action of gold thioglucose on pericapillary structures in the ventromedial hypothalamus

ACTION O F G O L D THIOGLUCOSE O N PERICAPILLARY STRUCTURES I N T H E VENTROMEDIAL HYPO- THALAMUS1

A. F. DEBONS, I. KRIMSKY, ANNETTE FROM, E. SICLARI, M. L. MAAYAN K. FAN, AND F. A. JIMENEZ

Department of Nuclear Medicine and Anatomical Pathology, Veterans Administration Medical Center, Brooklyn, New York, 11209, and State University of New York, Downstate Medical Center, Brooklyn, New York

PLATES XXXIX-XLIV

PARENTERAL injection of gold thioglucose (GTG) causes bilateral necrosis of a circumscribed area of the ventromedial portion of the hypothalamus (VMH) in mice (Marshall, Barrnett and Mayer, 1955). The glucose portion of the GTG molecule is required for production of necrosis of the VMH; a large number of other gold thio-compounds tested, including close structural analogues of GTG (gold thiogalactose, gold thiosorbitol), are inactive (Mayer, 1960). GTG-induced necrosis is prevented by the inhibitors of glucose transport, 2-deoxyglucose (Likuski, Debons and Cloutier, 1967) and phlorizin (Debons et al., 1974a).

The GTG-induced necrosis requires insulin : mice rendered diabetic with alloxan or anti-insulin serum do not get the lesion (Debons et al., 1968); administration of insulin to alloxan-diabetic mice or cessation of treatment with anti-insulin serum results in a return of sensitivity to GTG (Debons et al., 1969). The requirement for the glucose moiety, the action of inhibitors of glucose transport and the insulin-dependence suggest that there are present in this region of the brain certain cells with an insulin-dependent mechanism for the uptake of glucose.

Additional evidence in support of this hypothesis is the effect of glucocorticoids on GTG-induced necrosis of the VMH. Removal of glucocorticoids by adrenalectomy, which ameliorates the abnormal glucose metabolism of insulin deficiency (Long, Katzin and Fry, 1940; Long and Lukens, 1936), restores the sensitivity of the VMH of alloxan-diabetic mice to GTG-induced necrosis. Administration of cortisone to alloxan-diabetic mice rendered sensitive to GTG by adrenalectomy, restores resistance to GTG. The amount of cortisone required does not abolish the sensitivity of normal (i.e. non- diabetic) mice to GTG (Debons et al., 1974b). Similarly, hypophysectomy, which also counteracts the effects of insulin deficiency on glucose utilisation

Accepted for publication 6th Feb. 1979. 1 Some of the findings reported here have been presented in preliminary form in Federation

J . PATH.-VOL. 129 (1979) 73 Proceedings 1977; 36, 143.

74 A . F. DEBONS ET AL.

(Houssay and Biasotti, 1931), also abolishes the insulin requirement for GTG- induced necrosis (Debons et al., 1977). The foregoing observations indicate that, in contrast to the brain as a whole, there is in the VMH an insulin- dependent mechanism for the uptake of glucose.

The region of the VMH which undergoes necrosis after administration of GTG is known to be functionally and anatomically heterogeneous; identifica- tion of a specific insulin-sensitive cell type has yet to be made. It has been reported (Caffyn, 1971, 1972a) that, following intraperitoneal injection of a necrotising dose of GTG, the first change to occur was separation of the endo- thelial cells of the venules and capillaries in the portion of the VMH between the lateral border of the arcuate nucleus and the ventromedial border of the ventromedial nucleus; this occurred before any changes in neurons and glial cells. The consequent abnormal permeability of the capillaries (demonstrated by leakage of Evans’ blue or carbon particles from capillaries into tissue spaces) led to stoppage of blood flow and this in turn to necrosis of the structures in the region. It was also found (Caffyn, 19726) that certain anti- inflammatory agents inhibited, to varying degrees, GTG-induced damage as seen under the light microscope. On the basis of these findings, it was concluded that GTG acted primarily as a toxin on the small blood vessels of the region and also that GTG-induced necrosis of the VMH was not evidence for the presence of a neural glucoreceptor in this region.

