Cell Tiss. Res. 161,277--283 (1975) 9 by Springer-Verlag 1975
Correlations between Brain Catecholamines, Neurosecretion, and Serum Corticoid Levels in Osmotically Stressed Mallard Ducks (Anasplatyrhynchos) T. H. McNeill, J. H. Abel*, Jr., a n d G. P. Kozlowski Departments of Anatomy and Physiology-Biophysics, Colorado State University, Fort Collins, Colorado, U.S.A. Received March 18, 1975
Summary. The effects of depleting brain catecholamines with a combined treatment of reserpine and a-methyl-p-tyrosine on serum corticosterone levels and release of immunoreactive neurophysin from the median eminence, in osmotically stressed and unstressed mallard ducks, were studied. Corticoid levels in salt loaded birds were more than three times that of unstressed birds. The combined treatment of reserpine and cr significantly decreased the concentration of brain monoamines in all experimental groups and raised serum corticoid levels in non-stressed birds to the same level found in the osmotically stressed animals. Immunoreaetive neurophysin in the zona externa of the median eminence was depleted in all birds subjected to either osmotic stress and/or reserpine treatment but not in unstressed control birds. These preliminary data indicate that catecholamines may exert an inhibitory influence on both ACTH release from the anterior pituitary and neurophysin from the median eminence and that these two events may in some way be interrelated in the duck. Key words: Neurosecretion - - Osmotic stress - - Serum corticoid levels - - Catecholamine depletion - - Anas platyrhynchos (Ayes).
Introduction The extra-renal salt secreting system of euryhaline birds is controlled i n p a r t by the hormones of the adrenal a n d anterior p i t u i t a r y glands (Holmes, 1972). F o r example, recent work i n the m a l l a r d duck has shown t h a t 15 m i n u t e s after a n i n t r a p e r i t o n e a l i n j e c t i o n of h y p e r t o n i c saline (Allen et al., 1975a), serum corticoid levels are significantly increased over nomstressed birds a n d t h a t the salt gland takes u p a n d specifically b i n d s large q u a n t i t i e s of t r i t i a t e d corticosterone (Allen et al., 1975b). I t is n o t yet k n o w n how or to what e x t e n t the b r a i n exerts control over this endocrine response. I n m a m m a l s , however, recent evidence has shown t h a t catecholamines p r o b a b l y have a n i n h i b i t o r y influence on the release of A C T H from the h y p o p h y s i s (Ganong, 1972; V a n Loon, 1973; Scapaginini et al., 1972a, b ; H6kfelt et al., 1972). This has n o t been d e m o n s t r a t e d to be the case for birds nor is there precise evidence i n either birds or m a m m a l s i n d i c a t i n g what the corticotropin releasing factor (CRF) is, or where it is synthesized.
Send o//print requests to: Dr. John H. Abel, Jr., Dept. of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado 80523, U.S.A. This research was supported by research grant no. GB33321 from the National Science Foundation. We wish to express our thanks to Howard Funk, research leader, Colorado Division of Wildlife, for the use of the State's fine animal facilities.
278
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R e c e n t r e p o r t s from this l a b o r a t o r y , however, have shown t h a t t h e p a r a v e n t r i cular nucleus in t h e h y p o t h a l a m u s of t h e m a l l a r d duck is i n v o l v e d in t h e a d a p t i v e response to osmotic stress (Rhees et al., 1972), t h a t it is the site of d i r e c t corticoid feedback, t h a t it is r i c h l y i n n e r v a t e d b y m o n o a m i n e r g i c boutons, a n d t h a t it contains i m m u n o r e a c t i v e n e u r o p h y s i n as well as stainable n e u r o s e c r e t o r y m a t e r i a l (Abel et al., 1975). I n a d d i t i o n , stainable n e u r o s e c r e t o r y m a t e r i a l in t h e zona e x t e r n a of t h e a n t e r i o r m e d i a n eminence is r a p i d l y d e p l e t e d following t h e initiat i o n of osmotic stress a n d s t a y s d e p l e t e d in birds exposed to a h y p e r t o n i c saline e n v i r o n m e n t (Rhces et al., 1972). This p r e l i m i n a r y s t u d y was therefore, designed to d e t e r m i n e : 1) t h e influence of catecholamines oll t h e stress-induced rise in serum corticoid levels in t h e bird, 2) t h e effect of catecholamines u p o n t h e release of n e u r o p h y s i n from t h e zona e x t e r n a of t h e a n t e r i o r m e d i a n eminence, a n d 3) if t h e r e is a positive or n e g a t i v e correlation b e t w e e n these two responses.
