Sherman female rat o f the same age would weigh from 300 to 450 g. Increases in water intake also followed ventral N A B damage. Although the per cent changes from baseline were much less than those recorded for feeding, the effects were statistically significant (Table I).

Histochemical results The distribution of catecholamine fluorescent varicosities within the forebrain of the control animals corresponded to that c o m m o n l y seen in normal rats in this laboratory and was consistent with previous mapping studies30, 7s. In comparison, marked changes were observed in all rats in the ventral NAB-destruction groups (Fig. 2). A m o n g the electrolytically lesioned animals large but less than complete reductions o f catecholamine varicosities were noted in areas previously shown6, v8 to be the primary projection sites of the ventral N A B (hypothalamus, preoptic area,

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Fig. 2. Fluorescence photomicrographs representative of the effects of surgery in Experiment I. a: catecholamine varicosities surround fornix in the hypothalamus of the vehicle-injected rat. b: a marked, but less than complete reduction occurred in the electrolytically lesioned animal, while, c: a nearly total loss was noted in the 6-OH-DA-injected rat from group V. Parallel changes occurred in the medial preoptic area: d: vehicle-injected rat; e: lesioned; f: 6-OH-DA-injected animal. Losses were similar in all the 6-OH-DA group animals. Autofluorescent lipofucsin granules are present in the depleted brains ( x 63).

ventral p o r t i o n o f the interstitial nucleus o f the stria terminalis a n d regions o f the a m y g d a l a a n d septum). H o w e v e r , except f o r one animal, d o r s a l N A B - i n n e r v a t e d areas a p p e a r e d n o r m a l (cortex, t h a l a m u s a n d h i p p o c a m p u s ) . H i s t o c h e m i c a l analyses o f the 6 - O H - D A - i n j e c t e d rats revealed an a l m o s t t o t a l d e p l e t i o n o f c a t e c h o l a m i n e fluorescent varicosities in the p r o j e c t i o n areas o f b o t h the ventral a n d d o r s a l N A B , i.e. nearly all ascending n o r a d r e n e r g i c i n p u t to the f o r e b r a i n h a d been disrupted. The p a t t e r n o f d e p l e t i o n was similar in all 6 - O H - D A - i n j e c t e d

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animals except for those receiving the lateral injections (group III) in which losses in the dorsal NAB-innervated areas were only partial. It should be mentioned that a few restricted regions contained significant numbers of fluorescent varicosities (presumably noradrenergic), even when there was a nearly complete depletion in all other noradrenergically innervated areas. Very fine catecholamine varicosities in the posterolateral septum and large varicosities in the ventral supraoptic nucleus and in the rotondocellular portion of the paraventricular thalamic nucleus (medial to stria medullaris) appeared to be refractory to the effects of the 6-OH-DA injections. The paraventricular hypothalamic nucleus was also difficult to completely deplete, especially the anterior half, but a nearly total reduction was noted in a few animals. The forebrains of two electrolytically lesioned and two 6-OH-DA-injected rats which did not become markedly hyperphagic were also analyzed with fluorescence histochemical techniques. Despite the fact that none of these animals increased its feeding by as much as 10~, 3 of these 4 were markedly depleted of noradrenergic varicosities, similar to that seen in the hyperphagic animals. Damage to dopaminergic systems in the ventral NAB-damaged rats did not appear to be extensive. In the electrolytically lesioned animals no changes were detectable in the dopamine cell groups except for a loss of a few of the A-8 cells in the mesencephalic reticular formation (terminology of Dahlstr6m and Fuxe25). The terminal areas, in particular the striatum, olfactory tubercle, nucleus accumbens and central nucleus of the amygdala appeared normal. The animals injected with 6-OH-DA at the level of the rostral half of the oculomotor nucleus appeared to have lost more dopaminergic cells than the electrolytically lesioned rats although this reduction was still not extensive. The brains of these rostrally injected rats were devoid of the scattered cells in the central mesencephalic tegmentum (group A-8), while a partial loss also occurred in the posterior, dorsal region of the substantia nigra (A-9). Other catecholamine-containing cells in this region were unaffected; the mesolimbic group (A-10) as well as the catecholaminergic cells along the midline at more posterior levels were both intact. The loss of dopamine-containing cells did not manifest itself in marked changes in fluorescence intensity of the dopaminergically innervated forebrain areas such as the striatum (Fig. 3). A single exception to this pattern was noted in one animal with diminished fluorescence in portions of the striatum, accompanied by extensive cell loss in the substantia nigra. The animals which received the posterior 6-OH-DA injections at the trochlear level had a nearly normal distribution of dopamine-containing ceils. Only a few of the posterior cells of group A-8 in the central tegmentum were lost. Forebrain assay results

Forebrain norepinephrine levels in the 8 6-OH-DA-injected rats were reduced to less than 10% of the sham level (6-OH-DA group (4- S.E.M.) ~ 23 4- 11 ng norepinephrine/g; sham group = 395 4- 26 ng/g; t --~ 13.68, P < 0.01). Dopamine was slightly reduced in these 6-OH-DA animals, being 8 7 ~ of the sham value (6-OH-DA group = 833 ± 16 ng dopamine/g; sham group ----955 4- 40 ng/g; t =

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Fig. 3. Corpus striatum in animals from Experiment I. a: vehicle-injected; b: electrolytically lesioned; c: 6-OH-DA-injected. In general there were no marked losses of dopamine fluorescence in this region in any groups (x 63).

2.47, P < 0.05). Serotonin levels were nearly identical in both groups (6-OH-DA group = 622 ± 12 ng serotonin/g; sham group = 613 ± 29 ng/g; t = 0.13).

Histological results Light microscopic analysis of brains from 6-OH-DA-injected animals revealed areas of unspecific damage occurring at the cannula tip. A 0.9-1.8-ram region of gliosis was present in rats sacrificed 6 days after surgery; this was true whether the 8.0 fig of 6-OH-DA was injected in a concentrated (0.8 ffl) or a dilute (4.0/zl) dose. No actual tissue loss was noted in these animals, however, a small, 0.2-0.4-ram area of cell loss was present in the 6-OH-DA rats sacrificed immediately after surgery. Tissue loss following electrolytic lesions was considerably more extensive, being 1.01.2 m m in diameter just after the operation. Electrolytically lesioned animals sacrificed 6 days postoperatively had areas of gliosis similar in size to those injected with 6 - O H - D A but the reaction appeared more intense in the lesioned rats.

