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Effects of gonadectomy and thyroidectomy on tyrosine hydroxylase in discrete areas of the rat median eminence
396
Brain Research, 179 (1979) 396-400 (~) Elsevier/North-Holland Biomedical Press
Effects of gonadectomy and thyroidectomy on tyrosine hydroxylase in discrete areas of the rat median eminence
TATSUO NAKAHARA, HIDEYUKI UCHIMURA, MAKOTO HIRANO, MASASHI SAITO, JANG SO0 KIM and TAKASHI MATSUMOTO
Department of Chemistry, Faculty of Science, Kyushu University, ltakozaki, Fukuoka, 812 and Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga, 842-01 (Japan) (Accepted August 30th, 1979)
Key words: median eminence - - tyrosine hydroxylase - - thyroidectomy - - gonadectomy
Catecholamine (CA) neurons terminating in the median eminence (ME) are involved in the regulation of anterior pituitary. Fluorescence histochemical studies on the distribution of CA nerve terminals within the ME have shown that the subependyreal layer (SEL) contains mainly noradrenaline (NA) nerve terminals and the lateral palisade zone (LPZ) contains mainly dopamine (DA) nerve terminals, whereas the medial palisade zone (MPZ) receives both NA and DA nerve terminalsl,4,1L Moreover, it has been shown that the rates of CA turnover in these discrete areas of the ME respond differently to changes in the endocrine state. Thus, significant changes in CA turnover have been found in the SEL and LPZ during pregnancy13 or during estrous cycle 1°, in the SEL and LPZ ~1 or in the LPZ ~7 following treatment with estrogens, and in the LPZ and MPZ following treatment with prolactin ~7. Biochemical studies have indicated that the activity of tyrosine hydroxylase (TH), the rate-limiting step of CA synthesis, in the ME was changed by several endocrine manipulationsS,6,s, 15. However, it has been unclear as to which discrete areas of the ME include the endocrine-responsive TH. Recently, we subdivided the rat ME using the microdissection technique with freeze-dried sections and obtained reproducible values for TH activity in the internal and external areas of the ME ~6. In the present study, the rat ME was subdivided into 3 areas, the SEL, MPZ and LPZ, and TH activity in each discrete area was measured after gonadectomy or thyroidectomy to provide further information for the localization of endocrine-responsive CA nerve terminals within the ME. Male, Wistar-King rats, 3 months of age (230-300 g), were housed 5 per cage and maintained under controlled conditions (22 :k 2 °C, light on 06.00-18.00 h). Castration, thyroidectomy or sham-operations were performed under Nembutal anesthesia. The animals were kept under the same conditions as before operations and killed by decapitation at 14.00 h 21 days after operations. The brain was frozen in
397
/" ......(, LPZ
J \ SEL
•
M,PZ
"'~
~
, LPZ
I I
Fig. 1. Schematic drawing of the dissected areas of the rat median eminence. Abbreviations: SEL, subependimal layer; MPZ, medial palisade zone; LPZ, lateral palisade zone.
liquid nitrogen and cut into serial frontal sections of 100 #m thickness in a cryostat at --15 °C. The sections were freeze-dried overnight at --30 °C and 10-3 mm Hg, and stored in evacuated tubes at --20 °C until used. The ME and its discrete areas were dissected freehand with a fine microknife under a stereomicroscope ( × 40). The major part of the ME was removed from the sections between A 4700 #m and A 3900 #m planes with the guide of the rat brain atlas of KSnig and Klippela. The ME was subdivided into the SEL, MPZ and LPZ according to the definition of LSfstr6m et a1.12,t3 (Fig. 1). Each sample was weighed by an electronic microbalance (Type 4125, Sartorius) with a digital voltmeter (Type EO-12, Eto). The sensitivity of the balance is 0.1 pg. TH activity in the freeze-dried tissue was assayed by a modification of the method of Nagatsu et al. la. The incubation mixture contained 0.2 M sodium phosphate buffer (pH 6.0), 1 mM FeSOa, 90 U/#I catalase, 4 mM 6MPH4 (2-amino-4hydroxy-6-methyl-tetrahydropteridine), 0.1 M 2-mercaptoethanol, 0.05 ~o Triton X100, 200/~M L-[14C(U)]tyrosine (spec. act. 139 mCi/mmol) purified by passing through alumina columns at pH 8.6. Three/~1 of the incubation mixture was added to the freeze-dried tissue in a microtube. After preincubation for 30 rain at 0 °C, the assay mixture was incubated for 15 min at 30 °C. The reaction was terminated by adding 100 #1 of a solution composed of 2.5 #M L-DOPA, 0.1 mM L-tyrosine, 35 mM EDTA, 25 mM NaHSO3, 50 mM KH2PO4, and NaOH to give a final pH 8.9. The diluted samples were kept for 30 min in ice-water bath. The procedure gave the same TH activity as that determined by the method using perchloric acid for termination of the reaction. [laC]DOPA was isolated by a modification of the method of Coyle2. The sample (103 #1) was transferred to