Materials and Methods Thirty adult mallard ducks were administered 0.5% salt water ad libitum for one week and 1% salt water for a second week in order to activate the salt glands and pre-adapt the birds to osmotic stress (Rhees et al., 1972). The birds were taken completely off food, given fresh water and randomly divided into two groups : control (C), and depleted (D). The depleted group was pretreated with an intraperitoneal injection of 250 mg/Kg of a-methyl-p-tyrosine (Sigma) and 5 mg/Kg reserpine (Sigma) sixteen hours and four hours, respectively, before decapitation. This combined treatment has been shown to deplete monoamines from the central nervous system (Carr and Moore, 1968). In order to study the effects of short term osmotic stress on corticoid levels, half the birds in each group (5 birds) were administered an intraperitoneal (IP) injection of 20% salt water (20 ml/100 g body weight), and the other half an equal volume injection of isotonic saline. The birds were decapitated 20 minutes later, the trunk blood collected, and the brains quickly removed. The cerebellum, brain stem, optic lobes, and cerebral hemispheres were dissected from the brain leaving the diencephalon. Two brains from each group were placed in Bouin's fixative for eighteen hours, dehydrated, embedded in paraffin, and sectioned at 6 tzm on a rotary microtome. The remaining brains were wrapped in aluminum foil and placed in a Dewer flask containing liquid nitrogen. Serum blood samples were frozen and stored for quantitative measurements of corticoid content using the CBG protein assay of Murphy (1967). Neurophysin was localized using the immunocytochemical technique of Zimmerman and co-workers (1973b). Brain catecholamines were determined by a modification of the fluorometric assay of Anton and Sayre (1962). Results I n control birds, t h e i n t r a p e r i t o n e a l injection of h y p e r t o n i c saline induced g r e a t e r t h a n a threefold increase ( p < 0 . 0 1 ) in serum corticoid levels c o m p a r e d t o t h e isotonic-injected controls (Fig. 1). H y p o t h a l a m i c catecholamine levels in these two groups of birds d i d n o t differ significantly from each o t h e r (Fig. 2). W i t h reserpine a n d ~ - m e t h y l - p - t y r o s i n e t h e serum corticosterone levels in t h e o s m o t i c a l l y stressed birds were similar to those found in t h e o s m o t i c a l l y stressed controls. H o w e v e r , in unstressed birds t h a t received the m o n o a m i n e d e p l e t i n g drugs, t h e serum corticoid levels were t h r e e times g r e a t e r t h a n in non-stressed controls a n d were n o t significantly different from either t h e d r u g t r e a t e d stressed or control stressed groups. Thus, t h e d e p l e t i o n of b r a i n catecholamines i n d u c e d a rise in serum corticoid levels in unstressed birds similar to w h a t is observed
Catechotamines, Neurosecretion, and Serum Corticoid Levels in the Duck
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Fig. 1. Effect of catecholamine depletion and salt stress upon corticoid levels in the serum. C N S control nonstress, CS control stress, D N S depleted non-stress, D S depleted stress
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Fig. 2. Effect of reserpine (5 mg/kg) and a-methyl-p-tyrosine (250 mg/kg) on catecholamine levels in duck hypothalamus
n o r m a l l y o n l y in birds p l a c e d u n d e r intense osmotic stress. B r a i n levels of catechola m i n e s in b o t h t h e stressed a n d non-stressed groups which received reserpine a n d :r were significantly decreased ( p ~ 0 . 0 1 ) when c o m p a r e d
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Fig. 3. (A) Anterior median eminence of control non-stressed group. Neurophysin is localized in both zona interna (ZI) and zona externa (ZE). In the zona externa it is distributed between the portal capillaries (~) • 380. (B) Anterior median eminence from the depleted non-stressed group pretreated with reserpine and u-methyl-p-tyrosine. There is a loss of immuno-reactive neurophysin (~) between the capillaries of the zona externa. • 380
to the control non-stressed birds, but were not significantly different from each other. I n the anterior median eminence of the non-stressed control birds, both the fibrous layer of the zona interna and the palisade layer of the zona externa possessed a b u n d a n t immunoreactive neurophysin (Fig. 3a). Neurophysin in the palisade layer was predominately distributed between the capillaries of the porta] plexus. I n contrast, in the osmotically stressed birds t h a t received no drugs as well as in both the stressed and unstressed monoamine depleted groups, neurophysin was substantially depleted from the palisade layer of the median eminence. A correlation may, therefore, exist between the release of neurosecretory material into the portal vessels of the hypophysis and the stress induced rise in serum corticoid levels. A direct connection between these two events has yet to be proven, but the d a t a presented here makes such a hypothesis tenable. Discussion
The preliminary results obtained in this s t u d y on the pharmacological manilation of catecholamines indicate t h a t brain monoamines probably exert an inhibitory influence on the stress-induced rise in serum corticoid levels in the duck similar to what has been observed in mammals (Ganoug, 1972; Scapagnini et al., 1972; Van Loon etal., 1973).