Discussion Destruction of ascending noradrenergic pathways was associated with significant increases in both food intake and weight gain. The 6-OH-DA injections which produced hyperphagia abolished nearly all forebrain norepinephrine fluorescence. Brain assays of 8 hyperphagic 6-OH-DA animals indicated that norepinephrine levels were less than 1 0 ~ of normal. Destruction of dopamine-containing neurons probably did not contribute to the increased food intake since 6 - O H - D A injection at the trochlear level produced overeating with only slight loss of dopaminergic cells. Moreover, even the anterior (oculomotor level) injections which destroyed more

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dopamine-containing cells did not result in marked dopamine losses in the forebrain projection sites; levels were close to 90 ~ of normal. The histological analysis indicated that one cannot rule out the involvement of unspecific, non-catecholaminergic damage in mediating the feeding increases. Even after dilute (4.0 #1) injections there was considerable gliosis in the cannulation region. However, it should be noted that variation of the injection site, which varied the locus of unspecific damage, did not alter ventral forebrain norepinephrine losses or the development of hyperphagia. Also, forebrain levels of non-catecholamine, serotonin were normal in the assayed, 6-OH-DA, rats. Discrete electrolytic lesions of the ventral NAB, sparing the dorsal pathway, were as effective in producing the consummatory changes as were injections of 6OH-DA which destroyed nearly all forebrain noradrenergic innervation. This evidence suggests that it may be the specific loss of the ventral NAB which produces the feeding increases. However, these data do not necessarily preclude the additional involvement of the dorsal system or the smaller intermediate NAB. Apparently norepinephrine loss does not always lead to hyperphagia. Three of 4 animals which failed to increase their food consumption by at least 10~ could not be histochemically distinguished from similarly treated hyperphagic rats. Yet, it should be noted that the converse situation never occurred; hyperphagia without marked loss of norepinephrine varicosities was never observed. Water intake was also elevated by damage to the ventral NAB. However, the hyperdipsia may well have occurred in response to changed osmotic and oral factors accompanying increased food intake. The fact that the drinking increments were approximately half of the feeding changes is consistent with this suggestion. The results of the assays indicate that over 90 ~ of forebrain norepinephrine is derived from ascending systems passing through the midbrain; this is consistent with previous findingsS,vs. However, histochemical analyses revealed 3 areas, the posterolateral septum, the ventral supraoptic nucleus and the paraventricular thalamic nucleus, which may not be innervated by the ascending noradrenergic pathways. Midbrain 6-OH-DA injections which almost totally depleted the remaining noradrenergic areas of the forebrain left these regions virtually intact. However, an alternative explanation is that the noradrenergic neurons innervating these areas are not sensitive to 6-OH-DA. E X P E R I M E N T II

The results of Experiment I suggested that destruction of norepinephrinecontaining neurons leads to hyperphagia. However, the damage was not completely specific to noradrenergic systems in any of the experimental groups. 6-Hydroxydopamine caused non-selective destruction at the injection site in addition to producing small dopamine losses; these artifacts might have played a role in the feeding increases. The selectivity of 6-OH-DA is based upon its active uptake into catecholaminergic neuronslS,Z7,45,66. The unspecific effects are presumably due to passive diffusion

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of this toxic compound or result from extraneuronal reactions, i.e., are largely independent of active uptake. Desmethylimipramine (DMI) has been demonstrated to be a selective inhibitor of active transport into noradrenergic but not dopaminergic nerves 19,24,32,a9. Therefore, if the hyperphagia following injection of 6-OH-DA is specifically due to destruction of norepinephrine-containing neurons, then DMI pretreatment should block both the hyperphagia and the loss of forebrain noradrenergic terminals.

Me~o~ General procedure Subjects and measurements were similar to those of Experiment I.

Surgical procedures DMI/6-OH-DA (N = 10). Animals were pretreated with two 25 mg/kg intraperitoneal injections of DMI-HC1 20 rain apart followed by bilateral cannulation of 8.0/~g/0.8 #1 of 6-OH-DA-HC1 into the vicinity of the ventral NAB at the oculomotor level. Brain injection procedures and stereotaxic coordinates were identical to those described for group II, Experiment I. The intracerebral cannulation of 6-OH-DA followed the second DMI injection by 30-75 rain. 6-OH-DA (N = 8). Surgical procedure same as for the above group except that the DMI injections were omitted. DMI/vehicle (N = 8). Surgical procedure same as for the DMI/6-OH-DA group except that 6-OH-DA was not added to the vehicle. Vehicle (N = 8). Surgical procedure same as for 6-OH-DA group except that both the addition of 6-OH-DA to the injection vehicle and DMI pretreatment were omitted.

Histological and histochemical procedure Representative animals from each group were taken for fluorescence histochemical analysis in the following numbers: DMI/6-OH-DA - - 5, 6-OH-DA - - 3, DMI/vehicle - - 2, vehicle - - 2, vehicle - - 2. To compare unspecific damage in DMI-injected and unpretreated animals an additional 8 rats were intracerebrally injected with 8.0 #g/0.8 #1 6-OH-DA, 4 of which were each pretreated with a total of 50 mg/kg DMI; surgery and pretreatment were identical to the description given above. These animals were sacrificed 6 days postoperatively, stained with cresyl violet and examined under the light microscope.

Results Behavioral results DMI pretreatment did prevent the hyperphagia following cannulation of 6-OHDA into the area of the ventral NAB. Injection of 6-OH-DA without pretreatment led to feeding increases in all 8 animals; 7 of 8 consumed over 20 ~ more food follow-

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ing surgery; the mean group increase was 39.1 ~ (Table II). In contrast, the DMIpretreated 6-OH-DA group ate almost the same after surgery as before (Table II) and no animal increased its food intake by as much as 20 ~. DMI, alone, appeared to have no effect upon food intake; the feeding differences between the pretreated and unpretreated vehicle groups were trivial (P > 0.10, Table II). Statistical comparisons indicated no significant differences between the vehicle groups and the DMI/6-OH-DA group but a highly significant difference between these three groups and the unpretreated 6-OH-DA animals (P < 0.001, Table II). With respect to water intake, the changes were relatively small and the pattern did not conform entirely to what was expected (Table II). Postoperative changes in body weight gain paralleled the feeding effects but the differences between groups were not large enough to reach significance (Table II).