However, studies conducted in our laboratory have revealed that vascular damage and general necrosis following administration of GTG are secondary to damage to neural elements in a restricted area of the VMH. This was accomplished by dissociating neural damage from vascular damage. In the present paper we report morphological damage produced by GTG in neural elements in the absence of damage to blood vessels, and the influence of insulin and glucocorticoids on damage to the neural elements. In the accompanying paper we will present evidence that the vascular damage which follows the neural damage produced by GTG depends on exposure of the vessels to the oedema-producing action of serotonin normally present in this region.

MATERIALS AND METHODS

CFI female mice obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass., 6 to 8 weeks of age, were used in all experiments. They were maintained on Purina mouse chow and tap water. They were kept at 22.5”C with a 12 hr light and 12 hr dark cycle.

For production of necrosis of the VMH, GTG was given either in a dose of 800 mg/kg intraperitoneally as an 8 per cent. solution or in a dose of 1200 mg/kg intravenously as a 12 per cent. solution. For visualising the necrosis, mice were decapitated 24 hr after the administration of GTG and their brains were fixed in Bouin’s fluid, embedded in paraffin, sectioned transversely at 7 microns, stained with haematoxylin and eosin, and examined under the light microscope.

Abnormally increased capillary permeability as indicated by leakage of Evans’ blue from blood vessels into tissue spaces was visualised by injecting 0.15 ml of a 0.45 per cent. Evans’ blue solution into a tail vein 1 hr before the animal was killed. The brain was fixed in 7.5 per cent. trichloroacetic acid overnight and washed in 70 per cent. ethanol. The brain was sectioned transversely through the median eminence and examined at low magnification for the presence of blue-stained areas.

GTG ACTION O N HYPOTHALAMUS 75

The non-necrotising dose of GTG was 100 mg/kg. It was administered intravenously

Aspirin was administered intraperitoneally in a dose of 200 mg/kg as a 2 per cent. solu-

Hydrocortisone (Solu-Cortef, hydrocortisone sodium succinate, Upjohn) and cortisone

Guinea pig anti-insulin serum was prepared as previously described (Debons et al., 1968). For electron microscopy, mice were decapitated and the brains rapidly removed from

the skull, cut transversely through the middle of the median eminence and immersed in fixative. The fixative was either 10 per cent. formalin in isotonic phosphate buffer, pH 7.0 (cat. no. So-F-100, Fisher Scientific Co.) or 4 per cent. glutaraldehyde in isotonic phosphate buffer, pH 7.3. After fixation for 2 hr, a slab about 0.5 mm thick was obtained by cutting parallel to the initial transverse cut. A piece of the VMH, which included the cell-poor area lying between the ventromedial and arcuate nuclei and the borders of these nuclei adjacent to the cell-poor area, was removed and cut into approximately 0.5 mm cubes. The pieces were then post-fixed for 1 hr in 1 per cent. osmium tetroxide. The tissue was dehydrated in alcohol and embedded in epon. Thin sections were cut at a 60 nm setting and stained with uranyl acetate and lead citrate.

as a 2 per cent. solution.

tion adjusted to pH 5.0 with NaOH.

(Cortone acetate, Merck, Sharpe & Dohme) were given intraperitoneally.

RESULTS

Induction of prolonged resistance to GTG by pre-treatment with aspirin plus GTG or by a small (non-necrotising) dose of GTG

Caffjm (19726) found that aspirin given 30 min. before GTG completely inhibited the GTG-induced necrosis of the VMH. We have confirmed this finding, i.e. mice so treated show no departure from normal in appearance of the VMH under the light microscope. However, they differ from untreated mice in that they are resistant to a second necrotising dose of GTG for about 48 hr. The onset of resistance occurs at about 12 hr. Aspirin when given before GTG is effective for about 6 hr in preventing GTG necrosis (table I ,

TABLE I Onset and duration of resistance to GTG-induced necrosis produced by pretreatment with

aspirin plirs GTC

fig. 1).