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The fact that catecholamine levels do not change in osmotically stressed animals not treated with drugs seems at first to be inconsistent with this hypothesis. However, in mammals, stresses such as immobilization also induce no change in brain norepinephrine levels (Corrodi et al., 1968) even though stress of all types is known to increase the turnover of norepinephrine (Gordon et al., 1966; Thierry eta[., 1968; Javoy, 1968; Fuxe, 1968). Thus, in the undepleted but osmotically stressed ducks norepinephrine (NE) release is probably quickly compensated for by N E synthesis resulting in no significant net change. The depletion of neurophysin from the zona externa of the anterior median eminence in both osmotically stressed and catecholamine depleted birds also appears to be highly significant with respect to the adaptive response to osmotic stress. Neurophysin has been localized principally in the supraoptic and paraventricular nucleus (PVN) of the duck brain (Abel et al., 1975). Of these two nuclei, the PVN is by far the larger and more prominent. Thus, it is possible that much of the neurophysin within the zona externa of the median eminence originates in the neurons of the PVN although direct connections have not yet been discerned. In birds, the PVN has classically been associated with osmoregulation through the production and secretion of vasotocin (Kawashima et al., 1964). However, this m a y not be its sole function. Recent evidence from this laboratory has shown that the PVN undergoes a marked hypertrophy during osmotic stress (Rhees et al., 1972), that it is a direct site for corticoid feedback within the brain, and that both the small and large neurons of this nucleus contain neurophysin and are richly innervated by monoaminergic nerve terminals (Abel et al., 1975). In addition, m a n y investigators have found synapses between the neurosecretory perikarya and monoaminergic boutons (Peterson, 1965; Palenov et al., 1966), as well as high monoamine oxidase activity (Urano, 1968; Kobayashi et al., 1969). Since the zona externa of the median eminence in the duck exhibits, at best, sparse amine fluorescence (Calas et al., 1974), one must assume that the monoaminergic control of neurosecretion occurs principally at the level of the perikarya (Sharp and Follett, 1970; Warren Soest etal., 1973). Both neurophysin and aldehyde-fuchsin stainable material are observed in large amounts within the hypophyseal portal vessels shortly after the initiation of osmotic stress (unpublished data) indicating that neurophysin with its associated polypeptide probably does enter the adenohypophysis. What its effect here is, is still totally unknown. Considering, however, that, in mammals, ACTH release, NE turnover and stress are strongly correlated and that pressor substances are known to potentiate ACTH release (Ganong, 1963; Mangili, 1966; Javoy, 1968; Fuxe, 1970; Gold et al., 1967; Zimmerman et al., 1973a), we hypothesize that there is a direct correlation between the release of neurophysin into the hypophyseal portal vessels and the stress induced rise in serum corticoid levels.
References
Abel, J. H., Jr., Takemoto, D., Hoffman, D. L., McNeill, T. H., Kozlowski, G. P., Masken, J. F., Sheridan, P. : Corticoid uptake by the paraventricular nucleus in the hypothalamus of the duck, Arias platyrhynchos. Cell Tiss. Res. 161,285-291 (1975)
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Allen, J. C., Abel, J. H., Jr., Takemoto, D., Masken, J. F.: Effect of osmotic stress on serum corticoid and plasma glucose levels in the duck (Anas platyrhynchos). Gen. comp. Endocr. (in press) Allen, J. C., Abel, J. H., Jr., Takemoto, D. : Uptake and binding of labeled corticosterone by the salt gland of the duck (Anas platyrhynchos). Gem comp. Endoer. (in press) Anton, A. H., Sayer, D . F . : A study of the factors affecting aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. exp. Ther. 188, 360-375 (1962) Calas, A., Hartwig, H. G., Collin, J. P. : Noradrenergic innervation of the median eminence. Microspectrofluorimetrie and pharmacological study in the duck, Anas platyrhynchos. Z. Zellforsch. 147, 491-504 (1974) Carr, L. A., Moore, K. E. : Effects of reserpine and a-methyl-p-tyrosin on brain eatecholamines and the pituitary-adrenal response to stress. Neuroendocrinology 3, 285-302 (1968) Corrodi, H., Fuxe, K., HSkfelt, T.: The effect of immobilization stress on the activity of central monoamine neurons. Life Sci. 7, 107-112 (1968) Fuxe, K., Corrodi, H., H5kfelt, T., Jonsson, G.: Central monoamine neurons and pituitaryadrenal activity. In: Progr. Brain Res., vol. 32, p. 42-56 (D. DeWied and J. A. W. M. Weijnen, eds.). Amsterdam-London-New York: Elsevier Pub. Co. 1970 Ganong, W. F. : The central nervous system and the synthesis and release of adrenocortieotropic hormone. In: Advances in Neuroendocrinology, p. 92-149 (A. V. Nalbandov, ed.). Urbana: Univ. of Ill. Press 1963 Ganong, W. F. : Evidence for a central noradrenergic system that inhibits ACTH secretion. In: Brain-endocrine interaction. Median eminence: Structure and function (K. M. Knigge, D. E. Scott, A. Weindl, eds.), p. 254-266. Basel: Karger 1972 Gold, E. M., Ganong, W. F.: Effects of drugs on neuroendocrine processes. In: Neuroendocrinology (L. Martini, W. F. Ganong, eds.), vol. 2, p. 377-438. New York-London: Academic Press 1967 Gordon, R., Spector, S., Sjoerdsma, A., Udenfriend, S. : Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J. Pharmacol. exp. Ther. 153, 440-447 (1966) HSkfelt, T., Fuxe, K.: On the morphology and neurocndocrine role of the hypothalamic catecholamine neurons. In: Brain-endocrine interaction. Median eminence: Structure and function (K. M. Knigge, D. E. Scott, A. Weindl, eds.), p. 181-223. Basel: Karger 1972 Holmes, W. N. : Regulation of electrolyte balance in marine birds with special reference to the role of the pituitary-adrenal axis in the duck (Anas platyrhynchos). Fed. Proc. 31, 1578 1598 (1972) Javoy, F., Glowinski, J., Kordon, C.: Effect of adrenalectomy on the turnover of norepinephrine in the rat brain. Europ. J. Pharmacol. 