Histochemical and histological results Analysis of unpretreated 6-OH-DA-injected animals revealed a nearly total loss of fluorescent varicosities in noradrenergically innervated areas, as described in Experiment I. In comparison, animals which were pretreated with D M I before 6-OHDA injection appeared to have a normal or nearly normal distribution of fluorescent varicosities in all forebrain areas; the brains were almost indistinguishable from those of the control animals (Fig. 4). Dopaminergic cell loss was similar in both 6-OH-DA injection groups and corresponded to the pattern previously noted among animals injected with 6-OH-DA at the oculomotor level (Experiment I). In control animals fluorescent varicosities were noted in the mesencephalic tegmentum, extending close to the posterior substantia nigra; these have been identified as being noradrenergic vs. Among the unpretreated 6-OH-DA animals these varicosities disappeared together with neighboring nigral cells. In the DMI-pretreated, 6-OH-DA rats only the dopaminecontaining nigral cells disappeared. Examination of cresyl violet-stained brains from animals sacrificed 6 days after surgery indicated that DMI did not alter the pattern of unspecific damage produced by 6-OH-DA. Gliosis and cell loss around the injection site were identical in DMIpretreated and the unpretreated rats. Discussion Rats injected with 6-OH-DA into the vicinity of the ventral noradrenergic bundle became hyperphagic and histofluorescence analysis revealed a nearly complete loss of fluorescent varicosities in forebrain areas known to be noradrenergically innervated. Other rats which received systemic administration of the selective noradrenergic neuron uptake blocker, DMI, before 6-OH-DA, ate normally and appeared to have normal noradrenergic innervation of all forebrain areas. Cell losses in the dopaminergic areas A-8 and A-9 were similar in both groups. The area of unspecific damage was not altered by D M I pretreatment. These findings would apparently rule out the destruction of a dopaminergic system or unspecific damage as being responsible for the development of hyperphagia. Destruction of the ventral NAB and perhaps

Fig. 4. Fluorescence photomicrographs representing the effect of DMI pretreatment (50 mg/kg) on 6-OH-DA-induced loss of forebrain noradrenergic varicosities in Experiment II. a: the ventral region of the interstitial nucleus of the stria terminalis is heavily innervated in the control animal (the anterior commissure is at the top of the picture), b: rat injected with 6-OH-DA, alone, is completely devoid of catecholamine fluorescence in this area; the remaining bright particles are autofluorescent lipofucsin granules, c: in contrast, the rat pretreated with DMI before 6-OH-DA has a normal distribution of norepinephrine-containing varicosities here as well as in other forebrain areas. A similar pattern is illustrated in sections from the basal hypothalamus at the level of the paraventricular nucleus, d: a normal distribution of fluorescent varicosities is present in the animal injected with 6-OH-DA but pretreated with DMI. e: these are absent in the unpretreated, 6-OH-DA-injected animals ( × 63).

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other noradrenergic pathways appears to be the cause of the consummatory increases. E X P E R I M E N T III

The evidence from the first experiment suggests that destruction of the ventral NAB may be of primary importance to the development of hyperphagia. However, the dorsal NAB also contributes to some extent to the noradrenergic innervation of the hypothalamus51, 53 and this pathway may also be involved in the regulation of food intake. The present experiment tested whether selective destruction of the dorsal NAB without ventral NAB damage would affect feeding.

Methods General procedure Subjects and measurements were the same as in Experiment I. All subjects were identically treated except for surgery.

Surgical procedure 6-OH-DA/dorsal NAB (N = 12). Eight/zg of 6-OH-DA-HC1 in 0.8/zl of vehicle was stereotaxically injected according to the general procedure described in the first experiment. The tip of the injection needle was intended for the region directly above the dorsal NAB with corresponding brain coordinates: AP 2.8, L O.85, DV 5.6. 6-OH-DA/ventral NAB (N = 8). Eight#g/0.8/zl 6-OH-DA was bilaterally injected into the region of the ventral NAB using the same brain coordinates and procedures as described for group II, Experiment I. Vehicle/dorsal NAB (N = 8). Animals received the same treatment as the 6-OH-DA/dorsal NAB group except for the addition of 6-OH-DA to the injection vehicle.

Histochemical and histological procedure Analyses were conducted identically to Experiment I. Representative animals from each group for fluorescence histochemical analysis were as follows: 6-OH-DA/ dorsal NAB - - 6, 6-OH-DA/ventral NAB - - 3, vehicle/dorsal NAB - - 2.

Results Behavioral results Injection of 6-OH-DA into the vicinity of the dorsal NAB did not result in hyperphagia while similar injections into the ventral NAB produced overeating. The less than 2 ~ changes from baseline in both the 6-OH-DA/dorsal NAB and control groups were not significantly different from one another (P > 0.10, Table III); however, the 35.5 ~ increase in food intake among the ventral NAB animals was statistically different from the dorsal NAB and control values (P < 0.001, Table III).

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The 6-OH-DA injections near the dorsal bundle also failed to produce any notable decreases in food intake. Postsurgical aphagia was never observed in these animals and data from the first postoperative measurement period, covering days 2 and 3 after surgery, revealed no significant differences between the dorsal NAB- and the vehicle-injected groups (per cent change in food intake and standard error of the mean, postoperative days 2 and 3: 6-OH-DA/dorsal NAB group = - - 6 . 0 ± 6.4 ~ ; vehicle/dorsal NAB = --11.4 ± 5 . 5 ~ ; t = 0.59, d f = 18, P > 0.10). Postoperative changes in water intake were statistically similar in the dorsal NAB group and the controls (P > 0.10, Table III). In contrast, the 20~o increment in drinking noted in the ventral NAB animals was significantly different from the other two groups (P < 0.005, Table lII). Postsurgical changes in the rate of body weight gain tended to reflect the consummatory data. Both the dorsal NAB injected- and the control-group's rate of weight gain fell off after surgery while this measure increased in the ventral NAB group. Although the control group decrease was statistically greater than the rate decrement in the dorsal NAB group, this difference was only marginally significant; most of the variance in the statistical analysis was attributable to the large difference between ventral NAB animals versus the other two groups (Table III). Histochemical and histological results Fluorescence histochemical analysis of the 6-OH-DA/dorsal NAB animals revealed a nearly complete absence of catecholamine varicosities in forebrain areas known to be innervated by this dorsal pathway6,53,7s. Whole brain or smear 62 analysis of neocortex indicated that this region was totally devoid of noradrenergic innervation (Fig. 5); the fine varicosities of the hippocampus were likewise, completely absent. The thalamus, which also receives dorsal NAB input, was devoid of catecholamine fluorescence in most regions with the exceptions being the paraventricular thalamic nucleus and the antero-ventral nucleus. With respect to the former structure, no depletion was ever noted, while in the case of the antero-ventral nucleus, losses were less than complete and varied from animal to animal. There was also a generally uniform loss of fluorescent varicosities in the main projection areas of the ventral NAB in most of the 6-OH-DA/dorsal NAB rats. However, this reduction was considerably less than that seen after ventral NAB electrolytic lesions. Dopaminergic systems were unchanged in this group. The fluorescence pattern among the animals in the ventral NAB group was similar to that described in the first experiment. There was a nearly complete loss of fluorescent varicosities in all forebrain regions. Analyses were made relative to the brains of the sham-operated rats which appeared to have a normal distribution of catecholamine fluorescence. Discussion Animals injected with 6-OH-DA near the dorsal NAB neither increased nor decreased their food intake; dorsal NAB disruption was verified by complete loss

