BEHAVIORAL BIOLOGY 16, 365-372 (1976), Abstract No. 5225

BRIEF REPORT

Neurochemical Correlates of Hypothalamic Obesity in Rats I

DONALD V. COSCINA 2, DAMODAR D. GODSE,

and HARVEY C. STANCER

Section of Neurochemistry, Clarke Institute of Psychiatry and Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada

To assess the possibility that damage confined to brain noradrenergic systems is related to hyperphagia and obesity following medial hypothalamic injury, 40 female rats sustained radio-frequency lesions of varying size aimed rostral to the ventromedial nuclei. After two weeks of maintenance on a high-fat diet, forebrain levels of norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid were determined for all lesioned rats as well as for 20 sham-operated controls. Lesions produced 35% greater weight gain than in controls, and significant depletions of norepinephrine (-19%) and serotonin (-8%). Norepinephrine levels were inversely correlated with weight gain. Serotonin levels approached a similar inverse relationship. Norepine- phrine and serotonin levels were positively correlated with each other. 5-hydroxyindoleacetic acid levels were positively correlated with norepine- phrine levels and inversely correlated with weight gain. These findings confirm previous suggestions of an association between altered noradrenergic brain function and hypothalamic hyperphagia, but show that serotonin metabolism is altered in a similar fashion. They also suggest that a small but refiable alteration of brain amine(s) can be associated with profound behavioral changes.

Gold (1973) has recently suggested that the obesity which follows lesions aimed at the ventromedial hypothalamic nuclei (VMHN) is a conse- quence of damage to the nearby ventral noradrenergic bundle (VNAB) or its terminals.IThis hypothesis is in keeping with observations that electrolytic or

chemical lesions of the VNAB following intracerebral injection of 6- hydroxydopamine (60HDA) can produce obesity (Ahlskog and Hoebel, 1973; Ahlskog, 1974) as can unilateral lesions of the VNAB combined with contralateral parasaggital knife-cuts rostral to the VMHN (Kapatos and Gold,

1Supported by funds from the Clarke Institute of Psychiatry. Portions of this work were presented at the Fourth Annual Meeting of the Society for Neuroscience held in St. Louis, Missouri, ~ October, 1974. We thank R. McArthur, P. Chan, and M. Gutman for technical assistance throughout various phases of this work.

2Reprint requests to the first author at: Section of Neuroehemistry, Clarke Institute of Psychiatry, 250 College St., Toronto, Ontario, Canada M5T 1R8.

365

Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

366 COSCINA, GODSE, AND STANCER

1973). Gold (1973) has further stated that the degree of medial hypothalamic (MH) obesity seems correlated with the size of injury rostral to the VMHN, seemingly because larger lesions involve greater portions of the VNAB or its projections.

Discrete nuclei within the medial basal hypothalamus contain varying concentrations of putative neurotransmitters including epinephrine (Koslow and Schlumph, 1974), norepinephrine (NE) (Palkovits, Brownstein, Saavedra and Axelrod, 1974), dopamine (DA) (Palkovits et al., 1974) serotonin (5-hydroxytryptamine or 5HT) (Saavedra, Palkovits, Brownstein and Axelrod, 1974), histamine (Brownstein, Saavedra, Palkovits and Axelrod, 1974) and acetylcholine (Jacobowitz and Palkovits, 1974). Since NE is the putative neurotransmitter associated with the VNAB (Ungerstedt, 1971), lesions such as those described by Gold (1973) might be expected to produce significant inverse correlations between weight gain and endogenous levels of brain NE, but not with other endogenous brain amines. In partial support of this suggestion, Glick, Greenstein and Water (1973) have reported that weight gain either five or ten days after VMHN lesions in rats is inversely correlated with tel-diencephalic NE levels but not with striatal DA levels. However, Glick e t

al. gave no description of lesion size or locus to permit comparison with Gold's (1973) analysis, and fed their rats standard lab (pellet) chow which promotes less weight gain than the high-fat diet used by Gold. Of perhaps greater importance, Glick et al. made no other amine measures in brain aside from NE which likely would have been affected by MH injury (striatal DA levels being reflective of damage to the more laterally-situated nigro-neostriatal system). Previous work from our group (Coscina, Rosenblum-Blinick, Godse and Stancer, 1973) has shown that obesity-producing lesions aimed at the VMHN are capable of significantly depleting both NE and 5HT in forebrain. Therefore, several specific aspects of Gold's (1973) paradigm and resultant hypothesis remain to be validated neurochemically.