4, 103-104 (1968) Kawashima, S., Farner, D., Kobayashi, H., Oksehe, A., Lorenzen, L.: The effect of dehydration on acid-phosphatase activity, catheptic-proteinase activity and neurosecretion in the hypothalamo-hypophyseal system of the White-crowned Sparrow, Zonotrichia leucophrys garabelii. Z. Zellforsch. 62, 149-181 (1964) Kobayashi, H., Matsui, T.: Fine structure of the median eminence and its functional significance. In: Frontiers in neuroendocrinology (W. F. Ganong, L. Martini, eds.), p. 3 4 6 . New York: Oxford Univ. Press 1969 Mangili, G., Motta, M., Martini, L.: Control of adrenocorticotropic hormone secretion. In: Neuroendoerinology (L. Martini, W. F. Ganong, eds.), vol. 1, p. 297-370. New York: Academic Press 1966 Murphy, B. E. P. : Some studies on the protein binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive protein binding radioassay. J. clin. Endocr. 27, 973-990 (1970) Palenov, A. L., Senchik, J. I. : Synapses on neurosecretory cells of the supraoptic nucleus in white mice. Nature (Lond.) 211, 1423-1424 (1966) Peterson, R. P.: Synapses in rat supraoptic nucleus. Anat. Rec. 151, 399 (1965) Rhees, R. W., Abel, J. H. Jr., Frame, J. R.: Effect of osmotic stress and hormone therapy on the hypothalamus of the duck (Anas platyrhynchos). Neuroendoerinology 10, 1-22 (1972)
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Scapagnini, U., Van Loon, G. R., Moberg, A. P., Preziosi, P., Ganong, W. F.: Evidence for central norepinephrine-mediated inhibition of ACTH secretion in the rat. Neuroendocrinology 10, 155-160 (1972) Scapagnini, U., Preziosi, P. : Role of brain norepinephrine and serotonin in the tonic and phasic regulation of hypothalamic hypophysial adrenal axis. Arch. int. Pharmacodyn., Suppl. to 196, 205-220 (1972) Sharp, P. J., Follett, B.K.: The adrenergic supply within the avian hypothalamus. In: Aspects of neuroendocrinology (W. Bargmann, B. Scharrer, eds.), p. 95-103. BerlinHeidelberg-New York: Springer 1970 Thierry, A. M., Fekete, M., Glowinski, J. : Effects of stress on the metabolism of noradrenaline, dopamine, and serotonin (5-HT) in the central nervous system of the rat, (II). Modifications of serotonin metabolism. Enrop. J. Pharmacol. 4, 384-389 (1968) Urano, A. : Monoamine oxidase in the hypothalamus of the Japanese quail and the mouse. J. Fac. Sci. Univ. Tokyo, Sec. IV, 11, 431-451 (1968) Van Loon, G.R.: Brain catecholamines and ACTH secretion. In: Frontiers in neuroendocrinology (W. Ganong, L. Martini, eds.), p. 209-247. London-Toronto: Oxford Univ. Press. 1973 Warren Soest, S., Farner, D. S., Oksche, A.: Fluorescence microscopy of neurons containing primary catecholamines in the ventral hypothalamus of the white-crowned sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 141, 1-17 (1973) Zimmerman, E. A., Carmel, P. W., Husain, M. K., Tannenbaum, M., Robinson, A. G.: Vasopressin and neurophysin: high concentrations in monkey hypophyseal portal blood. Science 182,925-927 (1973) Zimmerman, E. A., Hsu, K. C., Robinson, A. G., Carmel, P. W., Frantz, A. G., Tannenbaum, M.: Studies of neurophysin secreting neuron with immuno-peroxidase techniques employing antibody to bovine neurophysin. I. Light microscopic findings in monkey and bovine tissue. Endocrinology 12, 93i-940 (1973)
Correlations between Brain Catecholamines, Neurosecretion, and Serum Corticoid Levels in Osmotically Stressed Mallard Ducks (Anasplatyrhynchos) T. H. McNeill, J. H. Abel*, Jr., a n d G. P. Kozlowski Departments of Anatomy and Physiology-Biophysics, Colorado State University, Fort Collins, Colorado, U.S.A. Received March 18, 1975
Summary. The effects of depleting brain catecholamines with a combined treatment of reserpine and a-methyl-p-tyrosine on serum corticosterone levels and release of immunoreactive neurophysin from the median eminence, in osmotically stressed and unstressed mallard ducks, were studied. Corticoid levels in salt loaded birds were more than three times that of unstressed birds. The combined treatment of reserpine and cr significantly decreased the concentration of brain monoamines in all experimental groups and raised serum corticoid levels in non-stressed birds to the same level found in the osmotically stressed animals. Immunoreaetive neurophysin in the zona externa of the median eminence was depleted in all birds subjected to either osmotic stress and/or reserpine treatment but not in unstressed control birds. These preliminary data indicate that catecholamines may exert an inhibitory influence on both ACTH release from the anterior pituitary and neurophysin from the median eminence and that these two events may in some way be interrelated in the duck. Key words: Neurosecretion - - Osmotic stress - - Serum corticoid levels - - Catecholamine depletion - - Anas platyrhynchos (Ayes).