FOOD INTAKE AND AMPHETAMINE ANOREXIA

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Fig. 5. Fluorescence photomicrographs depicting norepinephrine depletion patterns induced by 6-OH-DA injection (Experiment IIl). Forebrain projection areas of the dorsal NAB were devoid of fluorescent varicosities in animals injected near the dorsal or the ventral pathways, a: numerous varicosities are apparent in smear from the neocortex of the control animal, b: these are absent in the rat injected with 6-OH-DA near the dorsal NAB, and c: catecholamine fluorescence is also gone in smear from a ventral NAB-injected animal. Autofluorescent lipofucsin granules remain in the depleted smears (× 100). In contrast, the ventral forebrain (e.g., hypothalamus and preoptic area) was devoid of fluorescent varicosities only in animals injected with 6-OH-DA near the ventral NAB. d: catecholamine fluorescence in the subfornical hypothalamus of a control rat (level of the ventromedial nucleus; fornix toward the top of the picture), e: similar distribution of varicosities is present in 6-OH-DA/dorsal NAB rat. f: there is almost a complete loss of fluorescence in the 6-OH-DA/ ventral NAB animal (× 63).

o f cortical a n d h i p p o c a m p a l catecholamine varicosities. Similar injections near the ventral N A B in other animals p r o d u c e d hyperphagia. A p p a r e n t l y , the dorsal p a t h w a y does n o t play a significant role in c o n s u m m a t o r y regulation. The small reduction o f h y p o t h a l a m i c noradrenergic varicosities following dorsal N A B 6 - O H - D A injections is consistent with Loizou's 51 report o f h y p o t h a l a m i c losses after locus coeruleus lesions; the locus coeruleus is the sole nucleus o f origin o f the dorsal N A B 53,7s. However, in this study, h y p o t h a l a m i c losses m a y also have been due to destruction o f scattered fibers which ascend between the dorsal a n d ventral noradrenergic bundles. U n g e r s t e d t vs shows fibers travelling ventrolateral to the dorsal N A B while

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Maeda and Shimizu 53 describe a discrete bundle, the intermediate pathway, directly ventral to the dorsal system. The thalamus has been reported to be one of the primary projection areas of the dorsal NAB vs. In this study two thalamic nuclei still contained catecholamine varicosities when other dorsal NAB-innervated areas (e.g., neocortex and hippocampus) were completely depleted. The difficulty in denervating the paraventricular nucleus of the thalamus has previously been noted (Experiment I). In the case of the antero-ventral thalamic nucleus, at least a few varicosities remained after dorsal NAB disruption and this appears to be consistent with the finding of Maeda and Shimizu aa. They reported that antero-ventral thalamic varicosities were undiminished by locus coeruleus lesions. EXPERIMENT IV

Numerous studies have suggested that the noradrenergic input to the basal forebrain plays an important role in regulating specific segments of the female estrous cycle. Blocked ovulation and prolonged diestrus follow reserpine-induced depletion of monoamines s, norepinephrine receptor blockade 67-69 or destruction of catecholamine-containing neurons by intraventricular 6-OH-DA 43. These findings suggest that activity at noradrenergic synapses might be necessary for the increased gonadotropin release in proestrus which leads to ovulation. The appetite of female rats appears to be suppressed by the presence of circulating ovarian steroidsll,42,46,s2, sa. In particular, food intake and body weight vary with the estrous cycle and consumption is much lower during estrus than diestrus16,47,71.

Viewed together, these studies indicate that noradrenergic manipulations are capable of disrupting the estrous cycle and that such changes in ovarian steroid rhythms can lead to altered food consumption. Since all the rats reported in this series of experiments have been female, perhaps the hyperphagia following ventral NAB destruction was secondary to disruption of the estrous cycle. If this were the case, it would be expected that ventral NAB damage in male rats would not result in overeating.

Methods General procedure Subjects were all Sherman male rats weighing 340-450 g at the time of surgery. Consummatory and body weight measurements were taken as described in Experiment I.

Surgical procedure 6-OH-DA-injeeted (N = 10). 6-OH-DA-HC1 was bilaterally injected into the vicinity of the ventral NAB at the level of the rostral half of the oculomotor nucleus; procedures were identical to those described for group II, Experiment I.

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Vehicle-injected (N = 10). Treatment was the same as in the 6-OH-DA-injected group except that 6-OH-DA-HC1 was not added to the injection vehicle. Histochemical and histological procedure Brains were treated as in Experiment I. Five representative 6-OH-DA-injected and 3 vehicle-injected animals were used in the histochemical analysis.

Results Male rats injected with 6-OH-DA in the vicinity of the ventral NAB ate an average of 2 2 . 3 ~ more food following surgery; food consumption in the control group was essentially unchanged (comparison between groups, P < 0.001, Table IV). Postoperative water intake rose in both groups and the difference between the two groups was not significant (P ~ 0.10, Table IV).

Histochemical and histological results Analysis of the representative 6-OH-DA-injected rats revealed depletion of forebrain catecholamine varicosities similar to the animals injected with 6-OH-DA at the oculomotor level in Experiment I. The distribution of fluorescent varicosities in the 3 male control animals was identical to that previously observed in sham or unoperated female rats.

Discussion Male rats, like female rats, do become hyperphagic following ventral NAB destruction. This suggests that disruption of the estrous cycle cannot account for the observed feeding increments. EXPERIMENT V

Literature cited at the beginning of this article suggested that the ventral NAB is involved in the suppression of feeding and in addition mediates at least part of amphetamine's effects upon appetite. The work reported thus far supports the former point; loss of this noradrenergic influence results in overeating. It therefore seemed reasonable to expect that disruption of the ventral NAB would antagonize amphetamine's anorectic effects. This hypothesis was initially tested using rats which had undergone electrolytic destruction of this noradrenergic pathway. The study was subsequently replicated employing 6-OH-DA-injected animals; both of these experiments are reported here.

Me~o~ General procedure Identical procedures were used in both the lesion experiment and the 6-OH-DA

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replication, except for surgery. Subjects were singly housed, female Sherman rats weighing 250-340 g at the time of the operation. After surgery the animals were placed on a 20 h/day food deprivation schedule (Purina Laboratory Pellets) which was continued for the duration of the experiment; water was always available. After 2 weeks of experience on the feeding schedule the animals were intraperitoneally injected with saline on 2 successive days to habituate them to the injection procedure; the actual testing began 1 week later. Baseline tests using the saline vehicle were given on the day preceding the amphetamine administration. The injection preceded the presentation of food by 0.5 h and consumption was measured 1 h after food was made available. It was also measured 4 h later but these data will not be reported here. Spillage was caught and corrected for. Food intake after amphetamine was expressed as a per cent of the amount eaten after saline on the previous day. Five doses of D-amphetamine sulfate, dissolved in saline, were intraperitoneally injected (0.5, 1.0, 2.0, 4.0 and 8.0 mg/kg). The groups were divided in two so that approximately half the animals in each group received the amphetamine doses in the order opposite to the other half. A minimum period of 7 days elapsed between each amphetamine injection.

