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Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices
BrainResearch, 369(1986)391-394 Elsevier
391
BRE 21453
Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices JUN ARITA and FUKUKO KIMURA Department of Physiology, Yokohama City UniversitySchool of Medicine, Yokohama 232 (Japan) (Accepted November 26th, 1985) Key words: aging- - calcium ion - - 3,4-dihydroxyphenylalanine- - tuberoinfundibular dopaminergic neuron
The rates of basal and cyclicAMP-dependent DOPA accumulation in the median eminence of hypothalamic slices were not different between ovariectomized young and aged rats. However, the rate of Ca2+-dependent, depolarization-induced DOPA accumulation was smaller in aged rats, suggesting that the Ca2+ system in the regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic neurons is altered by aging.
Tuberoinfundibular dopaminergic (TIDA) neurons have cell bodies in the arcuate and periventricular nuclei of rat hypothalamus and axon terminals in the vicinity of the hypophysial portal vasculature in the median eminence 10. Dopamine released from the axon terminals into portal blood 5 has a direct inhibitory action on prolactin (PRL) secretion from the pituitary gland 13, serving as a PRL release inhibitory factor. It is known in rats that the concentration of PRL in serum increases progressively with agel2,14. It has been suggested that reduced functions of T I D A neurons contribute to the appearance of hyperprolactinemia in aged rats6-8,11,14-17.19. Especially, several studies have shown that the rate of dopamine biosynthesis in T I D A neurons, as estimated by the rate of in vivo 3,4-dibydroxyphenylalanine (DOPA) accumulation in the median eminence after injection of h D O P A decarboxylase inhibitor, is smaller in aged rats than in young rats6,7A5. Therefore, in the present study, we examined in vitro D O P A accumulation in the median eminence of aged rats using hypothalamic slices in order to know the intracellular mechanism for reduced dopamine biosynthesis in T I D A neurons of aged rats. Young (3 months of age) and aged female rats (18-
20 months of age) of the Wistar strain were used in this study. The aged rats used were multiparous and displayed constant cornification of vaginal epithelial cells when they were ovariectomized. The young and aged rats were decapitated 5 weeks after ovariectomy. Sagittal hypothalamic slices including the median eminence, arcuate nucleus and small parts of the ventromedial and periventricular nuclei were prepared as described previously2. The standard medium used was composed of 116 mM NaCl, 5 mM KCI, 2.5 mM CaCI2, 1 mM MgSO4, 1.25 mM KH2PO4, 26 mM NaHCO3, 10 mM glucose, 20/~M tyrosine and 6 mg/l phenol red, and saturated with an atmosphere of 95% 02/5% CO2, pH 7.4 at 37 °C. In vitro dopamine biosynthesis in T I D A neurons was estimated by measurement of D O P A accumulated in the median eminence after incubation of hypothalamic slices with 3-hydroxybenzylhydrazine (NSD 1015), a D O P A decarboxylase inhibitor. The amount of D O P A in the supernatant of homogenate of the median eminence was determined by high-performance liquid chromatography with electrochemical detection, as described elsewhereL PRL in serum obtained at the time of decapitation was quantified by radioimmunoassay with reagents provided by the N I A D D K of the National Institutes of Health
Correspondence: J. Arita, Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama, Kanagawa 232, Japan. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
392 ( U . S . A . ) . The values for P R L are expressed in terms of rat P R L RP-1 (RP, reference preparation). The concentrations of P R L in serum were 33 _+ 3 (mean + S . E . M . ) (n = 26) and 100 + 23 (n = 25) ng/ml in young and aged rats, respectively. Since they were ovariectomized to eliminate influences of ovarian hormones on P R L secretion, the higher concentration of P R L in aged rats is not due to constant estrus but due to an alteration in the function of the hypothalamo-hypophysial axis itself. The rates of in vivo D O P A accumulation in the median eminence of young and aged rats were measured to confirm the results r e p o r t e d by others6.7,1L The amount of in vivo D O P A accumulation in the median eminence, as determined by i.p. injection of 100 mg/kg N S D 1015, 45 min before decapitation, were 21.1 + 2.6 (n = 5) and 12.7 + 1.3 (n = 5) ng/mg protein in young and aged