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Starvation and dehydration: Effect on hypothalamic monoamines and serum LH and prolactin
Gen. Pharmac. Vol. 20, No. 1, pp. 111-113, 1989
0306-3623/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc
Printed in Great Britain. All rights reserved
STARVATION A N D DEHYDRATION: EFFECT ON HYPOTHALAMIC MONOAMINES A N D SERUM LH A N D PROLACTIN PABLO SCACCHI, SILVIA E. CARBONE, DORA C. DE RONDINA, IRMA O. MASTRONARDIand BERTA SZWARCFARB Instituto de Fisiologia "Profesor Dr Bemardo A. Houssay", Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina (Received 16 November 1987; received for publication 17 March 1988)
Abstract--1. Rats submitted to starvation or water deprivation showed a decrease in LH and prolactin serum levels. 2. 5-HT was increased without changes in DA and NA in cerebral cortex of starved rats. 3. Neurotransmitters did not change in hypothalamus of starved rats but water deprivation decreased NA and increased 5-HT.
INTRODUCTION
It is well known that undernutrition provokes several disorders being one of them reproduction alterations. In the rat the inanition or chronic desnutrition induced testicular and ovarian atrophy (Negro-Vilar et al., 1971; Mulinos and Pomerantz, 1941). Female dancers and gymnasts submitted to restricted food regimes suffer amenorrhea. On the other hand the neuroendocrine abnormalities which have been documented recently in primary anorexia nervosa suggest the presence of hypothalamic dysfunction during the cachectic phase of the illness (Silverman, 1974; Vigersky et al., 1976) and are consistent with an abnormality in sympathetic neuronal function (Ziegler et al., 1976, 1977). These patients also present abnormalities in t h e catecholaminergic metabolism. Gross et al. (1979) studied patients with primary anorexia nervosa all of which were amenorrheic. Plasma levels of tyrosine, norepinephrine (NE) and urinary excretion of its metabolites (homovanillic acid and 3-methoxy-4-hydroxyphenyl glycol) were determined and compared with those of normal females and they found significantly lower levels in all of the measured parameters. In that study it was found that the disturbances in the cardiovascular function (bradycardia, orthostatic hypotension) and in the catecholamine metabolism could be reversed by weight gain. The reversal of the biochemical abnormalities after weight gain suggested that they were secondary to the emaciation and general malnutrition characteristic of primary anorexia nervosa and were not aetiological factors. Many authors have studied the relation between desnutrition or dehydration and functional integrity of hypothalamus. They worked with different animal species, animals of different ages, normal, hemicastrated or castrated, male or female and the results were not in accord. Most of them agreed that underfeeding diminished pituitary LH and FSH and hypothalamic.LH-RH.
Nevertheless, Howland (1971) found that in female rats pituitary LH and FSH did not change while plasmatic LH diminished by 50% and suggested that synthesis and release of hormones were equally impaired and that this was consistent with the hypothesis that undernutrition acts on the pituitary gland by inhibiting hypothalamic production of LH releasing factor. Decrease in ovarian, uterine and anterior pituitary weight have been reported by other authors. The underfeeding resulted in cessation of oestrus cycles in all the animals and this reversed when the feeding was normalized (Bela et al., 1967). Negro-Vilar et al. (1971) studying the effects of complete food deprivation during 7 days in male adult rats found a large reduction in hypothalamic F S H - R H and in pituitary FSH levels; furthermore, there was a significant decrease in weight of anterior pituitary, seminal vesicles and prostate but testicular weight remained unchanged. These results suggested to the authors that starvation inhibits FSH release by decreasing hypothalamic FSH-RH activity, perhaps by reducing the availability of blood precursors for FSH synthesis. It is possible that hypothalamic neurons producing FSH-RH are sensitive to the levels of nutrients in the circulation, or that they may be influenced by the feeding centers in the hypothalamus. Since the deficiencies induced by severe underfeeding closely resembled those observed after hypophysectomy, Mulinos and Pomerantz (1940) called this condition "pseudohypophysectomy". However, few of the previous data in the literature revealed a neuroendocrine relation between hormonal variations and the possible changes of monoamine levels in the central nervous system. For this reason we decided to evaluate whether there was correlation between the variation in hypothalamic and corticocerebral monoamine levels and prolactin and LH plasmatic levels using as experimental model adult rats submitted to starvation or water deprivation.