No. of mice Pretreatment

6 Aspirin followed 6 by GTG 30 6 minutes later 6 5

25 10 35 5 5 5 5 Aspirin 5 5 5

Subsequent injection of GTG

+3 hr + 6 hr 1 9 hr 4- 12 hr +18 hr +24 hr +36 hr +48 hr +72 hr 1 9 6 hr + 120 hr + 3 hr + 6 hr +9 hr

+ I 2 hr

Necrosis of VMH (incidence,

per cent.) 33 50 83 0 0 0 0

20 100 100 100 0

60 100 100

Dosage: Aspirin, 200 mg/kg, ip; GTG, 800 mg/kg, ip.

76 A . F. DEBONS E T A .

Resistance to a necrotising dose of GTG can also be achieved by the prior injection of a small (non-necrotising) dose of GTG. The onset and duration of resistance induced to a necrotising dose of GTG is similar to that following the administration of aspirin plus GTG (table 11).

TABLE I1 Onset and duration of resistance to GTC-induced necrosis produced by pretreatment with a

non-necrotising dose of CTC*

Time of Necrosis of No. of second injection VMH (incidence, mice of GTG, 800 mg/kg per cent.)

7 1 3 hr 100 5 + 6 hr 80

12 +9 hr 50 6 + I 2 hr 0

25 +24 hr 0 5 +48 hr 20

10 +72 hr 50 8 +96 hr 100 7 +I20 hr 100

* The non-necrotising dose of GTG, 100 mg/kg was injected intravenously.

Failure of pre-treatment with a non-necrotising dose of GTG to produce resistance in the absence of insulin

Previous studies have shown that insulin is required for GTG-induced necrosis of the VMH (Debons et al., 1968, 1969). The following experiment was done to determine whether insulin was required for the production of desensitisation by pre-treatment with a non-necrotising dose of GTG.

Mice were made temporarily diabetic by administration of anti-insulin serum obtained from guinea pigs. At 24 hr before the hyperglycemia was to subside (as determined by preliminary experiments), the animals were injected with the small dose of GTG. Thirty-six hours after administration of the small dose of GTG (at which time they were normoglycemic) they were chal- lenged with the necrotising dose of GTG. As expected, normal mice were protected against the necrotising dose of GTG by a non-necrotising dose of GTG. In contrast, mice made diabetic by injection of anti-insulin serum and given the desensitising dose of GTG while they were diabetic were not resistant to a subsequent necrotising dose of GTG given when they had recovered from the diabetes. Normal guinea pig serum did not prevent desensitisation by the small dose of GTG (table 111).

Prevention by glucocorticoids of desensitisation by a non-necrotising dose of GTG Since it is known that glucocorticoids antagonise the effects of insulin on

glucose uptake, we investigated the effects of glucocorticoids on GTG action on the VMH. We have found that both hydrocortisone and cortisone prevent production of necrosis of the VMH by GTG (table IV). Furthermore, they

DEBONS, KRIMSKY, FROM, SICLARI, MAAYAN, FANI AND JIMENEZ

GTG ACTION ON HYPOTHALAMUS

a

PLATE XXXIX

b

C d

FIG. 1.-Transverse sections through the hypothalamus at the level of the median eminence: (a) untreated, x32 ; (b) untreated, x200; (c) GTG, x32 ; (d) GTG, ~ 2 0 0 ; (e) aspirin plus GTG 30 min. later, x 32; (f) aspirin plus GTG 30 min. later, X 200; (g) aspirin plus GTG 30 min. later plus GTG 24 hr later, 2: 32; (h) aspirin plus GTG 30 min. later plus GTG 24 hr later, x 200.

DEBONS, KRIMSKY, FROM, SICLARI, MAAYAN, FANI AND JIMINEZ


e

PLATE XL

f

h

DEBONS, KRIMSKY, FROM, SICLARI, MAAYAN, FANI AND JIMINEZ


PLATE XLI

a Fici. 2.-Sections through the VMH illustrating pericapillary damage following treatment with a

non-necrotising dose of GTG: (a) untreated, x 15,000; (b) and (c) treated with non-necrotising dose of GTG 24 hr before sacrifice- x 12000, x 8000.