an alumina column (Pasteur pipette, 0.7 x 15 cm, packed with 50 mg of alumina), and the column was then washed 4 times with 1.5 ml of water. The [14C]DOPA was eluted with 1.5 ml of 0.2 N acetic acid and the elute was counted in 10 ml of Triton phosphor. The recovery of [3H]DOPA was 59-63 ~o and blank values were about 250 cpm. In the preliminary measurements of TH activity in freeze-dried samples, the tissue pellets (5-15/zg) dissected from the similar limited region of caudate nucleus in 2-3 serial sections of 100 #m thickness were used. The reaction was linear for 20 min. The TH activities at the higher concentrations of tyrosine (up to 400/~M) or 6MPHa (up to 8 mM) were the same as the values under the standard assay conditions. The results are shown in Table I. Our methods were sensitive to permit
398 TABLE I
Effects of castration and thyroidectomy on tissue weight and tyrosine hydroxylase activity in the variou.~ areas of the rat median eminence
The results are expressed as values of mean 4- S.E.M. (number of animals). The abbreviations are as used in Fig. 1. Sham castration
Castration
Sham thyroidectomy
Thyroidectomy
7.2 zc 0.7 (5) 3.7 4- 0.4 (4) 7.2 ~ 0.4 (5)
7.6 ~ 0.5 (4) 3.7 4- 0.2 (4) 7.9 ± 0.3 (4)
Tissue weight (l~g dry wt./animal)
SEL MPZ LPZ
7.2 ~ 0.3 (5) 3.8 ~+ 0.4 (5) 7.0 4- 0.6 (5)
8.2:5 0.9 (5) 4.2 4- 0.1 (4) 7.9 4- 0.4 (5)
Tyrosine hydroxylase activity (nmol DOPA/h/mg dry wt.) SEL MPZ LPZ
4.6 4- 0.5 (5) 6.9 4- 0.3 (5) 7.5 4- 0.4 (5)
4.6 4- 0.3 (5) 7.7 4- 0.5 (4) 7.6 4- 0.4 (5)
5.3 4- 0.6 (5) 7.7 4- 0.6 (4) 8.4 ~ 0.7 (5)
6.6 ~ 0.5 (4) 10.5 ~ 0.6 (4)* 9.7 4- 1.1 (4)
* P < 0.05 (Student's t-test). measurement of T H activity in discrete areas of the ME dissected from a single animal. The experimental values of T H activity were 5-8-fold higher than the blank values. The weight of the ME was not significantly changed after castration or thyroidectomy. However, a small increase in weight was found in all the discrete areas of the ME after castration, which may be related to configurational changes in perivascular space during high neurosecretory states is. In the sham-operated rats, the M P Z and LPZ contained higher T H activity than the SEL. There was no significant difference in the activity between the M P Z and LPZ, although the M P Z contained slightly less activity than the LPZ. These results are similar to the distribution of T H reported in the bovine ME 7. On the other hand, quantitative microfluorimetric studies showed that the highest CA fluorescence intensity was recorded in the LPZ, the lowest in the SEL and the intermediate in the M p z 12. N o significant change in T H activity was found in all the discrete areas of the ME 21 days after castration. Previously, we observed a small but significant decrease in T H activity in the whole ME 8 days after castration is. This discrepancy may be due to the difference in post-castrational intervals. In contrast to our results, the increased T H activity has been found in the ME 7, 9 or 10 days after castration (see refs. 6, 8 and 5 respectively). A significant increase in T H activity (36 ~ of the control value) was found in the M P Z 21 days after thyroidectomy. The increased activity was also observed in the SEL and LPZ but the increase was not significant. The total T H activity in 3 discrete areas, the summation of specific enzyme activity multiplied by weight, was increased by 30 of the control value. This result is consistent with those reported in the whole MES, 6,s,15. Immunohistochemical studies ~ showed that the density of T R H (thyro-
399 t r o p i n - r e l e a s i n g h o r m o n e ) - p o s i t i v e fiber w i t h i n the M E was highest in the m e d i a l p a r t o f the external layer, a n d decreased t o w a r d s the lateral parts. Therefore, the increased T H activity f o u n d in the M P Z after t h y r o i d e c t o m y p r o v i d e s f u r t h e r a n a t o m i c a l evidence for the p a r t i c i p a t i o n o f C A nerve t e r m i n a l s in the M E to T R H secretion. R e g a r d i n g the T H within the M E , K i z e r et al. 6 r e p o r t e d t h a t b i l a t e r a l v e n t r a l N A b u n d l e lesions i n d u c e d a t o t a l d e p l e t i o n o f N A a n d d o p a m i n e - f l - h y d r o x y l a s e f r o m the M E w i t h o u t a n y changes in the T H activity. I f the m a j o r p a r t o f T H in the M P Z is c o n t a i n e d in D A nerve terminals, the increased T H activity f o u n d in the M P Z after t h y r o i d e c t o m y suggests a s t i m u l a t o r y role o f D A n e u r o n s in the r e g u l a t i o n o f T R H secretion at the level o f the M E . R e c e n t study i n d i c a t e d t h a t t h y r o i d e c t o m y increased the rate o f D A t u r n o v e r in the M E b u t d i d n o t c h a n g e the rate o f N A t u r n o v e r 5.