111
112
PABLO SCACCHIet al. MATERIALS AND METHODS
Eighteen male adult rats (strain of the Instituto "Bernardo A. Houssay", Facultad de Medicina, Universidad de Buenos Aires), weighing 240 + 5 g were used. The animals were distributed in three equal groups, housed in cages and kept under a 12 hr light-dark cycle (07.00-19.00 light) in a temperature and humidity controlled room. The control group had water and food ad libitum. The second group was submitted to starvation: without food but with water ad libitum and the third group was deprived of water: they received no water but food ad libitum. The animals were daily weighed. At the end of the experiment, that lasted seven days, the animals were killed with a guillotine and the blood was collected from the trunk and allowed to clot at 4°C in glass tubes, separated by centrifugation 10 rain at 2500 rpm and the serum was separated and frozen at -30°C until the estimations of LH and Pro levels were carried out by the radioimmunoassay. Serum levels of LH were determined in duplicate using the double antibody radioimmunoassay method of Niswender et al. (1968). Values are expressed in ng/ml in terms of the reference preparation, NIAMDD-RatLH-RP-1. The intra-assay and inter-assay coet~cients of variations were 6 and 11%, respectively. The minimum sensitivity of the assay was 2 ng/ml serum. Serum Pro was measured in duplicate using the double antibody radioimmunoassay with kits supplied by the NIAMDD-Rat Pituitary Hormone Distribution Program. Values are expressed in ng/ml, on the basis of the NIAMDD references preparation supplied with the kit (Rat Prolactin RP-I). Results were statistically evaluated using analysis of variance and Tukey's multiple range test. Plasma osmolality was measured by the depression of the freezing point (Osmette S, Precision Systems, INC), expressing in mOsm/ml. The skull was rapidly opened and the brain immediately removed. The cerebral cortex and hypothalamus were dissected and frozen until the spectrofluorometric determinations of monoamines. Dopamine (DA) and noradrenaline (NA) were measured by the method of Orsingher et al. (1980) expressing the results in #g/g fresh tissue, and serotonin (5-HT) by the technique of Curzon and Green (1970). Statistical analysis was performed using Student's "t"-test.
RESULTS AND DISCUSSION
As expected there was a significant decrease in body weight in both (deprived) groups in comparison with the control group (33% less in starved and 37% less in water deprivated rats). It should be noted that the water deprivated rats did not eat. Furthermore, we have found a significant increase of plasmatic osmolality in the water deprivated group (260 + 3 m O s m / m l vs 294 -I- 2 mOsm/ml; P < 0.01). Fig. 1 shows a significant reduction in L H level which was observed in both experimental groups. Similar results have been reported by Howland (1971). It could be explained by a decrease in the synthesis by starvation (decrease of carrier-proteins and/or the hormone precursors). As it may be seen in Fig. 2, there also was a significant decrease in Pro levels in both groups. It is known that plasmatic osmolality alters Pro levels. It was seen in man that the increase in osmolality values produces a decrease in Pro levels and vice versa (Scacchi, 1984). We have only found an increase in cerebral cortex serotonin levels in starved rats without alteration of
15,-
..~ I
.
(7l l-IControl ~ llStarvation V/~] ~Woter ~ deprivation ( } n* of cases * p (0.05
5
--
Fig. 1. Effects of starvation on dehydration on serum LH levels.
'
2O
!
T
/q ~E
(6)[
[] Control
IIStarvation []Water deprivation ( ) n" of cases ~ p
Fig. 2. Levels of prolactin in rats following the starvation or water deprivation. N A or D A levels. So far, therefore, it has not been possible to find out an explanation about this increase. We may postulate that perhaps on account of unfeeding, there was a lack of the necessary cofactors for its degradation (Fig. 3), The changes found in m o n o a m i n e levels in the hypothalamus are shown in Fig. 4. There were not variations in the monoamine content in underfed rats but in the water deprived group we found a significant decrease in N A level and a significant increase in 5-HT level. It is clear that monoamines are altered on different ways or by different mechanisms both in cerebral cortex and hypothalamus. D A and N A are aminergic neurotransmitters that have been reported to be implicated in the modulation of hypothalamic function. Rotsztejn et al. (1976) have observed that the in vitro release of L H - R H from the isolated anterior medial
DA NA [ ] Control IIStarvatIon [ ] Water deprivation
5-HT n =12 . p <005
Fig. 3. Effects of starvation or dehydration in monoamines on cerebral cortex in rats.