Introduction The extra-renal salt secreting system of euryhaline birds is controlled i n p a r t by the hormones of the adrenal a n d anterior p i t u i t a r y glands (Holmes, 1972). F o r example, recent work i n the m a l l a r d duck has shown t h a t 15 m i n u t e s after a n i n t r a p e r i t o n e a l i n j e c t i o n of h y p e r t o n i c saline (Allen et al., 1975a), serum corticoid levels are significantly increased over nomstressed birds a n d t h a t the salt gland takes u p a n d specifically b i n d s large q u a n t i t i e s of t r i t i a t e d corticosterone (Allen et al., 1975b). I t is n o t yet k n o w n how or to what e x t e n t the b r a i n exerts control over this endocrine response. I n m a m m a l s , however, recent evidence has shown t h a t catecholamines p r o b a b l y have a n i n h i b i t o r y influence on the release of A C T H from the h y p o p h y s i s (Ganong, 1972; V a n Loon, 1973; Scapaginini et al., 1972a, b ; H6kfelt et al., 1972). This has n o t been d e m o n s t r a t e d to be the case for birds nor is there precise evidence i n either birds or m a m m a l s i n d i c a t i n g what the corticotropin releasing factor (CRF) is, or where it is synthesized.
Send o//print requests to: Dr. John H. Abel, Jr., Dept. of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado 80523, U.S.A. This research was supported by research grant no. GB33321 from the National Science Foundation. We wish to express our thanks to Howard Funk, research leader, Colorado Division of Wildlife, for the use of the State's fine animal facilities.
278
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R e c e n t r e p o r t s from this l a b o r a t o r y , however, have shown t h a t t h e p a r a v e n t r i cular nucleus in t h e h y p o t h a l a m u s of t h e m a l l a r d duck is i n v o l v e d in t h e a d a p t i v e response to osmotic stress (Rhees et al., 1972), t h a t it is the site of d i r e c t corticoid feedback, t h a t it is r i c h l y i n n e r v a t e d b y m o n o a m i n e r g i c boutons, a n d t h a t it contains i m m u n o r e a c t i v e n e u r o p h y s i n as well as stainable n e u r o s e c r e t o r y m a t e r i a l (Abel et al., 1975). I n a d d i t i o n , stainable n e u r o s e c r e t o r y m a t e r i a l in t h e zona e x t e r n a of t h e a n t e r i o r m e d i a n eminence is r a p i d l y d e p l e t e d following t h e initiat i o n of osmotic stress a n d s t a y s d e p l e t e d in birds exposed to a h y p e r t o n i c saline e n v i r o n m e n t (Rhces et al., 1972). This p r e l i m i n a r y s t u d y was therefore, designed to d e t e r m i n e : 1) t h e influence of catecholamines oll t h e stress-induced rise in serum corticoid levels in t h e bird, 2) t h e effect of catecholamines u p o n t h e release of n e u r o p h y s i n from t h e zona e x t e r n a of t h e a n t e r i o r m e d i a n eminence, a n d 3) if t h e r e is a positive or n e g a t i v e correlation b e t w e e n these two responses.