The present work re-examined Gold's (1973) suggestion that MH lesions aimed rostral to the VMHN are efficacious in producing obesity and also in depleting forebrain NE. For this purpose, we used the lesion co-ordinates and high-fat diet reported by Gold. To assess the possible non-specificity of these lesions on other brain amine systems and the potential correlative relationship of such change to MH obesity, we measured forebrain levels of DA, 5HT, and serotonin's major metabolite, 5-hydroxyindoleacetic acid (5HIAA) in addition to NE levels.

Sixty-seven female albino rats (derived from Wistars at High Oaks Ranch, Ontario) were used. Rats weighed 230-270 g at surgery. All animals were housed in single cages maintained in a temperature (23-26 ° C) and light (on 0800-2000 hr) controlled colony. Food (see below) and water were freely available at all times.

The experiment was performed in two stages or replications. In the first

AMINE CORRELATES OF HYPOTHALAMIC OBESITY 367

replication, 23 rats sustained bilateral MH lesions and nine received sham operations. In the second replication, 24 rats were lesioned and 11 were sham-operated. In all cases, rats were pre-treated with atropine methyl nitrate (2rag in 0.2ml saline, i.p.) 10m in before anesthetization with sodium pentobarbital (Nembutal, 35 mg/kg/ml, i.p.), placed in a Kopf stereotaxic instrument, the scalp incised, and a 2-3 mm 2 section of skull removed along the appropriate midline axis. For lesioned rats (total N = 47), a Radionics Thermal Probe was lowered to the co-ordinates described by Gold (1973) and 50, 53, 55, 57 or 60 ° C was generated bilaterally for 1 min per animal (iV = 13, 9, 9, 9 or 7, respectively). This was done to produce nonirritative lesions of varying size. The lesioning electrode was not lowered into the brains of sham-operated rats (total N = 20). All skull defects produced by trephening were then filled with a nonantigenic matrix (Gelfoam, Upjohn Co.) and ineisions were closed with 9 mm stainless steel wound clips.

Following surgery, all rats were returned to homecages where fresh food pellets (Purina lab chow on cage floor) and tap water (in calibrated bottles with sipper tubes) were freely available for the first 24-hr postoperatively. From Day 2 until the end of the experiment on Day 14, all rats received Gold's (1973) high-fat diet (33% Crisco Vegetable Shortening, 67% powdered Purina chow, w/w) in large glass cups attached to the interior cage front. Water remained available as before. Rats in the first replication were moni- tored for 24-hr food and water intake as well as for bodyweight on days 2, 4, 6, 8, 10, 12 and 13 postoperatively. High positive correlations (P < .02) were found between 24-hr food consumption and weight gain for over 80% of these animals. Therefore, only bodyweights were recorded on the post-operative days cited above for rats in the second replication.

Between 0930 and 1130 hr on the fourteenth postoperative day, all animals except seven lesioned rats representative of weight gain range and lesion size (one 50, two 55 and one 60°C lesion-group from replication 1; one 50, one 53 and one 57 ° C lesion-group from replication 2) were sacrificed by decapitation within 30 sec of removal from homecages. Brains were rapidly removed from the calvaria, dissected, visually assessed for lesion size and locus, and frozen, all as previously described (Coscina et al., 1973). Fluoro- metric determinations of forebrain 5HT (all rats) and 5HIAA (rats in second replication) were determined by the method of Maickel, Cox, Saillant and Miller (1968). NE and DA (all rats) were determined by the method of Shellenberger and Gordon (1971). Recoveries of these amines with these methods have been previously reported by us (Coscina et al., 1973). The remaining seven MH-lesioned rats were overdosed with Nembutal, perfused transcardially with isotonic saline followed by 10% formalin, and their brains histologically prepared for determining lesion placement by light microscopy.

Bodyweight, expressed as percentage change from operation weight, was analyzed between groups and compared with all biochemical measures. The

368 COSCINA, GODSE AND STANCER

replicative nature of this experiment did not permit analysis of NE, DA or 5HT in one assay, i.e., several weeks intervened before samples from both replications were obtained. Samples are routinely analyzed in our laboratory within one week of freezing. To permit comparisons of data obtained from different assays, these results were expressed as a percentage of sham-operated control levels, which were obtained for each individual assay. This limitation did not apply to 5HIAA determinations since they were only analyzed for one replication. Therefore, these data were expressed in raw form (ng/g wet tissue wt). Between-groups comparisons were made by t-tests for independent samples (Hays, 1963). Pearson Product-Moment Correlation Coefficients were generated among variables by a PDP8 computer. All P values reported represent two-tailed distributions.