Starvation and dehydration
2.50
REFERENCES
I
~1.~ c-
I--IControl IStQrvation E~Water ~n=12 p{O.05 deprivation
"~ ~ l~
DA
NA
113
5-F~T
Fig. 4. Monoamine content in the hypothalamus of male rats submitted to starvation or dehydration. basal hypothalamus was enhanced by DA. Besides, the noradrenergic system stimulates L H - R H release and consequently influences pituitary L H secretion. It is known that N A acts exclusively at central levels as it has no effect on the pituitary gland. Our present results seem to demonstrate that the decrease of hypothalamic N A observed in water deprived rats may decrease the L H - R H hypothalamic release and the pituitary L H release. We speculate that the decrease in plasmatic L H observed by us may be a consequence of a decrease in L H release and not a decrease in its synthesis. On the other hand the serotoninergic system activation has an inhibitory effect on L H - R H (Schneider and McCann, 1970) and this agrees with our results as we found a serotonin increase. It is difficult for us to evaluate the serum prolactin decrease altogether with a D A normal level. Therefore, it is very likely that Pro decrease in underfeeding and water deprivation might not be influenced by neurotransmitters at central nervous system level. It would be another evidence that LH and P r o release are mainly controlled by different mechanisms. In the case of Pro, it may be thought about a direct metabolic effect. In fact, it is more realistic to suppose that underfeeding and water deprivation provoked two different effects: a direct metabolic effect on serum Pro level and an indirect effect on hypothalamic neurotransmitters at central nervous system level.
Bela E., Piacsek B. E. and Meites J. (1967) Reinitiation of gonadotrophin release in underfed rats by constant light or epinephrine. Endocrinology 81, 535-541. Curzon G. and Green A. R. (1970) Rapid method for the determination of 5-hydroxy-indole acetic acid in small regions of the rat brain. Br. J. Pharmac. Chemother. 39, 653-655. Gross H. A., Lake C. R., Ebert M. H., Ziegler M. G. and Kopin I. J. (1979) Catecholamine metabolism in primary anorexia nervosa. J. clin. Endocr. Metab. 49, 805-809. Howland B. E. (1971) Gonadotrophin levels in female rats subjected to restricted feed intake. J. Reprod. Fert. 27, 467470. Mulinos M. G. and Pomerantz L. (1940) Pseudohypophysectomy. A condition resembling hypophysectomy produced by malnutrition. J, Nutr. 19, 493-504. Mulinos M. G. and Pomerantz L. (1941) Pituitary replacement therapy in pseudohypophysectomy. Effects of pituitary implants upon organ weight of starved and underfed rats. Endocrinology 29, 558-563. Negro-Vilar A., Dickerman E. and Meites J. (1971) Effects of starvation on hypothalamic FSH-RF and pituitary FSH in male rats. Endocrinology 88, 1246-1249. Niswender G. H., Midgley A. R., Monroe S. E. and Reichert L. E. (1968) Radioimmunoassay for rat luteinizing hormone with antiovine serum and ovine LH TM I. Proc. Soc. exp. Biol. Med, 128, 807-811. Orsingher O. A., Marichich E. S., Molina V. A. and Ramirez O. A. (1980) A realiable and sensitive method for the simultaneous determinations of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indole acetic acid in small brain sample. Acta physiol, latinoam. 30, 111-115. Rotsztejn W. H., Charli J. L., Pattou E., Epelbaum J. and Kordon C. (1976) In vitro release of luteinizing hormonereleasing hormone (LH-RH) from rat mediobasal hypothalamus: effects of potassium, calcium and dopamine. Endocrinology 99, 1663-1669. Scacchi P. (1984) Prolactina I. In Neuroendocrinologia (Edited by Moguilevsky J. A. and Faig6n M. R.), pp. 173-189. L6pez Libreros, Buenos Aires. Schneider H. P. G. and McCann S. H. (1970) Mono and indolamines and control of LH secretion. Endocrinology 86, 1123-1127. Silverman J. A. (1974) Anorexia nervosa: clinical observations in a successful treatment plan. J. Pediatr. 84, 68-73. Vigersky R. A., Loriaux D. L., Anderson A. E. and Lipsett M. B. (1976) Anorexia nervosa: behavioral and hypothalamic aspects. J. clin. Endocr. Metab. 5, 51%535. Ziegler M. G., Lake C. R. and Kopin I. J. (1976) Deficient sympathetic nervous response in familial dysautonomia. New Engl. J. Med. 294, 630-633. Ziegler M. G,, Lake C. R. and Kopin I. J. (1977) The sympathetic nervous system defect in primary orthostatic hypotension. New Engl. J. Med. 296, 293-297.