rats, respectively. On the basis of the results of our previous studies using hypothalamic slices, the intracellular regulatory mechanism for d o p a m i n e biosynthesis in T I D A neurons seems to be c o m p o s e d of at least 3 different independent systems, that is basal, adenosine 3 ' , 5 ' cyclic m o n o p h o s p h a t e ( c A M P ) - d e p e n d e n t and Ca 2÷d e p e n d e n t systems3, 4. Therefore, these 3 systems for in vitro d o p a m i n e biosynthesis in the median eminence were examined in young and aged rats. First, the basal biosynthesis of d o p a m i n e in the median eminence, which was shown to be extracellular Ca2+-in d e p e n d e n t and tetrodotoxin-insensitive 3, was determined by incubation of hypothalamic slices in the standard m e d i u m containing N S D 10t5. The basal rates of D O P A accumulation in the median eminence were 11.9 + 1.4 (n = 5) in young rats and 11.4 + 1.8 (n = 5) ng/mg protein/h in aged rats (Fig. 1), and there was no difference between them ( P > 0.05). Next, the rate of c A M P - d e p e n d e n t d o p a m i n e biosynthesis was determined. The addition of the c A M P analogue d i b u t y r y l - c A M P ( d b - c A M P ) at a concentration of 5 m M into the m e d i u m containing NSD 1015 caused a 3.6-fold increase in the rate of D O P A accumulation in the median eminence of young rats. d b - c A M P - i n d u c e d D O P A accumulation in aged rats did not differ from that in young rats (P > 0.05). F u r t h e r m o r e , an increase in endogenous c A M P achieved by the addition of isobutylmethylxanthine ( I B M X ) , a phosphodiesterase inhibitor, at a concentration of 1 m M caused a similar increase in
u.J
F-"I YOUNG RATS
so
m
AGED RATS
mr Z
o
T 40 .~
~.~
~ 2op ~,0 O r-',, o
BASAL
db-cANIP
IBMX
Fig. 1. Basal and cAMP-dependent DOPA accumulation in the median eminence of young and aged rats. Hypothalamic slices of young and aged rats were preincubated at 37 °C for 40-60 min in the standard medium and then incubated for 60 min in the medium containing 10 mM NSD 1015. As for measurement of the rate of basal DOPA accumulation, hypothalamic slices were incubated in medium containing NSD 1015 with no additives. As for measurement of the rate of cAMP-dependent DOPA accumulation, either db-cAMP or IBMX was added to the medium containing NSD 1015 to obtain a concentration of 5 or 1 mM, respectively. After the end of incubation of slices in medium containing NSD 1015, the median eminence was dissected out from the slices and homogenized in 100 ktl of 0.1 N perchloric acid containing 3 mM EDTA and 5 mM sodium metabisulfite. The supernatant of the homogenate obtained after centrifugation of 10,000 g for 2 min was stored at -80 °C for DOPA determination. The values are expressed as ng DOPA accumulated/rag protein/h. Each column and its vertical line represent mean and S.E.M., based on 5 median eminences.
D O P A accumulation in the median eminence of young and aged rats (P > 0.05). Finally, Ca2+-de pendent d o p a m i n e biosynthesis was d e t e r m i n e d by causing depolarization in m e m b r a n e potentials of hypothalamic slices. A recent study has shown that depolarization achieved by high K ÷ in medium or veratridine elicits an increase in the rate of D O P A accumulation in the median eminence, which requires extracellular Ca 2+ and is blocked by Ca2+-channel blockers 3. Depolarization of slices by 50 m M K ÷ in m e d i u m m a r k e d l y increased the rate of D O P A accumulation in the median eminence of young rats (Fig. 2). The rate of D O P A accumulation induced by depolarization in aged rats was 74% of that in young rats, and this decrease in the rate of D O P A accumulation was statistically significant ( P < 0.01, one-way analysis of variance followed by D u n c a n ' s multiplerange test). The result that the rate of Ca2+-dependent, depolarization-induced in vitro D O P A accumulation in
393
I"-"] t.l.I
5 mM K* 50mMK +
~00 re'w*
K
75
o
50
least in part, for the reduced dopamine biosynthesis in T I D A n e u r o n s of aged rats as observed in vivo. This view is compatible with the result that the secretion of dopamine into portal blood was reduced in aged rats 11,16.19, since dopamine secretion in the median eminence is also Ca2+-dependent 1,9. Therefore, it is of interest to speculate that responses to Ca z+ stimulus of the regulatory mechanisms for T I D A neuronal functions, such as the synthesis and secretion of dopamine, are altered by aging.