1 Bj6rklund, A., Moore, R. Y., Nobin, A. and Stenevi, U., The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain, Brain Research, 51 (1973) 171-191. 2 Coyle, J. T., Tyrosine hydroxylase in rat brain - - cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 3 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Europ. J. Pharmacol., 34 (1975) 389-392. 4 Jonsson, G., Fuxe, K. and H6kfelt, T., On the catecholamine innervation of the hypothalamus, with special reference to the median eminence, Brain Research, 40 (1972) 271-281. 5 Kizer, J. S., Humm, J., Nicholson, G., Greeley, G. and Youngblood, W., The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic properties of tyrosine hydroxylase in discrete hypothalamic nuclei of the male rat, Brain Research, 146 (1978) 95-107. 6 Kizer, J. S., Muth, E. and .]acobowitz, D. M., The effect of bilateral lesions of the ventral noradrenergic bundle on endocrine-induced changes of tyrosine hydroxylase in the rat median eminence, Endocrinology, 98 (1976) 886--893. 7 Kizer, J. S., Palkovits, M., Tappaz, M., Kebabian, J. and Brownstein, M. J., Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence, Endocrinology, 98 (1976) 685-695. 8 Kizer, J. S., Palkovits, M., Zivin, J., Brownstein, M., Saavedra, J. M. and Kopin, I. J., The effect of endocrinological manipulations on tyrosine hydroxylase and dopamine-fl-hydroxylaseactivities in individual hypothalamic nuclei of the adult rat, Endocrinology, 95 (1974) 799-812. 9 KSnig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain andLower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 10 L6fstr6m, A., Catecholamine turnover alterations in discrete areas of the median eminence of the 4- and 5-day cyclic rat, Brain Research, 120 (1977) 113-131. 11 L6fstr6m, A., Eneroth, P., Gustafsson, J.-A. and Skett, P., Effects of estradiol benzoate on catecholamine levels and turnover in discrete areas of the median eminence and limbic forebrain, and on serum luteinizing hormone, follicle stimulating hormone and prolactin concentrations in the ovariectomized female rat, Endocrinology, 101 (1977) 1559-1569. 12 L6fstr6m, A., Jonsson, G. and Fuxe, K., Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. I. Aspects of the distribution of dopamine and noradrenaline nerve terminals, J. Histochem. Cytochem., 24 (1976) 415--429. 13 Lffstr6m, A., Jonsson, G., Wiesel, F.-A. and Fuxe, K., Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states, J. Histochem. Cytochem., 24 (1976) 430-442. 14 Nagatsu, T., Sudo, Y. and Nagatsu, I., Tyrosine hydroxylase in bovine caudate nucleus, J. Neurochem., 18 (1971) 2179-2189. 15 Nakahara, T., Uchimura, H., Hirano, M., Saito, M. and Ito, M., Effects of gonadectomy and thyroidectomy on the tyrosine hydroxylase activity in individual hypothalamic nuclei and lower brain stem catecholaminergic cell groups of the rat, Brain Research, 117 (1976) 351-356.