PLATE XLlI

b



C

DEBONS, K R I M S K Y , FROM, SICLARI, M A A Y A N , FANI A N D JIMENEZ

GTG ACTION O N HYPOTHALAMUS

PLATE XLIV

b

C d

FIG. 3.--Transverse sections at the level of the median eminence of brains of mice injected intravenously with Evans’ blue 1 hr before decapitation. ~ 4 . 5 . Treatment before Evans’ blue (GTG, where given, was injected 4 hr before Evans’ blue) : (a) none; (b) GTG, 800 mg/kg ip; (c) aspirin, 200 nig/kg ip followed 30 min. later by GTG, 800 mg/kg ip; (d) GTG, 100 mg/kg iv. Staining with Evans’ blue occurs only in (b) and the stain is confined to a small part of the ventromedial hypothalamus.

GTG ACTION ON HYPOTHALAMUS 77

also prevent desensitisation by a non-necrotising dose of GTG to a subsequent necrotising dose of GTG (table V). It is notable that cortisone, in amounts

TABLE I11 Resistance to GTG-induced necrosis produced by pretreatment with a non-necrotising dose of

GTG: dependence on insulin for development of resistance

Schedule of treatments at time of Necrosis of

Treatment Time mg, per cent. per cent.)

Blood glucose A , treatment VMH (incidence,

GTG, 100 mg/kg, iv 0 hr 102& 3

AIS, 0.5 ml ip Ohr 108& 3 0.5 m1 ip + 6 hr 375; 2

GTG, 100 mg/kg, iv +21 hr 3251 4 GTG, 800 mg/kg, ip +57 hr 8 6 1 6 86 AIS, 0.5 ml ip Ohr 104A 2

0.5 ml ip + 6 hr 3711 6 None $21 hr 280127

NS, 0.5 ml ip Ohr 125& 4

GTG, 100 mg/kg, iv +21 hr 1131 6

NS, 0.5 ml ip Ohr 107* 4

None 1-21 hr 1161 5

GTG, 800 mg/kg, ip +36 hr 8 9 1 3 0

GTG, 800 mg/kg, ip +57 hr 150&33 100

0.5 ml ip $ 6 hr

GTG, 800 mg/kg, ip +57 hr 104h 5 0

0 5 ml ip + 6 hr

GTG, 800 mg/kg, ip + 57 hr 103 f 5 100

guinea pig serum. Abbreviations: AIS, guinea pig anti-insulin serum; NS, normal

Each group consisted of seven to ten mice.

TABLE IV Prevention of GTG-induced necrosis by glucocorricoids

Necrosis of No: of VMH (incidence, mice Treatment per cent.)

8 GTG 100 8 Hydrocortisone+GTG 0

12 Hydrocortisone+GTG 100

6 Cortisone+GTG 30 0

8 Cortisone+GTG 24 hr 75

Dosage: GTG, 800 mg/kg, ip;

30 min. later

24 hr later

min. later

later

hydrocortisone, 250 mg/kg, ip; cortisone, 450 mg/kg, ip.

insufficient to prevent GTG-induced necrosis in normal animals, is effective in alloxan-diabetic adrenalectomised animals (these animals in contrast to alloxan-diabetic animals are sensitive to GTG (Debons et al., 1974)). These findings indicate that cortisone is acting as an anti-insulin agent (as distinguished from an anti-inflammatory agent) in preventing GTG action on the VMH.

78 A . F. DEBONS ET AL.

It is apparent that the effect of the non-necrotising dose of GTG (i.e. desensitisation to a subsequent necrotising dose) is dependent on the presence of insulin and is counteracted by glucocorticoids. These effects of the hormones thus parallel their action in altering the sensitivity of the VMH to the necrotising dose of GTG.