oQ 0 YOUNG
AGED
Fig. 2. Depolarization-induced DOPA accumulation in the median eminence of young and aged rats. Hypothalamic slices were incubated either for 60 min in medium containing 10 mM NSD 1015 and 5 mM KCI or for 20 min in medium containing 10 mM NSD 1015 and 50 mM KCI substituted for equimolar amounts of NaCI. See Fig. 1 (legend) for further detailed procedures. Each column and its vertical line represent mean and S.E.M., based on 5-6 median eminences.
At present, the cause for the reduced Ca2+-de pendent dopamine biosynthesis in T I D A neurons is unknown. It has recently been reported that shortterm hyperprolactinemia (e.g. for several days) stimulates T I D A n e u r o n a l functions while prolonged hyperprolactinemia (e.g. for several weeks or months) suppresses them18, z0. It is likely that long-term exposure of T I D A neurons to PRL in the process of aging reduces Ca2÷-dependent dopamine biosynthesis.
the median eminence was reduced in aged rats is suggestive of the view that the reduction in Ca2+-de pendent dopamine biosynthesis is responsible, at
search project on aging and age-related diseases (Yokohama City).
1 Annunziato, L., di Renzo, G., Amoroso, S. and Quattrone, A., Release of endogeneous dopamine from tuberoinfundibular neurons, Life Sci., 35 (1984) 399-407. 2 Arita, J. and Kimura, F., Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices, Neuroendocrinology, 39 (1984) 524-529. 3 Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvement of voltage-dependent Ca2+-channels, Brain Research, 347 (1985) 299-305. 4 Arita, J. and Kimura, F., Adenosine 3',5'-cyclic monophosphate stimulates dopamine biosynthesis in the median eminence of rat hypothalamic slices, Brain Research, in press. 5 Ben-Jonathan, N., Oliver, C., Weiner, H.J., Mical, R.S. and Porter, J.C., Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy, Endocrinology, 100 (1977) 452-458. 6 Demarest, K.T., Moore, K.E. and Riegle, G.D., Dopaminergic neuronal function, anterior pituitary dopamine content, and serum concentrations of prolactin, luteinizing hormone and progesterone in the aged female rat, Brain Research, 247 (1982) 347-354. 7 Demarest, K.T., Riegle, G.D. and Moore, K.E., Characteristics of dopaminergic neurons in the aged male rat, Neuroendocrinology, 31 (1980)222-227. 8 Estes, K.S. and Simpkins, J.W., Age-related alterations in dopamine and norepinephrine activity within microdis-
sected brain regions of ovariectomized Long-Evans rats, Brain Research, 298 (1984) 209-218, 9 Foreman, M.M. and Porter, J.C., Prolactin augmentation of dopamine and norepinephrine release from superfused medial basal hypothalamic fragments, Endocrinology, 108 (1981) 800-804. 10 Fuxe, K., Cellular localization of monoamines in the median eminence and in the infundibular stem of some mammals, Acta Physiol. Scand., 58 (1963) 383-384. 11 Gudelsky, G.A., Nansel, D.D. and Porter, J.C., Dopaminergic control of prolactin secretion in the aging male rat, Brain Research, 204 (1981) 446-450. 12 Huang, H.H., Marshall, S. and Meites, J., Capacity of old versus young female rats to secrete LH, FSH and prolactin, Biol. Reprod., 14 (1976) 538-543. 13 MacLeod, R.M., Fontham, E.H. and Lehmeyer, J.E., Prolactin and growth hormone production as influenced by catecholamines and agents that affect brain catecholamines, Neuroendocrinology, 6 (1970) 283-294. 14 Meites, J., Huang, H.H. and Simpkins, J.W., Recent studies on neuroendocrine control of reproductive senescence in rats. In E.L. Schneider (Ed.), The Aging Reproductive System, Raven Press, New York, 1978, pp. 213-235. 15 Reymond, M.J., Arita, J., Dudley, C.A., Moss, R.L. and Porter, J.C., Dopaminergic neurons in the mediobasal hypothalamus of old rats: evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor, Brain Research, 304 (1984) 215-223. 16 Reymond, M.J. and Porter, J.C., Secretion of hypothalam-
This study was supported partly by the special re-