400 16 Saito, M., Hirano, M., Uchimura, l-L, Nakahara, T. and Ito, M., Tyrosine hydroxylase activity in the catecholamine nerve terminals and cell bodies of the rat brain, J. Neurochem., 29 (1977) 161-165. 17 Wiesel, F.-A., Fuxe, K., H6ckfelt, T. and Agnati, L. F., Studies on dopamine turnover in ovariectomized or hypophysectomized female rats. Effects of 17fl-estradiol benzoate, ethynodioldiacetate and ovine prolactin, Brain Research, 148 (1978) 399-4l 1.
Brain Research, 179 (1979) 396-400 (~) Elsevier/North-Holland Biomedical Press
Effects of gonadectomy and thyroidectomy on tyrosine hydroxylase in discrete areas of the rat median eminence
TATSUO NAKAHARA, HIDEYUKI UCHIMURA, MAKOTO HIRANO, MASASHI SAITO, JANG SO0 KIM and TAKASHI MATSUMOTO
Department of Chemistry, Faculty of Science, Kyushu University, ltakozaki, Fukuoka, 812 and Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga, 842-01 (Japan) (Accepted August 30th, 1979)
Key words: median eminence - - tyrosine hydroxylase - - thyroidectomy - - gonadectomy
Catecholamine (CA) neurons terminating in the median eminence (ME) are involved in the regulation of anterior pituitary. Fluorescence histochemical studies on the distribution of CA nerve terminals within the ME have shown that the subependyreal layer (SEL) contains mainly noradrenaline (NA) nerve terminals and the lateral palisade zone (LPZ) contains mainly dopamine (DA) nerve terminals, whereas the medial palisade zone (MPZ) receives both NA and DA nerve terminalsl,4,1L Moreover, it has been shown that the rates of CA turnover in these discrete areas of the ME respond differently to changes in the endocrine state. Thus, significant changes in CA turnover have been found in the SEL and LPZ during pregnancy13 or during estrous cycle 1°, in the SEL and LPZ ~1 or in the LPZ ~7 following treatment with estrogens, and in the LPZ and MPZ following treatment with prolactin ~7. Biochemical studies have indicated that the activity of tyrosine hydroxylase (TH), the rate-limiting step of CA synthesis, in the ME was changed by several endocrine manipulationsS,6,s, 15. However, it has been unclear as to which discrete areas of the ME include the endocrine-responsive TH. Recently, we subdivided the rat ME using the microdissection technique with freeze-dried sections and obtained reproducible values for TH activity in the internal and external areas of the ME ~6. In the present study, the rat ME was subdivided into 3 areas, the SEL, MPZ and LPZ, and TH activity in each discrete area was measured after gonadectomy or thyroidectomy to provide further information for the localization of endocrine-responsive CA nerve terminals within the ME. Male, Wistar-King rats, 3 months of age (230-300 g), were housed 5 per cage and maintained under controlled conditions (22 :k 2 °C, light on 06.00-18.00 h). Castration, thyroidectomy or sham-operations were performed under Nembutal anesthesia. The animals were kept under the same conditions as before operations and killed by decapitation at 14.00 h 21 days after operations. The brain was frozen in
397
/" ......(, LPZ
J \ SEL
•
M,PZ
"'~
~
, LPZ
I I
Fig. 1. Schematic drawing of the dissected areas of the rat median eminence. Abbreviations: SEL, subependimal layer; MPZ, medial palisade zone; LPZ, lateral palisade zone.