Electron microscopic changes in the VMH after aspirin plus GTG or a non- necrotising dose of GTG

Although both desensitising treatments (a non-necrotising dose of GTG or aspirin plus a necrotising dose of GTG) produce no visible morphological change in the VMH under the light microscope, the fact that they induce resistance to a necrotising dose of GTG suggested that they might produce

TABLE V

Prevention by glucocorticoids of desensitisation to a necrotising dose of GTG by pretreatment with a non-necrotising dose of GTG

Subsequent Necrosis of injection of VMH (incidence,

Pretreatment GTG, 800 mg/kg per cent.) GTG, 100 mg/kg, iv +24 hr 0 Hydrocortisone, 250 mg/kg, +24 hr 100

ipSGTG, 100 mg/kg, iv 30 min. later

100 mg/kg, iv 30 min. later Cortisone, 450 mg/kg, ip+GTG, +24 hr 71

Each group consisted of seven mice.

changes visible at magnifications obtainable with the electron microscope. Accordingly, 20 mice were subjected to the desensitising treatments, killed 24 hr later, and the region of interest in the VMH was examined under the electron microscope. Ten untreated mice were prepared in the same manner and used as controls. It was found that the desensitising treatments had produced morphological changes visible under the electron microscope in all of the treated animals. These changes consisted of fragmentation and dissolution of structures around many of the capillaries without any appearance of oedema or changes in the capillary wall (figs. 2b, c). These changes were confined to a small area of the VMH. This area consisted of the cell-poor zone between the arcuate and ventromedial nuclei and the borders of these nuclei adjacent to the cell-poor area. In the untreated animal, the capillaries in this region are surrounded by densely packed cell processes (fig. 2a). Whether these processes are neuronal or glial (or both) in nature has not been established.

Electron microscope examination of the hypothalami of 5 mice killed 5 days after desensitising with a non-necrotising dose of GTG revealed no abnormalities. This is consistent with the return of sensitivity to a necrotising dose of GTG by this time.


GTG-induced oedema of the VMH and prevention of this oedema by treatments which prevent GTG-induced necrosis of the VMH

It has been reported (Caffyn, 1971, 1972a) that necrosis of the VMH by GTG is preceded and is caused by increased capillary permeability with consequent oedema. We have shown above that pre-treatment with aspirin plus GTG or with a non-necrotising dose of GTG confers resistance to a necrotising dose of GTG ; the pretreatment produces morphological changes, visible under the electron microscope, in pericapillary structures. The question arose whether the pretreatments prevent oedema as well as the necrosis. Leakage of Evans blue from the blood vessels into the tissue spaces was used as an index of increased capillary permeability and consequent oedema. The results of the experiments are summarised in table VI and illustrated in figs. 3a-d.

TABLE VI

Leakage of Evans blue into VMH after administration of GTG

Treatment before injection of Evans blue*

None GTG, 800 mg/kg, ip Aspirin, 290 mg/kg, ip+GTG, 800 mg/kg,

10 30 min. later G f G , 100 mg/kg, iv Aspirin+GTG, 800 mg/kg, ip 30 rnin. later

GTG, 1 0 0 mg/kg, iv then GTG, 800 mg/kg, then GTG, 800 mg/kg, ip 24 hr later

ip 24 hr later

Leakage of Evans blue into VMH

(incidence, per cent.) 0

100 0

0 0

0

See Methods section for details of estimating increased abnormal capillary permeability by Evans blue. Mice were killed 5 hr after the last injection of GTG. Each group consisted of eight mice.

In agreement with Caffyn (1972a), we found that GTG causes oedema of the VMH. It should be noted (fig. 3b) that the median eminence is relatively free of oedema compared with the adjacent portion of the hypothalamus. The treatments which prevent GTG-induced-necrosis also prevent oedema. Since the non-necrotising dose of GTG, without producing oedema, desensitises the VMH to a subsequent necrotising dose of GTG, the desensitisation does not depend on the production of oedema. However, the non-necrotising dose causes damage to pericapillary structures (visible under the electron microscope) and therefore the desensitisation appears to be a consequence of this damage. The production of damage to the pericapillary neural structures by treatment with aspirin plus GTG or with a non-necrotising dose of GTG, accompanied by absence of observable damage to the capillaries either under the electron microscope or measured by exudation of Evans blue indicates that the primary targets of GTG are certain pericapillary structures in a restricted portion of the VMH.