Examination of histologically prepared brain sections as well as fresh tissue later used for fluorometric assays revealed comparable findings. All lesioned rats possessed bilateral MH injury which was usually symmetrical. On occasion, slight (up to 0.4 mm) lateral deviations from the midline were observed in both hemispheres in the same direction (i.e., midline determina- tions were slightly off-center). These slight deviations did not prevent over- eating and weight gain. Regardless of lesion size, electrode tracts were centered between the posterior aspects of the optic chiasma and the anterior portions of the VMHN. The ventral aspects of even the largest lesions were always situated at least 0.5 mm above the base of the MH. In terms of lesion size, very small (50 ° C) lesions often produced little more cavitation than that attributable to electrode insertion, i.e., no larger than 1 mm diam. Medium- sized (53 ° C) lesions seemed to extend from the posterior one-third of the optic chiasma to the anterior one-third of the VMHN. These lesions typically spared the VMHN proper but always involved aspects of the dorsomedial hypothalamic nuclei and, more rostro-caudally, the paraventricular nuclei. Large (55-60 ° C) lesions produced massive cavitation, encompassing all MH tissue from the anterior hypothalamic region up to and including the posterior aspects of the VMHN. These lesions extended from 0.5 mm above the base of the MH up to and sometimes including the reuniens nucleus of the thalamus. At their widest extent, such lesions damaged the perifornical aspects of the lateral hypothalamus and extended through the midline, obliterating the walls of the third ventrical. However, even in these cases of widespread injury, damage was never observed to the optic chiasma, globus pallidus, internal capsule or cerebral peduncles.

Figure 1 depicts mean bodyweight curves for both groups over the two-week observation period. MH-lesioned rats showed profound, consistent weight gains over the entire post-operative interval compared to sham-operated controls. On the average, lesioned rats gained 7.4 g/day (range = 3-12) which was significantly more (P< .001) than the 1.1 g/day (range=0.4-2.2)gain observed for controls. This magnitude of weight gain after lesioning resulted in

AMINE CORRELATES OF HYPOTHALAMIC OBESITY 369

140 [ ° M H Lesion, N = 40 e J e

-r 130 •

,v, j o / 420

~ /o/° < I10 •

~: ~ 0 / 0 ~ 0._..__.0 ~ 0 ..,....._ 0 ~ 0 0 I 00

m 90

I I I I I I I I

Op 2 4 6 8 I0 12 13

DAYS POST - LESION

Fig. 1. Bodyweight (BW), expressed as percentage of operation weight, of MH- lesioned and sham-operated rats over 13 days of maintenance on high-fat diet. Each symbol represents the combined mean and standard error of the mean per group. See graph for definition of symbols and N per group.

almost a 40% increase in weight over the experimental period which was

considerably greater ( P < . 0 0 1 ) than the 5% increase seen after sham surgery. MH lesions produced significant ( P < . 0 1 ) depletions of forebrain NE

and 5HT levels without affecting DA or 5HIAA (see Table 1). The weight gain produced by MH lesions was inversely correlated with levels of brain NE (Table 2). Such weight gain was also inversely correlated with brain 5HIAA levels. Levels of brain 5HT approached a similar inverse trend (.10 > P > .05)

with weight and were positively correlated with NE, DA and 5HIAA levels. In

addition, brain NE was positively correlated with brain 5HIAA levels.

TABLE 1

Comparisons of MH-Lesioned and Sham-Op Controls for Bodyweight (% Increase Over Operation Weight on Day 13 Post-Op), Levels of Brain Amines NE, DA, 5HT (% of

Sham-OP Control Values), and Levels of Brain 5HIAA (ng/g wet tissue wt) a

Group % Op. BW NE DA 5HT 5HIAA

Sham-Op .~ +5.04 100.0 100.0 100.0 541 SEM .632 1.64 2.09 2.95 51.4

n 20 20 20 20 9 MH-lesion X +39.40** 81.0"* 96.3 92.6* 531

SEM 1.91 1.79 1.46 1.13 14.7 n 40 40 40 40 19

*P < .01 compared to Sham-Op Controls. **P < .001 compared to Sham-Op Controls. aAll tissues for biochemical determinations obtained at sacrifice on day 14 post-op.