Surgical procedure, lesion experiment Lesion (N = 9). Electrolytic lesions intended to destroy the ventral NAB were made according to the procedure described for group I, Experiment I, with slight modifications (brain coordinates: AP 1.0, L 1.5, DV 6.8; lesion parameters: 0.65 mA for 30 sec). Sham (N = 8). The skull flap was removed, the dura pricked, but the brain was not invaded. Surgical procedure, 6-OH-DA experiment 6-OH-DA (N = 10). The animals were injected in the vicinity of the ventral NAB at the level of the trochlear nucleus. The surgical technique was similar to the description given for group V, Experiment I, except that a more dilute dose of 6-OH-DA was employed (8.0 #g/4.0/A). Sham (N = 10). The dura was pricked after removal of the skull flap. Histochemical and histological procedure All of the electrolytically lesioned animals were perfused with formalin and the brains stained with cresyl violet. Fluorescence histochemical analysis of cerebral catecholamines was performed on 5 injected and 5 sham animals from the 6-OH-DA experiment; the histochemical method was described in Experiment I. Results Behavioral results Noradrenergic damage did attenuate amphetamine-induced anorexia and shift

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the dose-response curve to the right (Fig. 6). Comparison across all doses of the electrolytically lesioned animals versus the lesion controls revealed a highly significant difference (F = 13.61; d f = 1,15; P < 0.005). A similarly significant main effect was also obtained when the 6-OH-DA-injected animals were evaluated relative to their control group (F = 11.38; d f ~- 1,18; P < 0.005). There was a statistically reliable effect of dose in both experiments (P < 0.001), however, the interaction between group and dose reached significance only in the 6-OH-DA study (P < 0.005). This latter finding probably resulted from the floor effect at the highest amphetamine levels; no significant interactions but undiminished group effects were found when the statistics were recomputed eliminating data from the two highest amphetamine doses. Histochemical and histological results

Lesion placement was verified in all the electrolytically lesioned animals. The 6-OH-DA-injected rats were nearly totally devoid of varicosities in all the noradrenergically innervated forebrain areas identical to the description given in Experiment I for similarly operated animals. Almost no reduction of dopaminergic neurons was noted in these rats. Discussion

Electrolytic lesions placed in the path of the ventral NAB attenuated amphetamine anorexia. In a second experiment, 6-OH-DA injection near the ventral system similarly antagonized amphetamine's effect upon appetite. Fluorescence histochemistry verified that after the 6-OH-DA injections almost all the ascending noradrenergic input to the forebrain had been destroyed. Dopamine-containing neurons did not seem to be involved since the relatively posterior injections used here caused almost no dopaminergic cell losses. Therefore, facilitation of transmission at noradrenergic

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synapses appears to be one of the ways in which amphetamine suppresses eating. It should be noted, however, that surgically induced motivational changes could also have mediated the reduction in amphetamine potency, independent of an interaction between this drug and noradrenergic systems. It is apparent that activation of norepinephrine-containing neurons cannot account for the entire amphetamine effect upon appetite; in the present experiments the dose-response curves were only shifted by the norepinephrine loss. Obviously other systems or mechanisms are also operating. Central dopaminergic neurons may well be involved in view of Ungerstedt's findings76. It is also possible that amphetamine has a direct action upon receptors, particularly at higher dosesZ% GENERAL DISCUSSION

This series of experiments indicates that the main noradrenergic pathway to the hypothalamus, the ventral NAB, contributes to the inhibitory control of food intake and, in addition, is probably involved in the mediation of amphetamine anorexia. Destruction of this system results in hyperphagia, often leading to obesity and also antagonizes amphetamine-induced appetite loss. These results are diametrically opposed to what one would have expected based upon the previous work of numerous investigators. Repeatedly, it has been shown that hypothalamically injected norepinephrine elicits feeding rather than inhibiting it (e.g., refs. 13, 38 and 57). In an effort to explain these differences, a number of possibilities are considered below. Denervation supersensitivity might account for the present findings, however, two points argue against this. First, although the development of supersensitivity could conceivably return a deficient system to normal, there are no physiological precedents to suggest that a supra-normal response could occur after denervation (in the absence of exogenous pharmacological agents). Second, in view of the nearly total depletion of forebrain norepinephrine noted in many of the 6-OH-DA animals, it seems unlikely that the few remaining terminals could support a supra-normal response. Previous studies demonstrating norepinephrine elicited eating, rather than satiety, are subject to alternative explanations. The implicit assumption of studies involving cannulated norepinephrine is that the drug-receptor interaction is occurring at noradrenergic synapses. However, it is not clear that noradrenoceptive sites are found only at noradrenergic synapses. Pertinent to this issue, Bloom et al. 1° have noted that cerebellar Purkinje cells respond to iontophoretically injected serotonin despite a complete absence of serotonergic afferents. It is also conceivable that the presence of unphysiological amounts of norepinephrine, as occur following cannulation, produces an opposite postsynaptic effect to that of the endogenous transmitter. This could be mediated by: (a) depolarization blockade, as has been noted in other systems29,6a; (b) dual receptors with different thresholds located at a single synapse, similar to those found in Aplysiaaa,s°,sl; or (c) a recurrent inhibitory circuit (the essential condition being that the recurrent