394 ic dopamine into pituitary stalk blood of aged female rats, Brain Res. Bull., 7 (1981) 69-73. 17 Sarkar, D.K., Gottschall, P.E. and Meites, J., Damage to hypothalamic dopaminergic neurons is associated with development of prolactin-secreting pituitary tumors, Science, 218 (1982) 684-686. 18 Sarkar, D.K., Gottschall, P.E. and Meites, J., Decline of tuberoinfundibular dopaminergic function resulting from chronic hyperprolactinemia in rats, Endocrinology, 115
(1984) 1269-1274. 19 Sarkar, D.K., Oottschall, P.E., Xie, O.-W. and Meites, J., Reduced tuberoinfundibular dopaminergic neuronal function in rats with in situ prolactin-secreting pituitary tumors. Neuroendocrinology, 38 (1984) 498-503. 20 Simpkins, J.W. and Gabriel, S.M., Chronic hyperprolactinemia causes progressive changes in hypothalamic dopaminergic and noradrenergic neurons, Brain Research, 309 (1984) 277-282.
391
BRE 21453
Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices JUN ARITA and FUKUKO KIMURA Department of Physiology, Yokohama City UniversitySchool of Medicine, Yokohama 232 (Japan) (Accepted November 26th, 1985) Key words: aging- - calcium ion - - 3,4-dihydroxyphenylalanine- - tuberoinfundibular dopaminergic neuron
The rates of basal and cyclicAMP-dependent DOPA accumulation in the median eminence of hypothalamic slices were not different between ovariectomized young and aged rats. However, the rate of Ca2+-dependent, depolarization-induced DOPA accumulation was smaller in aged rats, suggesting that the Ca2+ system in the regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic neurons is altered by aging.
Tuberoinfundibular dopaminergic (TIDA) neurons have cell bodies in the arcuate and periventricular nuclei of rat hypothalamus and axon terminals in the vicinity of the hypophysial portal vasculature in the median eminence 10. Dopamine released from the axon terminals into portal blood 5 has a direct inhibitory action on prolactin (PRL) secretion from the pituitary gland 13, serving as a PRL release inhibitory factor. It is known in rats that the concentration of PRL in serum increases progressively with agel2,14. It has been suggested that reduced functions of T I D A neurons contribute to the appearance of hyperprolactinemia in aged rats6-8,11,14-17.19. Especially, several studies have shown that the rate of dopamine biosynthesis in T I D A neurons, as estimated by the rate of in vivo 3,4-dibydroxyphenylalanine (DOPA) accumulation in the median eminence after injection of h D O P A decarboxylase inhibitor, is smaller in aged rats than in young rats6,7A5. Therefore, in the present study, we examined in vitro D O P A accumulation in the median eminence of aged rats using hypothalamic slices in order to know the intracellular mechanism for reduced dopamine biosynthesis in T I D A neurons of aged rats. Young (3 months of age) and aged female rats (18-
20 months of age) of the Wistar strain were used in this study. The aged rats used were multiparous and displayed constant cornification of vaginal epithelial cells when they were ovariectomized. The young and aged rats were decapitated 5 weeks after ovariectomy. Sagittal hypothalamic slices including the median eminence, arcuate nucleus and small parts of the ventromedial and periventricular nuclei were prepared as described previously2. The standard medium used was composed of 116 mM NaCl, 5 mM KCI, 2.5 mM CaCI2, 1 mM MgSO4, 1.25 mM KH2PO4, 26 mM NaHCO3, 10 mM glucose, 20/~M tyrosine and 6 mg/l phenol red, and saturated with an atmosphere of 95% 02/5% CO2, pH 7.4 at 37 °C. In vitro dopamine biosynthesis in T I D A neurons was estimated by measurement of D O P A accumulated in the median eminence after incubation of hypothalamic slices with 3-hydroxybenzylhydrazine (NSD 1015), a D O P A decarboxylase inhibitor. The amount of D O P A in the supernatant of homogenate of the median eminence was determined by high-performance liquid chromatography with electrochemical detection, as described elsewhereL PRL in serum obtained at the time of decapitation was quantified by radioimmunoassay with reagents provided by the N I A D D K of the National Institutes of Health