Materials and Methods Thirty adult mallard ducks were administered 0.5% salt water ad libitum for one week and 1% salt water for a second week in order to activate the salt glands and pre-adapt the birds to osmotic stress (Rhees et al., 1972). The birds were taken completely off food, given fresh water and randomly divided into two groups : control (C), and depleted (D). The depleted group was pretreated with an intraperitoneal injection of 250 mg/Kg of a-methyl-p-tyrosine (Sigma) and 5 mg/Kg reserpine (Sigma) sixteen hours and four hours, respectively, before decapitation. This combined treatment has been shown to deplete monoamines from the central nervous system (Carr and Moore, 1968). In order to study the effects of short term osmotic stress on corticoid levels, half the birds in each group (5 birds) were administered an intraperitoneal (IP) injection of 20% salt water (20 ml/100 g body weight), and the other half an equal volume injection of isotonic saline. The birds were decapitated 20 minutes later, the trunk blood collected, and the brains quickly removed. The cerebellum, brain stem, optic lobes, and cerebral hemispheres were dissected from the brain leaving the diencephalon. Two brains from each group were placed in Bouin's fixative for eighteen hours, dehydrated, embedded in paraffin, and sectioned at 6 tzm on a rotary microtome. The remaining brains were wrapped in aluminum foil and placed in a Dewer flask containing liquid nitrogen. Serum blood samples were frozen and stored for quantitative measurements of corticoid content using the CBG protein assay of Murphy (1967). Neurophysin was localized using the immunocytochemical technique of Zimmerman and co-workers (1973b). Brain catecholamines were determined by a modification of the fluorometric assay of Anton and Sayre (1962). Results I n control birds, t h e i n t r a p e r i t o n e a l injection of h y p e r t o n i c saline induced g r e a t e r t h a n a threefold increase ( p < 0 . 0 1 ) in serum corticoid levels c o m p a r e d t o t h e isotonic-injected controls (Fig. 1). H y p o t h a l a m i c catecholamine levels in these two groups of birds d i d n o t differ significantly from each o t h e r (Fig. 2). W i t h reserpine a n d ~ - m e t h y l - p - t y r o s i n e t h e serum corticosterone levels in t h e o s m o t i c a l l y stressed birds were similar to those found in t h e o s m o t i c a l l y stressed controls. H o w e v e r , in unstressed birds t h a t received the m o n o a m i n e d e p l e t i n g drugs, t h e serum corticoid levels were t h r e e times g r e a t e r t h a n in non-stressed controls a n d were n o t significantly different from either t h e d r u g t r e a t e d stressed or control stressed groups. Thus, t h e d e p l e t i o n of b r a i n catecholamines i n d u c e d a rise in serum corticoid levels in unstressed birds similar to w h a t is observed
Catechotamines, Neurosecretion, and Serum Corticoid Levels in the Duck
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Fig. 1. Effect of catecholamine depletion and salt stress upon corticoid levels in the serum. C N S control nonstress, CS control stress, D N S depleted non-stress, D S depleted stress
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Fig. 2. Effect of reserpine (5 mg/kg) and a-methyl-p-tyrosine (250 mg/kg) on catecholamine levels in duck hypothalamus
n o r m a l l y o n l y in birds p l a c e d u n d e r intense osmotic stress. B r a i n levels of catechola m i n e s in b o t h t h e stressed a n d non-stressed groups which received reserpine a n d :r were significantly decreased ( p ~ 0 . 0 1 ) when c o m p a r e d
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Fig. 3. (A) Anterior median eminence of control non-stressed group. Neurophysin is localized in both zona interna (ZI) and zona externa (ZE). In the zona externa it is distributed between the portal capillaries (~) • 380. (B) Anterior median eminence from the depleted non-stressed group pretreated with reserpine and u-methyl-p-tyrosine. There is a loss of immuno-reactive neurophysin (~) between the capillaries of the zona externa. • 380
to the control non-stressed birds, but were not significantly different from each other. I n the anterior median eminence of the non-stressed control birds, both the fibrous layer of the zona interna and the palisade layer of the zona externa possessed a b u n d a n t immunoreactive neurophysin (Fig. 3a). Neurophysin in the palisade layer was predominately distributed between the capillaries of the porta] plexus. I n contrast, in the osmotically stressed birds t h a t received no drugs as well as in both the stressed and unstressed monoamine depleted groups, neurophysin was substantially depleted from the palisade layer of the median eminence. A correlation may, therefore, exist between the release of neurosecretory material into the portal vessels of the hypophysis and the stress induced rise in serum corticoid levels. A direct connection between these two events has yet to be proven, but the d a t a presented here makes such a hypothesis tenable. Discussion
The preliminary results obtained in this s t u d y on the pharmacological manilation of catecholamines indicate t h a t brain monoamines probably exert an inhibitory influence on the stress-induced rise in serum corticoid levels in the duck similar to what has been observed in mammals (Ganoug, 1972; Scapagnini et al., 1972; Van Loon etal., 1973).