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portion of the system only be functional at high rates of firing, i.e., have a high threshold). Recent electrophysiological evidence suggests that one of these latter two mechanisms does, indeed, occur within the rat hypothalamus5s. Leibowitz4s,49 has found evidence for two adrenoceptive components within the hypothalamus, a lateral beta-satiety and a medial alpha-feeding system. If one assumed the lateral beta-system to be prepotent in the control of food intake this could explain why forebrain norepinephrine loss led to hyperphagia. However, this explanation meets with two objections. First, although quite high concentrations of norepinephrine are required for the elicitation of feeding from medial and perifornical regions, even much larger and extremely unphysiological quantities must be injected into the lateral area to produce satiety. Second, fluorescence histochemical analysis of normal brains in this laboratory reveals that the bulk of the noradrenergic innervation of the hypothalamus is contained in medial and perifornical regions, where injected norepinephrine elicits feeding. Thus, although possible, it would be surprising if the lateral system predominated to the extent necessary to explain the present findings. Norepinephrine-elicited eating may also involve changes in the hypotha|amic blood supply. Alpha-adrenergic receptors mediating vasoconstriction have been demonstrated in the mammalian hypothalamus65. Perhaps vasoconstriction due to alpha-adrenergic activation creates a state of local hypoglycemia, hypoxia, etc., within the medial hypothalamic satiety region and thus leads to eating. The disparity between the present findings and previous reports of norepinephrine-elicited eating might also be reconciled by assuming that hyperphagia in these two cases is mediated by different regions of the brain, or through the same extrahypothalamic area. Ventral NAB destruction may have had predominant effects at projection sites other than the ventral forebrain where injected norepinephrine elicits feeding. It is not inconceivable that retrograde disruption of, for example, hindbrain noradrenergic innervation was the critical factor in the production of hyperphagia. By the same token, hypothalamic cannulation of norepinephrine may induce eating through interactions occurring at distant noradrenergic synapses. Pharmacological evidence indicates that activation of monoamine receptors decreases turnover of endogenous monoamines while blockers produce an opposite effect4. Therefore, hypothalamically cannulated norepinephrine may be reducing activity in the entire presynaptic noradrenergic nerve and disruption of transmission in other branches of this neuron could be the cause of eating. An additional possibility involves the suggestion by Margules et al. 56 that intracerebrally injected norepinephrine only elicits eating during the day while reducing food intake at night. Since rats are nocturnal animals and do most of their eating at night, the net effect of noradrenergic neuron destruction, according to this model, should be increased food intake. Noradrenergic effects upon consumption have generally been assumed to be the result of changes in the electrophysiological state of a neural hunger or satiety system. The fact that the manipulations have occurred in the vicinity of the hypophysiotropic zone which regulates pituitary function has received little attention.

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Feeding changes following noradrenergic denervation or after hypothalamic cannulation of norepinephrine could be secondary to hormonal effects. For example, brain catecholamines appear to influence adrenocorticotropic33, 50 as well as somatotropic 59-61 hormone secretion. Both are intimately involved in metabolic functions and perhaps might participate in the regulation of food intake 37,4°,64. Whatever the mechanism, the present experiments do indicate that disruption of the ventral NAB, specifically, or all ascending noradrenergic pathways to the forebrain results in overeating, weight gain and attenuation of amphetamine anorexia. Concomitant damage to non-noradrenergic neurons was not associated with hyperphagia; both 6-OH-DA-induced overeating and loss of norepinephrine varicosities were blocked by D M I pretreatment which did not affect the size or pattern of the unspecific injection damage. These results indicate that the norepinephrine influence on feeding in the normal animal is predominantly inhibitory. ACKNOWLEDGEMENTS

Some of the experiments reported here were included in the dissertation submitted by J. Eric Ahlskog to the Department of Psychology, Princeton University in partial fulfillment of the requirements for a Ph.D. degree 1. The author wishes to thank Professor Bartley G. Hoebel for encouragement, support and advice while this research was being conducted. The author is additionally indebted to Patrick K. Randall for performing the neurochemical assays. This study was conducted while the author was a Spencer Neuroscience Fellow; additionally supported by USPHS Grants MG-08493 and MH-08493-09 and NSF Grants GB-8431X1 and GB-8431X2 to B. G. Hoebel.

REFERENCES 1 AHLSKOG,J. E., Brain Norepinephrine and its Involvement in the Regulation of Food Consumption, doctoral dissertation submitted to Princeton University, 1973. 2 AHLSKOG,J.E., AND HOEBEL,B.G., Hyperphagia resulting from selective destruction of an ascending adrenergic pathway in the rat brain, Fed. Proe., 31 (1972) 1002. 3 AHLSKO~,J. E., AND HOEBEL,B. G., Overeating and obesity from damage to a noradrenergic system in the brain, Science, 182 (1973) 166-169. 4 ANDI~N, N.-E., CORRODI, H., AND FUXE, K., Turnover studies using synthesis inhibition. In G. HOOPER(Ed.), Metabolism of Amines in the Brain, MacMillan, London, 1969, pp. 3847. 5 AND~N, N.-E., DAHLSTROM, A., FUXE, L., LARSSON, K., OLSSON, L., AND UNGERSTEDT, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physiol, scand., 67

(1966) 313-326. 6 ARBUTUNOTT,G. W., CROW,T. J., FUXE, K., OLSSON,L., AND UNGERSTEDT,U., Depletion of catecholamines in vivo induced by electrical stimulation of central monoamine pathways, Brain Research, 24 (1970) 471-483. 7 BAEZ,L., The Role of Brain Catecholamines in the Anorectie Response to Amphetamine, unpublished doctoral dissertation, Princeton University, 1973. 8 BARRACLOUGH,C. A., AND SAWYER,C. H., Induction of pseudopregnancy in the rat by reserpine and chlorpromazine, Endocrinology, 65 (1959) 563-571. 9 BLOOM,F. E., ALGERI, S., GROPPETTI,A., REVUELTA,A., AND COSTA,E., Lesions of central norepinephrine terminals with 6-OH-DA: biochemistry and fine structure, Science, 166 (1969) 1284-

1286.