Correspondence: J. Arita, Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama, Kanagawa 232, Japan. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
392 ( U . S . A . ) . The values for P R L are expressed in terms of rat P R L RP-1 (RP, reference preparation). The concentrations of P R L in serum were 33 _+ 3 (mean + S . E . M . ) (n = 26) and 100 + 23 (n = 25) ng/ml in young and aged rats, respectively. Since they were ovariectomized to eliminate influences of ovarian hormones on P R L secretion, the higher concentration of P R L in aged rats is not due to constant estrus but due to an alteration in the function of the hypothalamo-hypophysial axis itself. The rates of in vivo D O P A accumulation in the median eminence of young and aged rats were measured to confirm the results r e p o r t e d by others6.7,1L The amount of in vivo D O P A accumulation in the median eminence, as determined by i.p. injection of 100 mg/kg N S D 1015, 45 min before decapitation, were 21.1 + 2.6 (n = 5) and 12.7 + 1.3 (n = 5) ng/mg protein in young and aged rats, respectively. On the basis of the results of our previous studies using hypothalamic slices, the intracellular regulatory mechanism for d o p a m i n e biosynthesis in T I D A neurons seems to be c o m p o s e d of at least 3 different independent systems, that is basal, adenosine 3 ' , 5 ' cyclic m o n o p h o s p h a t e ( c A M P ) - d e p e n d e n t and Ca 2÷d e p e n d e n t systems3, 4. Therefore, these 3 systems for in vitro d o p a m i n e biosynthesis in the median eminence were examined in young and aged rats. First, the basal biosynthesis of d o p a m i n e in the median eminence, which was shown to be extracellular Ca2+-in d e p e n d e n t and tetrodotoxin-insensitive 3, was determined by incubation of hypothalamic slices in the standard m e d i u m containing N S D 10t5. The basal rates of D O P A accumulation in the median eminence were 11.9 + 1.4 (n = 5) in young rats and 11.4 + 1.8 (n = 5) ng/mg protein/h in aged rats (Fig. 1), and there was no difference between them ( P > 0.05). Next, the rate of c A M P - d e p e n d e n t d o p a m i n e biosynthesis was determined. The addition of the c A M P analogue d i b u t y r y l - c A M P ( d b - c A M P ) at a concentration of 5 m M into the m e d i u m containing NSD 1015 caused a 3.6-fold increase in the rate of D O P A accumulation in the median eminence of young rats. d b - c A M P - i n d u c e d D O P A accumulation in aged rats did not differ from that in young rats (P > 0.05). F u r t h e r m o r e , an increase in endogenous c A M P achieved by the addition of isobutylmethylxanthine ( I B M X ) , a phosphodiesterase inhibitor, at a concentration of 1 m M caused a similar increase in
u.J
F-"I YOUNG RATS
so
m
AGED RATS
mr Z
o
T 40 .~
~.~
~ 2op ~,0 O r-',, o
BASAL
db-cANIP
IBMX
Fig. 1. Basal and cAMP-dependent DOPA accumulation in the median eminence of young and aged rats. Hypothalamic slices of young and aged rats were preincubated at 37 °C for 40-60 min in the standard medium and then incubated for 60 min in the medium containing 10 mM NSD 1015. As for measurement of the rate of basal DOPA accumulation, hypothalamic slices were incubated in medium containing NSD 1015 with no additives. As for measurement of the rate of cAMP-dependent DOPA accumulation, either db-cAMP or IBMX was added to the medium containing NSD 1015 to obtain a concentration of 5 or 1 mM, respectively. After the end of incubation of slices in medium containing NSD 1015, the median eminence was dissected out from the slices and homogenized in 100 ktl of 0.1 N perchloric acid containing 3 mM EDTA and 5 mM sodium metabisulfite. The supernatant of the homogenate obtained after centrifugation of 10,000 g for 2 min was stored at -80 °C for DOPA determination. The values are expressed as ng DOPA accumulated/rag protein/h. Each column and its vertical line represent mean and S.E.M., based on 5 median eminences.