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The fact that catecholamine levels do not change in osmotically stressed animals not treated with drugs seems at first to be inconsistent with this hypothesis. However, in mammals, stresses such as immobilization also induce no change in brain norepinephrine levels (Corrodi et al., 1968) even though stress of all types is known to increase the turnover of norepinephrine (Gordon et al., 1966; Thierry eta[., 1968; Javoy, 1968; Fuxe, 1968). Thus, in the undepleted but osmotically stressed ducks norepinephrine (NE) release is probably quickly compensated for by N E synthesis resulting in no significant net change. The depletion of neurophysin from the zona externa of the anterior median eminence in both osmotically stressed and catecholamine depleted birds also appears to be highly significant with respect to the adaptive response to osmotic stress. Neurophysin has been localized principally in the supraoptic and paraventricular nucleus (PVN) of the duck brain (Abel et al., 1975). Of these two nuclei, the PVN is by far the larger and more prominent. Thus, it is possible that much of the neurophysin within the zona externa of the median eminence originates in the neurons of the PVN although direct connections have not yet been discerned. In birds, the PVN has classically been associated with osmoregulation through the production and secretion of vasotocin (Kawashima et al., 1964). However, this m a y not be its sole function. Recent evidence from this laboratory has shown that the PVN undergoes a marked hypertrophy during osmotic stress (Rhees et al., 1972), that it is a direct site for corticoid feedback within the brain, and that both the small and large neurons of this nucleus contain neurophysin and are richly innervated by monoaminergic nerve terminals (Abel et al., 1975). In addition, m a n y investigators have found synapses between the neurosecretory perikarya and monoaminergic boutons (Peterson, 1965; Palenov et al., 1966), as well as high monoamine oxidase activity (Urano, 1968; Kobayashi et al., 1969). Since the zona externa of the median eminence in the duck exhibits, at best, sparse amine fluorescence (Calas et al., 1974), one must assume that the monoaminergic control of neurosecretion occurs principally at the level of the perikarya (Sharp and Follett, 1970; Warren Soest etal., 1973). Both neurophysin and aldehyde-fuchsin stainable material are observed in large amounts within the hypophyseal portal vessels shortly after the initiation of osmotic stress (unpublished data) indicating that neurophysin with its associated polypeptide probably does enter the adenohypophysis. What its effect here is, is still totally unknown. Considering, however, that, in mammals, ACTH release, NE turnover and stress are strongly correlated and that pressor substances are known to potentiate ACTH release (Ganong, 1963; Mangili, 1966; Javoy, 1968; Fuxe, 1970; Gold et al., 1967; Zimmerman et al., 1973a), we hypothesize that there is a direct correlation between the release of neurophysin into the hypophyseal portal vessels and the stress induced rise in serum corticoid levels.
References
Abel, J. H., Jr., Takemoto, D., Hoffman, D. L., McNeill, T. H., Kozlowski, G. P., Masken, J. F., Sheridan, P. : Corticoid uptake by the paraventricular nucleus in the hypothalamus of the duck, Arias platyrhynchos. Cell Tiss. Res. 161,285-291 (1975)
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Allen, J. C., Abel, J. H., Jr., Takemoto, D., Masken, J. F.: Effect of osmotic stress on serum corticoid and plasma glucose levels in the duck (Anas platyrhynchos). Gen. comp. Endocr. (in press) Allen, J. C., Abel, J. H., Jr., Takemoto, D. : Uptake and binding of labeled corticosterone by the salt gland of the duck (Anas platyrhynchos). Gem comp. Endoer. (in press) Anton, A. H., Sayer, D . F . : A study of the factors affecting aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. exp. Ther. 188, 360-375 (1962) Calas, A., Hartwig, H. G., Collin, J. P. : Noradrenergic innervation of the median eminence. Microspectrofluorimetrie and pharmacological study in the duck, Anas platyrhynchos. Z. Zellforsch. 