See text for abbreviations.

370 COSCINA, GODSE AND STANCER

TABLE 2

Matrix of Pearson Product-Moment Correlation Coefficients for all Variables in Table la

BW NE DA 5HT (N)

NE -.4005* 40 DA +.0170 +.3036 40 5HT -.2773 +.6846** +.4173"* 40 5HIAA -.5315' +.4709* +.3735 +.4843* 21

*P < .05. **P < .01. aSee text for abbreviations. The number (N) of comparisons per cell are also indi-

cated. All comparisons were made on data from lesioned rats.

Our data show that MH lesions damaging tissue rostral to the VMHN are effective in producing hyperphagia and obesity. These findings support Gold's (1973) suggestion that the VMHN may be merely a "target" within a larger MH axis whose integrity is important to normal bodyweight regulation. Our added observation of significant (19%) depletion of brain NE (see also Coscina et al., 1973; Poncy, Bernard and Chernov, 1972) plus an inverse relationship between forebrain NE levels and obesity are consistent with the data of Glick et al. (1973) and in keeping with the "VNAB hypothesis" of hypothalamic hyperphagia (Ahlskog, 1974; Ahlskog & Hoebel, 1973; Gold, 1973; Kapatos & Gold, 1973). However, the added finding of significant 5HT depletion (8%) after MH lesions (see also Coscina e t al., 1973; Coscina, 1975) along with high positive correlations between NE and 5HT depletions shows that at least one other brain amine besides NE is simultaneously affected by hypothalamic lesions producing obesity. It must be remembered that while both NE and 5HT depletions were quantitatively small (i.e., < 25%), these values are ex- pressed as variations from intact forebrain concentrations. In all liklihood, they represent considerable regional (e.g., hypothalamic) amine reductions. When considered in this perspective, these data imply that regional rather than general brain amine content is more notably associated with MH hyperphagia.

Brain levels of 5HT's major metabolite, 5HIAA, could not be distin- guished between groups. Nevertheless, 5HIAA levels were inversely correlated with weight gain and positively correlated with 5HT levels in lesioned rats. We have previously observed decrements in brain 5HT without changes in 5HIAA after MH lesions (Coscina et al., 1973). The consistency of this finding may mean that these lesions increase forebrain 5HT turnover. Such a turnover change could be due to compensatory metabolic increments in undamaged 5HT neurons (see Agid, Javoy and Glowinski, 1973, for similar suggestion in DA neurons after subtotal destruction by 60HDA treatment) and/or to lesion-induced damage to NE systems whose neurons may normally innervate 5HT neurons thereby affecting 5HT metabolism. Within the limitations of the

AMINE CORRELATES OF HYPOTHALAMIC OBESITY 371

present experimental paradigm, explanations of this potential turnover increase must be considered speculative.

Our observations of inverse correlations between brain 5HIAA levels and weight gain after MH lesions, linked with a similar trend between 5HT levels and weight gain, may suggest some functional role for brain serotonin in the mediation of feeding. In keeping with this suggection, we have recently observed that 5HT-depleting lesions of the dorsal and median raphe nuclei produce almost complete blockade of hyperphagia and subsequent obesity following MH lesions (Coscina, 1975). More biochemical and pharmacological analyses are required to determine the possible dynamic aspects of 5HT metabolism in these animal models before a clear role can be ascribed to this biogenic amine in central mechanisms mediating feeding behaviors.

In summary, our findings lend partial support to the suggestions of Gold (1973). MH lesions producing obesity do not have to be restricted to the VMHN proper. At the same time, such lesions do reduce forebrain NE levels in a manner inversely related to obesity (see also Glick et al., 1973). However, the significance o f such NE depletion seems vague since concomitant decre- ments in 5HT levels occur and 5HT metabolism seems affected in the same correlative direction with weight gain. These findings, linked with a recent report (Lorden and Oltmans, 1975) in which NE depletion following electro- lytic or 60HDA lesions o f either the ascending VNAB or rostral VMHN did not produce obesity, question an exclusive role for hypothalamic and/or forebrain NE systems in the etiology of MH hyperphagia and obesity.

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