D O P A accumulation in the median eminence of young and aged rats (P > 0.05). Finally, Ca2+-de pendent d o p a m i n e biosynthesis was d e t e r m i n e d by causing depolarization in m e m b r a n e potentials of hypothalamic slices. A recent study has shown that depolarization achieved by high K ÷ in medium or veratridine elicits an increase in the rate of D O P A accumulation in the median eminence, which requires extracellular Ca 2+ and is blocked by Ca2+-channel blockers 3. Depolarization of slices by 50 m M K ÷ in m e d i u m m a r k e d l y increased the rate of D O P A accumulation in the median eminence of young rats (Fig. 2). The rate of D O P A accumulation induced by depolarization in aged rats was 74% of that in young rats, and this decrease in the rate of D O P A accumulation was statistically significant ( P < 0.01, one-way analysis of variance followed by D u n c a n ' s multiplerange test). The result that the rate of Ca2+-dependent, depolarization-induced in vitro D O P A accumulation in
393
I"-"] t.l.I
5 mM K* 50mMK +
~00 re'w*
K
75
o
50
least in part, for the reduced dopamine biosynthesis in T I D A n e u r o n s of aged rats as observed in vivo. This view is compatible with the result that the secretion of dopamine into portal blood was reduced in aged rats 11,16.19, since dopamine secretion in the median eminence is also Ca2+-dependent 1,9. Therefore, it is of interest to speculate that responses to Ca z+ stimulus of the regulatory mechanisms for T I D A neuronal functions, such as the synthesis and secretion of dopamine, are altered by aging.
oQ 0 YOUNG
AGED
Fig. 2. Depolarization-induced DOPA accumulation in the median eminence of young and aged rats. Hypothalamic slices were incubated either for 60 min in medium containing 10 mM NSD 1015 and 5 mM KCI or for 20 min in medium containing 10 mM NSD 1015 and 50 mM KCI substituted for equimolar amounts of NaCI. See Fig. 1 (legend) for further detailed procedures. Each column and its vertical line represent mean and S.E.M., based on 5-6 median eminences.
At present, the cause for the reduced Ca2+-de pendent dopamine biosynthesis in T I D A neurons is unknown. It has recently been reported that shortterm hyperprolactinemia (e.g. for several days) stimulates T I D A n e u r o n a l functions while prolonged hyperprolactinemia (e.g. for several weeks or months) suppresses them18, z0. It is likely that long-term exposure of T I D A neurons to PRL in the process of aging reduces Ca2÷-dependent dopamine biosynthesis.
the median eminence was reduced in aged rats is suggestive of the view that the reduction in Ca2+-de pendent dopamine biosynthesis is responsible, at
search project on aging and age-related diseases (Yokohama City).