147, 491-504 (1974) Carr, L. A., Moore, K. E. : Effects of reserpine and a-methyl-p-tyrosin on brain eatecholamines and the pituitary-adrenal response to stress. Neuroendocrinology 3, 285-302 (1968) Corrodi, H., Fuxe, K., HSkfelt, T.: The effect of immobilization stress on the activity of central monoamine neurons. Life Sci. 7, 107-112 (1968) Fuxe, K., Corrodi, H., H5kfelt, T., Jonsson, G.: Central monoamine neurons and pituitaryadrenal activity. In: Progr. Brain Res., vol. 32, p. 42-56 (D. DeWied and J. A. W. M. Weijnen, eds.). Amsterdam-London-New York: Elsevier Pub. Co. 1970 Ganong, W. F. : The central nervous system and the synthesis and release of adrenocortieotropic hormone. In: Advances in Neuroendocrinology, p. 92-149 (A. V. Nalbandov, ed.). Urbana: Univ. of Ill. Press 1963 Ganong, W. F. : Evidence for a central noradrenergic system that inhibits ACTH secretion. In: Brain-endocrine interaction. Median eminence: Structure and function (K. M. Knigge, D. E. Scott, A. Weindl, eds.), p. 254-266. Basel: Karger 1972 Gold, E. M., Ganong, W. F.: Effects of drugs on neuroendocrine processes. In: Neuroendocrinology (L. Martini, W. F. Ganong, eds.), vol. 2, p. 377-438. New York-London: Academic Press 1967 Gordon, R., Spector, S., Sjoerdsma, A., Udenfriend, S. : Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J. Pharmacol. exp. Ther. 153, 440-447 (1966) HSkfelt, T., Fuxe, K.: On the morphology and neurocndocrine role of the hypothalamic catecholamine neurons. In: Brain-endocrine interaction. Median eminence: Structure and function (K. M. Knigge, D. E. Scott, A. Weindl, eds.), p. 181-223. Basel: Karger 1972 Holmes, W. N. : Regulation of electrolyte balance in marine birds with special reference to the role of the pituitary-adrenal axis in the duck (Anas platyrhynchos). Fed. Proc. 31, 1578 1598 (1972) Javoy, F., Glowinski, J., Kordon, C.: Effect of adrenalectomy on the turnover of norepinephrine in the rat brain. Europ. J. Pharmacol. 4, 103-104 (1968) Kawashima, S., Farner, D., Kobayashi, H., Oksehe, A., Lorenzen, L.: The effect of dehydration on acid-phosphatase activity, catheptic-proteinase activity and neurosecretion in the hypothalamo-hypophyseal system of the White-crowned Sparrow, Zonotrichia leucophrys garabelii. Z. Zellforsch. 62, 149-181 (1964) Kobayashi, H., Matsui, T.: Fine structure of the median eminence and its functional significance. In: Frontiers in neuroendocrinology (W. F. Ganong, L. Martini, eds.), p. 3 4 6 . New York: Oxford Univ. Press 1969 Mangili, G., Motta, M., Martini, L.: Control of adrenocorticotropic hormone secretion. In: Neuroendoerinology (L. Martini, W. F. Ganong, eds.), vol. 1, p. 297-370. New York: Academic Press 1966 Murphy, B. E. P. : Some studies on the protein binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive protein binding radioassay. J. clin. Endocr. 27, 973-990 (1970) Palenov, A. L., Senchik, J. I. : Synapses on neurosecretory cells of the supraoptic nucleus in white mice. Nature (Lond.) 211, 1423-1424 (1966) Peterson, R. P.: Synapses in rat supraoptic nucleus. Anat. Rec. 151, 399 (1965) Rhees, R. W., Abel, J. H. Jr., Frame, J. R.: Effect of osmotic stress and hormone therapy on the hypothalamus of the duck (Anas platyrhynchos). Neuroendoerinology 10, 1-22 (1972)
Catecholamines, Neurosecretion, and Serum Corticoid Levels in the Duck
283
Scapagnini, U., Van Loon, G. R., Moberg, A. P., Preziosi, P., Ganong, W. F.: Evidence for central norepinephrine-mediated inhibition of ACTH secretion in the rat. Neuroendocrinology 10, 155-160 (1972) Scapagnini, U., Preziosi, P. : Role of brain norepinephrine and serotonin in the tonic and phasic regulation of hypothalamic hypophysial adrenal axis. Arch. int. Pharmacodyn., Suppl. to 196, 205-220 (1972) Sharp, P. J., Follett, B.K.: The adrenergic supply within the avian hypothalamus. In: Aspects of neuroendocrinology (W. Bargmann, B. Scharrer, eds.), p. 95-103. BerlinHeidelberg-New York: Springer 1970 Thierry, A. M., Fekete, M., Glowinski, J. : Effects of stress on the metabolism of noradrenaline, dopamine, and serotonin (5-HT) in the central nervous system of the rat, (II). Modifications of serotonin metabolism. Enrop. J. Pharmacol. 4, 384-389 (1968) Urano, A. : Monoamine oxidase in the hypothalamus of the Japanese quail and the mouse. J. Fac. Sci. Univ. Tokyo, Sec. IV, 11, 431-451 (1968) Van Loon, G.R.: Brain catecholamines and ACTH secretion. In: Frontiers in neuroendocrinology (W. Ganong, L. Martini, eds.), p. 209-247. London-Toronto: Oxford Univ. Press. 1973 Warren Soest, S., Farner, D. S., Oksche, A.: Fluorescence microscopy of neurons containing primary catecholamines in the ventral hypothalamus of the white-crowned sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 141, 1-17 (1973) Zimmerman, E. A., Carmel, P. W., Husain, M. K., Tannenbaum, M., Robinson, A. G.: Vasopressin and neurophysin: high concentrations in monkey hypophyseal portal blood. Science 182,925-927 (1973) Zimmerman, E. A., Hsu, K. C., Robinson, A. G., Carmel, P. W., Frantz, A. G., Tannenbaum, M.: Studies of neurophysin secreting neuron with immuno-peroxidase techniques employing antibody to bovine neurophysin. I. Light microscopic findings in monkey and bovine tissue. Endocrinology 12, 93i-940 (1973)