0306-3623/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc
Printed in Great Britain. All rights reserved
STARVATION A N D DEHYDRATION: EFFECT ON HYPOTHALAMIC MONOAMINES A N D SERUM LH A N D PROLACTIN PABLO SCACCHI, SILVIA E. CARBONE, DORA C. DE RONDINA, IRMA O. MASTRONARDIand BERTA SZWARCFARB Instituto de Fisiologia "Profesor Dr Bemardo A. Houssay", Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina (Received 16 November 1987; received for publication 17 March 1988)
Abstract--1. Rats submitted to starvation or water deprivation showed a decrease in LH and prolactin serum levels. 2. 5-HT was increased without changes in DA and NA in cerebral cortex of starved rats. 3. Neurotransmitters did not change in hypothalamus of starved rats but water deprivation decreased NA and increased 5-HT.
INTRODUCTION
It is well known that undernutrition provokes several disorders being one of them reproduction alterations. In the rat the inanition or chronic desnutrition induced testicular and ovarian atrophy (Negro-Vilar et al., 1971; Mulinos and Pomerantz, 1941). Female dancers and gymnasts submitted to restricted food regimes suffer amenorrhea. On the other hand the neuroendocrine abnormalities which have been documented recently in primary anorexia nervosa suggest the presence of hypothalamic dysfunction during the cachectic phase of the illness (Silverman, 1974; Vigersky et al., 1976) and are consistent with an abnormality in sympathetic neuronal function (Ziegler et al., 1976, 1977). These patients also present abnormalities in t h e catecholaminergic metabolism. Gross et al. (1979) studied patients with primary anorexia nervosa all of which were amenorrheic. Plasma levels of tyrosine, norepinephrine (NE) and urinary excretion of its metabolites (homovanillic acid and 3-methoxy-4-hydroxyphenyl glycol) were determined and compared with those of normal females and they found significantly lower levels in all of the measured parameters. In that study it was found that the disturbances in the cardiovascular function (bradycardia, orthostatic hypotension) and in the catecholamine metabolism could be reversed by weight gain. The reversal of the biochemical abnormalities after weight gain suggested that they were secondary to the emaciation and general malnutrition characteristic of primary anorexia nervosa and were not aetiological factors. Many authors have studied the relation between desnutrition or dehydration and functional integrity of hypothalamus. They worked with different animal species, animals of different ages, normal, hemicastrated or castrated, male or female and the results were not in accord. Most of them agreed that underfeeding diminished pituitary LH and FSH and hypothalamic.LH-RH.
Nevertheless, Howland (1971) found that in female rats pituitary LH and FSH did not change while plasmatic LH diminished by 50% and suggested that synthesis and release of hormones were equally impaired and that this was consistent with the hypothesis that undernutrition acts on the pituitary gland by inhibiting hypothalamic production of LH releasing factor. Decrease in ovarian, uterine and anterior pituitary weight have been reported by other authors. The underfeeding resulted in cessation of oestrus cycles in all the animals and this reversed when the feeding was normalized (Bela et al., 1967). Negro-Vilar et al. (1971) studying the effects of complete food deprivation during 7 days in male adult rats found a large reduction in hypothalamic F S H - R H and in pituitary FSH levels; furthermore, there was a significant decrease in weight of anterior pituitary, seminal vesicles and prostate but testicular weight remained unchanged. These results suggested to the authors that starvation inhibits FSH release by decreasing hypothalamic FSH-RH activity, perhaps by reducing the availability of blood precursors for FSH synthesis. It is possible that hypothalamic neurons producing FSH-RH are sensitive to the levels of nutrients in the circulation, or that they may be influenced by the feeding centers in the hypothalamus. Since the deficiencies induced by severe underfeeding closely resembled those observed after hypophysectomy, Mulinos and Pomerantz (1940) called this condition "pseudohypophysectomy". However, few of the previous data in the literature revealed a neuroendocrine relation between hormonal variations and the possible changes of monoamine levels in the central nervous system. For this reason we decided to evaluate whether there was correlation between the variation in hypothalamic and corticocerebral monoamine levels and prolactin and LH plasmatic levels using as experimental model adult rats submitted to starvation or water deprivation.