1 Annunziato, L., di Renzo, G., Amoroso, S. and Quattrone, A., Release of endogeneous dopamine from tuberoinfundibular neurons, Life Sci., 35 (1984) 399-407. 2 Arita, J. and Kimura, F., Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices, Neuroendocrinology, 39 (1984) 524-529. 3 Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvement of voltage-dependent Ca2+-channels, Brain Research, 347 (1985) 299-305. 4 Arita, J. and Kimura, F., Adenosine 3',5'-cyclic monophosphate stimulates dopamine biosynthesis in the median eminence of rat hypothalamic slices, Brain Research, in press. 5 Ben-Jonathan, N., Oliver, C., Weiner, H.J., Mical, R.S. and Porter, J.C., Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy, Endocrinology, 100 (1977) 452-458. 6 Demarest, K.T., Moore, K.E. and Riegle, G.D., Dopaminergic neuronal function, anterior pituitary dopamine content, and serum concentrations of prolactin, luteinizing hormone and progesterone in the aged female rat, Brain Research, 247 (1982) 347-354. 7 Demarest, K.T., Riegle, G.D. and Moore, K.E., Characteristics of dopaminergic neurons in the aged male rat, Neuroendocrinology, 31 (1980)222-227. 8 Estes, K.S. and Simpkins, J.W., Age-related alterations in dopamine and norepinephrine activity within microdis-
sected brain regions of ovariectomized Long-Evans rats, Brain Research, 298 (1984) 209-218, 9 Foreman, M.M. and Porter, J.C., Prolactin augmentation of dopamine and norepinephrine release from superfused medial basal hypothalamic fragments, Endocrinology, 108 (1981) 800-804. 10 Fuxe, K., Cellular localization of monoamines in the median eminence and in the infundibular stem of some mammals, Acta Physiol. Scand., 58 (1963) 383-384. 11 Gudelsky, G.A., Nansel, D.D. and Porter, J.C., Dopaminergic control of prolactin secretion in the aging male rat, Brain Research, 204 (1981) 446-450. 12 Huang, H.H., Marshall, S. and Meites, J., Capacity of old versus young female rats to secrete LH, FSH and prolactin, Biol. Reprod., 14 (1976) 538-543. 13 MacLeod, R.M., Fontham, E.H. and Lehmeyer, J.E., Prolactin and growth hormone production as influenced by catecholamines and agents that affect brain catecholamines, Neuroendocrinology, 6 (1970) 283-294. 14 Meites, J., Huang, H.H. and Simpkins, J.W., Recent studies on neuroendocrine control of reproductive senescence in rats. In E.L. Schneider (Ed.), The Aging Reproductive System, Raven Press, New York, 1978, pp. 213-235. 15 Reymond, M.J., Arita, J., Dudley, C.A., Moss, R.L. and Porter, J.C., Dopaminergic neurons in the mediobasal hypothalamus of old rats: evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor, Brain Research, 304 (1984) 215-223. 16 Reymond, M.J. and Porter, J.C., Secretion of hypothalam-
This study was supported partly by the special re-
394 ic dopamine into pituitary stalk blood of aged female rats, Brain Res. Bull., 7 (1981) 69-73. 17 Sarkar, D.K., Gottschall, P.E. and Meites, J., Damage to hypothalamic dopaminergic neurons is associated with development of prolactin-secreting pituitary tumors, Science, 218 (1982) 684-686. 18 Sarkar, D.K., Gottschall, P.E. and Meites, J., Decline of tuberoinfundibular dopaminergic function resulting from chronic hyperprolactinemia in rats, Endocrinology, 115
(1984) 1269-1274. 19 Sarkar, D.K., Oottschall, P.E., Xie, O.-W. and Meites, J., Reduced tuberoinfundibular dopaminergic neuronal function in rats with in situ prolactin-secreting pituitary tumors. Neuroendocrinology, 38 (1984) 498-503. 20 Simpkins, J.W. and Gabriel, S.M., Chronic hyperprolactinemia causes progressive changes in hypothalamic dopaminergic and noradrenergic neurons, Brain Research, 309 (1984) 277-282.