liquid nitrogen and cut into serial frontal sections of 100 #m thickness in a cryostat at --15 °C. The sections were freeze-dried overnight at --30 °C and 10-3 mm Hg, and stored in evacuated tubes at --20 °C until used. The ME and its discrete areas were dissected freehand with a fine microknife under a stereomicroscope ( × 40). The major part of the ME was removed from the sections between A 4700 #m and A 3900 #m planes with the guide of the rat brain atlas of KSnig and Klippela. The ME was subdivided into the SEL, MPZ and LPZ according to the definition of LSfstr6m et a1.12,t3 (Fig. 1). Each sample was weighed by an electronic microbalance (Type 4125, Sartorius) with a digital voltmeter (Type EO-12, Eto). The sensitivity of the balance is 0.1 pg. TH activity in the freeze-dried tissue was assayed by a modification of the method of Nagatsu et al. la. The incubation mixture contained 0.2 M sodium phosphate buffer (pH 6.0), 1 mM FeSOa, 90 U/#I catalase, 4 mM 6MPH4 (2-amino-4hydroxy-6-methyl-tetrahydropteridine), 0.1 M 2-mercaptoethanol, 0.05 ~o Triton X100, 200/~M L-[14C(U)]tyrosine (spec. act. 139 mCi/mmol) purified by passing through alumina columns at pH 8.6. Three/~1 of the incubation mixture was added to the freeze-dried tissue in a microtube. After preincubation for 30 rain at 0 °C, the assay mixture was incubated for 15 min at 30 °C. The reaction was terminated by adding 100 #1 of a solution composed of 2.5 #M L-DOPA, 0.1 mM L-tyrosine, 35 mM EDTA, 25 mM NaHSO3, 50 mM KH2PO4, and NaOH to give a final pH 8.9. The diluted samples were kept for 30 min in ice-water bath. The procedure gave the same TH activity as that determined by the method using perchloric acid for termination of the reaction. [laC]DOPA was isolated by a modification of the method of Coyle2. The sample (103 #1) was transferred to an alumina column (Pasteur pipette, 0.7 x 15 cm, packed with 50 mg of alumina), and the column was then washed 4 times with 1.5 ml of water. The [14C]DOPA was eluted with 1.5 ml of 0.2 N acetic acid and the elute was counted in 10 ml of Triton phosphor. The recovery of [3H]DOPA was 59-63 ~o and blank values were about 250 cpm. In the preliminary measurements of TH activity in freeze-dried samples, the tissue pellets (5-15/zg) dissected from the similar limited region of caudate nucleus in 2-3 serial sections of 100 #m thickness were used. The reaction was linear for 20 min. The TH activities at the higher concentrations of tyrosine (up to 400/~M) or 6MPHa (up to 8 mM) were the same as the values under the standard assay conditions. The results are shown in Table I. Our methods were sensitive to permit
398 TABLE I
Effects of castration and thyroidectomy on tissue weight and tyrosine hydroxylase activity in the variou.~ areas of the rat median eminence
The results are expressed as values of mean 4- S.E.M. (number of animals). The abbreviations are as used in Fig. 1. Sham castration
Castration
Sham thyroidectomy
Thyroidectomy
7.2 zc 0.7 (5) 3.7 4- 0.4 (4) 7.2 ~ 0.4 (5)
7.6 ~ 0.5 (4) 3.7 4- 0.2 (4) 7.9 ± 0.3 (4)
Tissue weight (l~g dry wt./animal)
SEL MPZ LPZ
7.2 ~ 0.3 (5) 3.8 ~+ 0.4 (5) 7.0 4- 0.6 (5)
8.2:5 0.9 (5) 4.2 4- 0.1 (4) 7.9 4- 0.4 (5)
Tyrosine hydroxylase activity (nmol DOPA/h/mg dry wt.) SEL MPZ LPZ
4.6 4- 0.5 (5) 6.9 4- 0.3 (5) 7.5 4- 0.4 (5)
4.6 4- 0.3 (5) 7.7 4- 0.5 (4) 7.6 4- 0.4 (5)
5.3 4- 0.6 (5) 7.7 4- 0.6 (4) 8.4 ~ 0.7 (5)
6.6 ~ 0.5 (4) 10.5 ~ 0.6 (4)* 9.7 4- 1.1 (4)
* P < 0.05 (Student's t-test). measurement of T H activity in discrete areas of the ME dissected from a single animal. The experimental values of T H activity were 5-8-fold higher than the blank values. The weight of the ME was not significantly changed after castration or thyroidectomy. However, a small increase in weight was found in all the discrete areas of the ME after castration, which may be related to configurational changes in perivascular space during high neurosecretory states is. In the sham-operated rats, the M P Z and LPZ contained higher T H activity than the SEL. There was no significant difference in the activity between the M P Z and LPZ, although the M P Z contained slightly less activity than the LPZ. These results are similar to the distribution of T H reported in the bovine ME 7. On the other hand, quantitative microfluorimetric studies showed that the highest CA fluorescence intensity was recorded in the LPZ, the lowest in the SEL and the intermediate in the M p z 12. N o significant change in T H activity was found in all the discrete areas of the ME 21 days after castration. Previously, we observed a small but significant decrease in T H activity in the whole ME 8 days after castration is. This discrepancy may be due to the difference in post-castrational intervals. In contrast to our results, the increased T H activity has been found in the ME 7, 9 or 10 days after castration (see refs. 6, 8 and 5 respectively). A significant increase in T H activity (36 ~ of the control value) was found in the M P Z 21 days after thyroidectomy. The increased activity was also observed in the SEL and LPZ but the increase was not significant. The total T H activity in 3 discrete areas, the summation of specific enzyme activity multiplied by weight, was increased by 30 of the control value. This result is consistent with those reported in the whole MES, 6,s,15. Immunohistochemical studies ~ showed that the density of T R H (thyro-