111
112
PABLO SCACCHIet al. MATERIALS AND METHODS
Eighteen male adult rats (strain of the Instituto "Bernardo A. Houssay", Facultad de Medicina, Universidad de Buenos Aires), weighing 240 + 5 g were used. The animals were distributed in three equal groups, housed in cages and kept under a 12 hr light-dark cycle (07.00-19.00 light) in a temperature and humidity controlled room. The control group had water and food ad libitum. The second group was submitted to starvation: without food but with water ad libitum and the third group was deprived of water: they received no water but food ad libitum. The animals were daily weighed. At the end of the experiment, that lasted seven days, the animals were killed with a guillotine and the blood was collected from the trunk and allowed to clot at 4°C in glass tubes, separated by centrifugation 10 rain at 2500 rpm and the serum was separated and frozen at -30°C until the estimations of LH and Pro levels were carried out by the radioimmunoassay. Serum levels of LH were determined in duplicate using the double antibody radioimmunoassay method of Niswender et al. (1968). Values are expressed in ng/ml in terms of the reference preparation, NIAMDD-RatLH-RP-1. The intra-assay and inter-assay coet~cients of variations were 6 and 11%, respectively. The minimum sensitivity of the assay was 2 ng/ml serum. Serum Pro was measured in duplicate using the double antibody radioimmunoassay with kits supplied by the NIAMDD-Rat Pituitary Hormone Distribution Program. Values are expressed in ng/ml, on the basis of the NIAMDD references preparation supplied with the kit (Rat Prolactin RP-I). Results were statistically evaluated using analysis of variance and Tukey's multiple range test. Plasma osmolality was measured by the depression of the freezing point (Osmette S, Precision Systems, INC), expressing in mOsm/ml. The skull was rapidly opened and the brain immediately removed. The cerebral cortex and hypothalamus were dissected and frozen until the spectrofluorometric determinations of monoamines. Dopamine (DA) and noradrenaline (NA) were measured by the method of Orsingher et al. (1980) expressing the results in #g/g fresh tissue, and serotonin (5-HT) by the technique of Curzon and Green (1970). Statistical analysis was performed using Student's "t"-test.
RESULTS AND DISCUSSION
As expected there was a significant decrease in body weight in both (deprived) groups in comparison with the control group (33% less in starved and 37% less in water deprivated rats). It should be noted that the water deprivated rats did not eat. Furthermore, we have found a significant increase of plasmatic osmolality in the water deprivated group (260 + 3 m O s m / m l vs 294 -I- 2 mOsm/ml; P < 0.01). Fig. 1 shows a significant reduction in L H level which was observed in both experimental groups. Similar results have been reported by Howland (1971). It could be explained by a decrease in the synthesis by starvation (decrease of carrier-proteins and/or the hormone precursors). As it may be seen in Fig. 2, there also was a significant decrease in Pro levels in both groups. It is known that plasmatic osmolality alters Pro levels. It was seen in man that the increase in osmolality values produces a decrease in Pro levels and vice versa (Scacchi, 1984). We have only found an increase in cerebral cortex serotonin levels in starved rats without alteration of
15,-
..~ I
.
(7l l-IControl ~ llStarvation V/~] ~Woter ~ deprivation ( } n* of cases * p (0.05
5
--
Fig. 1. Effects of starvation on dehydration on serum LH levels.
'
2O
!
T
/q ~E
(6)[
[] Control
IIStarvation []Water deprivation ( ) n" of cases ~ p
Fig. 2. Levels of prolactin in rats following the starvation or water deprivation. N A or D A levels. So far, therefore, it has not been possible to find out an explanation about this increase. We may postulate that perhaps on account of unfeeding, there was a lack of the necessary cofactors for its degradation (Fig. 3), The changes found in m o n o a m i n e levels in the hypothalamus are shown in Fig. 4. There were not variations in the monoamine content in underfed rats but in the water deprived group we found a significant decrease in N A level and a significant increase in 5-HT level. It is clear that monoamines are altered on different ways or by different mechanisms both in cerebral cortex and hypothalamus. D A and N A are aminergic neurotransmitters that have been reported to be implicated in the modulation of hypothalamic function. Rotsztejn et al. (1976) have observed that the in vitro release of L H - R H from the isolated anterior medial
DA NA [ ] Control IIStarvatIon [ ] Water deprivation
5-HT n =12 . p <005
Fig. 3. Effects of starvation or dehydration in monoamines on cerebral cortex in rats.