238

J.E. AHLSKOG

10 BLOOM, F. E., HOFFER, B. J., SIGGINS, G. R., BARKER, J. L., AND NICOLL, R. A., Effects of serotonin on central neurons: microiontophoretic administration, Fed. Proc., 31 (1972) 97-106. 11 BOGART, R., LASLEY, J. F., AND MAYER, D. T., Influence of reproductive hormones in ovariectomized female rats, Endocrinology, 35 (1944) 173-181. 12 BOOTH, D. A., Amphetamine anorexia by direct action on the adrenergic feeding system of rat hypothalamus, Nature (Lond.), 217 (1968) 869-870. 13 BOOTH, D. A., Mechanism of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus, J. Pharmacol. exp. Ther., 160 (1968) 336-348. 14 BREESE, G. R., AND TRAYLOR, T. D., Depletion of brain norepinephrine and dopamine: evidence for selective degeneration of catecholamine neurons, J. Pharmacol. exp. Ther., 174 (1970) 413-420. 15 BREESE,G. R., AND TRAYLOR, T. D., Depletion of brain norepinephrine and dopamine by 6-hydroxydopamine, Brit. J. Pharmacol., 42 (1971) 88-92. 16 BROBECK,J. R., WHEATLAND, M., AND STROMINGER,J. L., Variations in regulation of energy exchange associated with estrus, diestrus and pseudopregnancy in rats, Endocrinology, 40 (1947) 65-72. 17 BURKARD, W. P., JALFRE, M., AND BLUM, J., Effect of 6-hydroxydopamine on behavior and cerebral amine content in rats, Experientia (Basel), 25 (1969) 1295-1296. 18 CARLISLE,H. J., Differential effects of amphetamine of food and water intake in rats with lateral hypothalamic lesions, J. comp. physiol. Psychol., 58 (1964) 47-54. 19 CARLSSON, A., CORRODI, H., FVXE, K., AND HOKFELT, T., Effects of some antidepressant drugs on the depletion of intraneuronal brain catecholamine stores caused by 4,alpha-dimethyl-metatyramine, Europ. J. PharmacoL, 5 (1969) 367-373. 20 CARLSSON, A., FUXE, K., HAMBERGER,B., AND LINDQVIST, M., BiD-chemical and histochemical studies on the effects of imipramine-like drugs and (--)-amphetamine on central and peripheral catecholamine neurons, Acta physiol, scand., 67 (1966) 481-497. 21 CARR, L. A., AND MOORE, K. E., Norepinephrine: release from brain by D-amphetamine in vivo, Science, 164 (1969) 322-323. 22 CHANG, C. C., A sensitive method for spectrophotofluorometric assay of catecholamines, Int. J. Neuropharmacol., 3 (1964) 643-649. 23 CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 6 5 ~ 8 . 24 COYLE, J. Z., AND SNYDER, S. H., Antiparkinson drugs: inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action, Science, 166 (1969) 899-901. 25 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. Demonstration of monoamines in the cell bodies of brain stem neurons. I, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 26 EPSTEIN, A., Suppression of eating and drinking by amphetamine and other drugs in normal and hyperphagic rats, J. comp. physiol. Psychol., 52 (1959) 37-45. 27 EVETTS, K. D., AND IVERSEN, L. L., Effects of protriptyline on the depletion of catecholamines induced by 6-hydroxydopamine in the brain of the rat, J. Pharm. Pharmacol., 22 (1970) 540--543. 28 FALCK, B., HILLARP, N. A., THIEME, G., AND TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 29 FELTZ, P., AND DE CHAMPLAIN, J., Enhanced sensitivity of caudate neurones to microiontophoretic injections of dopamine in 6-hydroxydopamine treated cats, Brain Research, 43 (1972) 601-605. 30 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system, IV, Acta physiol, scand., 64, Suppl. 247 0965) 39-85. 31 FuxE, K., H(3KFELT, G., JONSSON, G., AND UNGERSTEDT, U., Fluorescence microscopy in neuroanatomy. In J. H. NAUTA AND S. O. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, 1969, pp. 275-314. 32 FUXE, K., AND UNGERSTEDT, U., Histochemical studies on the effect of (+)-amphetamine, drugs of the imipramine group and tryptamine on central neurons after intraventricular injection of catecholamines and 5-hydroxytryptamine, Europ. J. Pharmacol., 4 (1969) 135-144. 33 GANONG, W. F., Evidence for a central noradrenergic system that inhibits A C T H secretion. In K. KNIGGE, D. SCOTT AND A. WEINDL (Eds.), Brain-Endocrine Interaction - - Median Eminence: Structure and Function, Karger, New York, 1972, pp. 254-266. 34 GARDNER, K., AND KANDEL, E. R., Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition, Science, 176 (1972) 675-678.

FOOD INTAKE AND AMPHETAMINE ANOREXIA

239

35 GLOWINSKI,J., AND AXELROD,J., Effects of drugs on the uptake, release and metabolism of 3Hnorepinephrine in the rat brain, J. Pharmacol. exp. Ther., 149 (1965) 43-49. 36 GLOWINSKI, J., IVERSEN, L., AND AXELROD,J., Storage and synthesis of norepinephrine in the reserpine treated rat brain, J. Pharmacol. exp. Ther., 151 (1966) 385-399. 37 GROSSIE,J., AND TURNER, C. W., Effect of thyro-parathyroidectomy and adrenalectomy on food intake in rats, Proe. Soc. exp. Biol. (N. Y.), 118 (1965) 28-30. 38 GROSSMAN,S. P., Eating or drinking elicited by direct adrenergic or cholinergic stimulation of the hypothalamus, Science, 132 (1960) 301-302. 39 HAGGENDAL, J., AND HAMBERGER, B., Quantitative in vitro studies on noradrenaline uptake and its inhibition by amphetamine, desipramine and chloropromazine, Acta physiol, scand., 70 (1967) 277-280. 40 HAN, P. W., Hypothalamic obesity in rats without hyperphagia, Trans. N. Y. Acad. Sci., 30 (1967) 229-243. 41 HAUBRICH,D. R., AND DENZER, J. S., Simultaneous extraction and fluorometric measurement of brain serotonin, catecholamines, 5-hydroxy-indoleacetic acid and homovanillic acid, Anal. Biochem., 55 (1973) 306-312. 42 HERVEY, E., AND HERVEY, G. R., The relationship between the effects of ovariectomy and of progesterone treatment on body weight and composition in the female rat, J. Physiol. (Lond.), 187 (1966) 44. 43 H()KFELT,T., AND FUXE, K., On the morphology and the neuroendocrine role of the hypothalamic catecholamine neurons. In K. KNIGGE, D. SCOTT AND A. WEINDL (Eds.), Brain-Endocrine Interaction - - Median Eminence: Structures and Function, Karger, New York, 1972, pp. 181-233. 44 JACKS, B. R., DE CHAMPLAIN, J., AND CORDEAU, J. P., Effects of 6-hydroxydopamine on putative transmitter substances in the central nervous system, Europ. J. Pharmacol., 18 (1972) 353-360. 45 JONSSON, G., AND SACHS, C. H., Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons, Europ. J. Pharmacol., 9 (1970) 141-155. 46 KAKOLEWSKI, J. W., COX, V. C., AND VALENSTEIN, E. S., Sex differences in body-weight change following gonadectomy of rats, Psychol. Rep., 22 (1968) 547-554. 47 KENNEDY, G. C., AND MITRA, J., Hypothalamic control of energy balance and the reproductive cycle in the rat, J. Physiol. (Lond.), 166 (1963) 395-407. 48 LEIBOWITZ, S. F., Reciprocal hunger-regulating circuits involving alpha- and beta-adrenergic receptors located, respectively, in the ventromedial and lateral hypothalamus, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1063-1070. 49 LEmOWITZ, S. F., Hypothalamic norepinephrine as an alpha- and beta-adrenergic neurotransmitter active in the regulation of normal hunger, Proc. 79th Ann. Cony. Amer. Psychol. Ass., (1971) 741-742. 50 LIPPA, A. S., ANTELMAN, S. M., FAHRINGER,E. E., AND REDGATE, E. S., Relationship between catecholamines and ACTH: effects of 6-hydroxydopamine, Nature New Biol., 241 (1973)24-25. 51 LoIzou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. 52 LYTLE, L., The Ontogeny o f Lateral Hypothalamic Control o f Feeding and Drinking in the Rat, Unpublished doctoral dissertation, Princeton University, 1970. 53 MAEDA, T., ET SHIMIZU, N., Projections ascendantes du locus coeruleus et d'autres neurones aminergiques au niveau du prosenc6phale du rat, Brain Research, 36 (1972) 19-35. 54 MAICKEL, R. P., Cox, R. H., SAILLANT,J., AND MILLER, F. P., A method for the determination of serotonin and norepinephrine in discrete areas of rat brain, Int. J. Neuropharmacol., 7 (1968) 275-281. 55 MALMFORS,T., AND SACHS, C. H., Degeneration of adrenergic nerve produced by 6-hydroxydopamine, Europ. J. Pharmacol., 3 (1968) 89-92. 56 MARGULES,D. L., LEWIS, M.J., DRAGOVlCH,J. A., AND MARGULES,A. S., Hypothalamic norepinephrine: circadian rhythms and the control of feeding behavior, Science, 178 (1972) 640-643. 57 MILLER, N. E., Chemical coding of behavior in the brain, Science, 148 (1965) 328-338. 58 MILLER, J. J., AND MOGENSON, G. J., Effect of septal stimulation on lateral hypothalamic unit activity in the rat, Brain Research, 32 (1971) 125-142. 59 MULLER,E. E., DAL PRA, P., AND PECILE,A., Influence of brain neurohormones injected into the lateral ventricle of the rat on growth hormone release, Endocrinology, 83 (1968) 893-896. 60 MULLER,E. E., PECILE,A., FELICI, M., AND COCCHI, D., Norepinephrine and dopamine injection into lateral brain ventricle of the rat and growth hormone-releasing activity in the hypothalamus and plasma, Endocrinology, 86 (1970) 1376-1382.