80 A . F. DEBONS ET AL..

DISCUSSION Before the findings presented in this paper were made, the known effect of

GTG on the VMH was the production of death and dissolution of all the structures in this region (Marshall et al.). This was consistent with a primary attack by GTG on the microvasculature of the region, as proposed by Caffyn (1971, 1972a, 19726), with general necrosis resulting from circulatory stasis.

The demonstration in the present paper of clear-cut morphological effects by GTG on extravascular structures in the absence of vascular damage and general necrosis indicates that vascular damage is secondary. When the capillary wall is protected by administration of aspirin before the otherwise necrotising dose of GTG, or when the dose of GTG is too small to cause necrosis, damage to structures around many of the capillaries in a restricted area of the VMH occurs regularly. Either of these two treatments results in desensitisation to a subsequent necrotising dose of GTG; this indicates that the primary target of the necrotising dose is the same as that of the desensitising dose. This is supported by the finding that both desensitisation by the non- necrotising dose and production of necrosis by the necrotising dose are prevented by insulin deficiency and by glucocorticoids.

The influence of insulin and glucocorticoids on GTG-induced necrosis makes it unlikely that the primary target of GTG is the capillary wall, since there is no evidence that the capillaries of the VMH, where necrosis occurs, are different from capillaries in the rest of the brain with respect to these hormones. It is much more likely that certain cells of the VMH, with its multi- plicity of regulatory functions, are responsive to insulin and glucocorticoids.

The findings in this paper are consistent with the hypothesis that damage to the pericapillary cell processes releases from them a substance injurious to the adjacent capillary walls. The following paper presents evidence that this substance is serotonin.

Caffyn’s hypothesis (that the first site of attack of GTG is the capillary endothelium in the VMH) has apparently been accepted without reservation by some workers; they have concluded that GTG cannot serve as a useful probe of hypothalamic function (Miselis and Epstein, 1975; Mogenson, 1976). On the basis of the evidence available to date, this conclusion is clearly unwarranted.

SUMMARY The administration of GTG to mice leads to death of all structures in a

circumscribed area of the VMH as a result of loss of blood circulation. The loss of circulation is due to damage by GTG of neural processes adjacent to some of the capillaries in this area; damage to these processes leads to abnormal capillary permeability. Pericapillary damage occurs under conditions where capillary damage and consequent necrosis are prevented. Abnormal capillary permeability appears to follow release of a vasoactive substance from the damaged neural processes. Damage to the pericapillary neural processes by GTG is. insulin-dependent and is counteracted by glucocorticoids.


We wish to thank Dr M. Steinberg of the Schering Corporation, Bloomfield, New

This work was supported by Veterans Administration Medical Research Funds MRIS Jersey, for supplying us with gold thioglucose for these studies.

1788 and by U.S.P.H.S. Grant A.M. 12479.

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induced hypothalamic lesions by anti-inflammatory agents. J. Pathol., 106, 57. DEBONS, A. F., KRIMSKY, I., FROM, ANNETTE, AND CLOUTIER, R. J. 1969. Rapid effects of

insulin on the hypothalamic satiety center. Am. J. Physiol., 217, 1 1 14. DEBONS, A. F., KRIMSKY, I., FROM, ANNETTE, AND PATTINIAN, H. 1974a. Phlorizin inhibi-

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DEBONS, A. F., KRIMSKY, I., MAAYAN, M. L., FANI, K., AND JIMENEZ, F. A. 1977. Gold thioglucose obesity syndrome. Federation Proceedings, 36, 143.

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LIKUSKI, H. J., DEBONS, A. F., AND CLOUTIER, R. J. 1967. Inhibition of gold thioglucose- induced hypothalamic obesity by glucose analogues. Am. J. Physiol., 212, 669.

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LONG, C. N. H., AND LUKENS, F. D. W. 1936. The effects of adrenalectomy and hypophysectomy upon experimental diabetes in the cat. J. Exp. Med., 63, 465.

MARSHALL, N. B., BARRNETT, R. J., AND MAYER, J. 1955. Hypothalamic lesions in gold thioglucose injected mice. Proc. SOC. Exp. Biol. Med., 90, 240.

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action of gold thioglucose on pericapillary structures in the ventromedial hypothalamus

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