Starvation and dehydration
2.50
REFERENCES
I
~1.~ c-
I--IControl IStQrvation E~Water ~n=12 p{O.05 deprivation
"~ ~ l~
DA
NA
113
5-F~T
Fig. 4. Monoamine content in the hypothalamus of male rats submitted to starvation or dehydration. basal hypothalamus was enhanced by DA. Besides, the noradrenergic system stimulates L H - R H release and consequently influences pituitary L H secretion. It is known that N A acts exclusively at central levels as it has no effect on the pituitary gland. Our present results seem to demonstrate that the decrease of hypothalamic N A observed in water deprived rats may decrease the L H - R H hypothalamic release and the pituitary L H release. We speculate that the decrease in plasmatic L H observed by us may be a consequence of a decrease in L H release and not a decrease in its synthesis. On the other hand the serotoninergic system activation has an inhibitory effect on L H - R H (Schneider and McCann, 1970) and this agrees with our results as we found a serotonin increase. It is difficult for us to evaluate the serum prolactin decrease altogether with a D A normal level. Therefore, it is very likely that Pro decrease in underfeeding and water deprivation might not be influenced by neurotransmitters at central nervous system level. It would be another evidence that LH and P r o release are mainly controlled by different mechanisms. In the case of Pro, it may be thought about a direct metabolic effect. In fact, it is more realistic to suppose that underfeeding and water deprivation provoked two different effects: a direct metabolic effect on serum Pro level and an indirect effect on hypothalamic neurotransmitters at central nervous system level.
Bela E., Piacsek B. E. and Meites J. (1967) Reinitiation of gonadotrophin release in underfed rats by constant light or epinephrine. Endocrinology 81, 535-541. Curzon G. and Green A. R. (1970) Rapid method for the determination of 5-hydroxy-indole acetic acid in small regions of the rat brain. Br. J. Pharmac. Chemother. 39, 653-655. Gross H. A., Lake C. R., Ebert M. H., Ziegler M. G. and Kopin I. J. (1979) Catecholamine metabolism in primary anorexia nervosa. J. clin. Endocr. Metab. 49, 805-809. Howland B. E. (1971) Gonadotrophin levels in female rats subjected to restricted feed intake. J. Reprod. Fert. 27, 467470. Mulinos M. G. and Pomerantz L. (1940) Pseudohypophysectomy. A condition resembling hypophysectomy produced by malnutrition. J, Nutr. 19, 493-504. Mulinos M. G. and Pomerantz L. (1941) Pituitary replacement therapy in pseudohypophysectomy. Effects of pituitary implants upon organ weight of starved and underfed rats. Endocrinology 29, 558-563. Negro-Vilar A., Dickerman E. and Meites J. (1971) Effects of starvation on hypothalamic FSH-RF and pituitary FSH in male rats. Endocrinology 88, 1246-1249. Niswender G. H., Midgley A. R., Monroe S. E. and Reichert L. E. (1968) Radioimmunoassay for rat luteinizing hormone with antiovine serum and ovine LH TM I. Proc. Soc. exp. Biol. Med, 128, 807-811. Orsingher O. A., Marichich E. S., Molina V. A. and Ramirez O. A. (1980) A realiable and sensitive method for the simultaneous determinations of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indole acetic acid in small brain sample. Acta physiol, latinoam. 30, 111-115. Rotsztejn W. H., Charli J. L., Pattou E., Epelbaum J. and Kordon C. (1976) In vitro release of luteinizing hormonereleasing hormone (LH-RH) from rat mediobasal hypothalamus: effects of potassium, calcium and dopamine. Endocrinology 99, 1663-1669. Scacchi P. (1984) Prolactina I. In Neuroendocrinologia (Edited by Moguilevsky J. A. and Faig6n M. R.), pp. 173-189. L6pez Libreros, Buenos Aires. Schneider H. P. G. and McCann S. H. (1970) Mono and indolamines and control of LH secretion. Endocrinology 86, 1123-1127. Silverman J. A. (1974) Anorexia nervosa: clinical observations in a successful treatment plan. J. Pediatr. 84, 68-73. Vigersky R. A., Loriaux D. L., Anderson A. E. and Lipsett M. B. (1976) Anorexia nervosa: behavioral and hypothalamic aspects. J. clin. Endocr. Metab. 5, 51%535. Ziegler M. G., Lake C. R. and Kopin I. J. (1976) Deficient sympathetic nervous response in familial dysautonomia. New Engl. J. Med. 294, 630-633. Ziegler M. G,, Lake C. R. and Kopin I. J. (1977) The sympathetic nervous system defect in primary orthostatic hypotension. New Engl. J. Med. 296, 293-297.