240

J. E. AHLSKOG

61 MULLER, E. E., SAWANO, S., ARIMURA,A., AND SCHALLY, A. V., Blockade of growth hormone by brain norepinephrine depletors, Endocrinology, 80 (1967) 471-476. 62 OLSON, L., AND UNGERSTEDT,U., Monoamine fluorescence in CNS smears: sensitive and rapid visualization of nerve terminals without freeze-drying, Brain Research, 17 (1970) 343-347. 63 PATON, W. D. M., AND PERRY, W. L. M., The relationship between depolarization and block in the cat's superior cervical ganglion, J. Physiol. (Lond.), 119 (1953) 43-57. 64 PFAFF, D., Sex differences in food intake changes following pituitary growth hormone or prolactin injections, Proc. 77th Ann. Cony. Amer. Psychol. Ass., 4 (1969) 211-212. 65 ROSENDORFF, C., The measurement of local cerebral blood flow and the effect of amines. In J. MEYER AND J. SCHADI~(Eds.), Cerebral Blood Flow, Progr. Brain Res., Vol. 35, Elsevier, Amsterdam, 1972, pp. 114-156. 66 SACHS, C. H., Effect of 6-hydroxydopamine in vitro on the adrenergic neuron. In T. MALMFORS AND H. THOENEN (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, North-Holland Publ., Amsterdam, 1971, pp. 59-74. 67 SAWYER, C.H., Nervous control of ovulation. In C. W. LLOYD (Ed.), Recent Progress in the Endocrinology of Reproduction, Academic Press, New York, 1959, pp. 1-20. 68 SAWYER,C. H., MARKEE, J. E., AND HOLLINSHEAD, W. H., Inhibition of ovulation in the rabbit by the adrenergic blocking agent dibenamine, Endocrinology, 41 (1947) 395-402. 69 SAWYER, C. H., MARKEE, J. E., AND TOWNSEND, B. F., Cholinergic and adrenergic components in the neurohormonal control of the release of LH in the rabbit, Endocrinology, 44 (1949) 18-37. 70 SIMS, K. L., AND BLOOM, F. E., Rat brain 1-3-4-dihydroxyphenylalanine and 1-5-hydroxytryptophan decarboxylase activities: differential effect of 6-hydroxydopamine, Brain Research, 49 (1973) 165-175. 71 SLONAKER, J. R., The effect of copulation, pregnancy, pseudopregnancy and lactation on the voluntary activity and food consumption on the albino rat, Amer. J. Physiol., 71 (1925) 362-394. 72 STOWE, F. R., AND MILLER, A. T., The effect of amphetamine on food intake in rats with hypothalamic hyperphagia, Experientia (Basel), 13 (1957) 114-115. 73 TAVLOR,K. M., AND SNYDER, S. H., Amphetamine: differentiation by D- and L-isomers in behavior involving brain norepinephrine or dopamine, Science, 168 (1970) 1487-1489. 74 TRANZER,J. P., AND THOENEN, H., An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-OH-DA, Experientia (Basel), 24 (1968) 155-156. 75 UNOERSTEDT, U., 6-Hydroxydopamine induced degeneration of central monoamine neurons, Europ. J. Pharmacol., 5 (1968) 107-110. 76 UNGERSTEDT, U., Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta physiol, scand., Suppl. 367 (1971) 95-122. 77 UNGERSTEDT, U., Histochemical studies on the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in rat brain. In T. MALMFORSAND H. THOENEN (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, North-Holland Publ., Amsterdam, 1971, pp. 101-127. 78 UNGERSTEDT,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., Suppl. 367 (1971) 1-48. 79 URETSKY, N. J., AND IVERSEN, t . L., Effects of 6-hydroxydopamine on catecholamine containing neurons in the rat brain, J. Neurochem., 17 (1970) 269-278. 80 WACHTEL,H., AND KANDEL, E. R., A direct synaptic connection mediating both excitation and inhibition, Science, 158 (1967) 1206-1208. 81 WACHTEL, H., AND KANDEL, E. R., Conversion of synaptic excitation to inhibition at a dual chemical synapse, J. Neurophysiol., 34 (1971) 56-58. 82 WADE, G. N., AND ZUCKER, I., Development of hormonal control over food intake and body weight in female rats, J. comp. physiol. Psychol., 70 (1970) 213-220. 83 WADE, G. N., AND ZUCI,ZER, I., Modulation of food intake and locomotor activity in female rats by diencephalic hormone implants, J. comp. physiol. Psychol., 72 (1970) 328-336. 84 WEISSMAN,A., KOE, B., AND TENEN, S., Anti-amphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. exp. Ther., 151 (1966) 339-352. 85 WOLF, G., AND DICARA, L. V., Progressive morphologic changes in electrolytic brain lesions, Exp. Neurol., 23 (1969) 529-536.