399 t r o p i n - r e l e a s i n g h o r m o n e ) - p o s i t i v e fiber w i t h i n the M E was highest in the m e d i a l p a r t o f the external layer, a n d decreased t o w a r d s the lateral parts. Therefore, the increased T H activity f o u n d in the M P Z after t h y r o i d e c t o m y p r o v i d e s f u r t h e r a n a t o m i c a l evidence for the p a r t i c i p a t i o n o f C A nerve t e r m i n a l s in the M E to T R H secretion. R e g a r d i n g the T H within the M E , K i z e r et al. 6 r e p o r t e d t h a t b i l a t e r a l v e n t r a l N A b u n d l e lesions i n d u c e d a t o t a l d e p l e t i o n o f N A a n d d o p a m i n e - f l - h y d r o x y l a s e f r o m the M E w i t h o u t a n y changes in the T H activity. I f the m a j o r p a r t o f T H in the M P Z is c o n t a i n e d in D A nerve terminals, the increased T H activity f o u n d in the M P Z after t h y r o i d e c t o m y suggests a s t i m u l a t o r y role o f D A n e u r o n s in the r e g u l a t i o n o f T R H secretion at the level o f the M E . R e c e n t study i n d i c a t e d t h a t t h y r o i d e c t o m y increased the rate o f D A t u r n o v e r in the M E b u t d i d n o t c h a n g e the rate o f N A t u r n o v e r 5.
1 Bj6rklund, A., Moore, R. Y., Nobin, A. and Stenevi, U., The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain, Brain Research, 51 (1973) 171-191. 2 Coyle, J. T., Tyrosine hydroxylase in rat brain - - cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 3 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Europ. J. Pharmacol., 34 (1975) 389-392. 4 Jonsson, G., Fuxe, K. and H6kfelt, T., On the catecholamine innervation of the hypothalamus, with special reference to the median eminence, Brain Research, 40 (1972) 271-281. 5 Kizer, J. S., Humm, J., Nicholson, G., Greeley, G. and Youngblood, W., The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic properties of tyrosine hydroxylase in discrete hypothalamic nuclei of the male rat, Brain Research, 146 (1978) 95-107. 6 Kizer, J. S., Muth, E. and .]acobowitz, D. M., The effect of bilateral lesions of the ventral noradrenergic bundle on endocrine-induced changes of tyrosine hydroxylase in the rat median eminence, Endocrinology, 98 (1976) 886--893. 7 Kizer, J. S., Palkovits, M., Tappaz, M., Kebabian, J. and Brownstein, M. J., Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence, Endocrinology, 98 (1976) 685-695. 8 Kizer, J. S., Palkovits, M., Zivin, J., Brownstein, M., Saavedra, J. M. and Kopin, I. J., The effect of endocrinological manipulations on tyrosine hydroxylase and dopamine-fl-hydroxylaseactivities in individual hypothalamic nuclei of the adult rat, Endocrinology, 95 (1974) 799-812. 9 KSnig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain andLower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 10 L6fstr6m, A., Catecholamine turnover alterations in discrete areas of the median eminence of the 4- and 5-day cyclic rat, Brain Research, 120 (1977) 113-131. 11 L6fstr6m, A., Eneroth, P., Gustafsson, J.-A. and Skett, P., Effects of estradiol benzoate on catecholamine levels and turnover in discrete areas of the median eminence and limbic forebrain, and on serum luteinizing hormone, follicle stimulating hormone and prolactin concentrations in the ovariectomized female rat, Endocrinology, 101 (1977) 1559-1569. 12 L6fstr6m, A., Jonsson, G. and Fuxe, K., Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. I. Aspects of the distribution of dopamine and noradrenaline nerve terminals, J. Histochem. Cytochem., 24 (1976) 415--429. 13 Lffstr6m, A., Jonsson, G., Wiesel, F.-A. and Fuxe, K., Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states, J. Histochem. Cytochem., 24 (1976) 430-442. 14 Nagatsu, T., Sudo, Y. and Nagatsu, I., Tyrosine hydroxylase in bovine caudate nucleus, J. Neurochem., 18 (1971) 2179-2189. 15 Nakahara, T., Uchimura, H., Hirano, M., Saito, M. and Ito, M., Effects of gonadectomy and thyroidectomy on the tyrosine hydroxylase activity in individual hypothalamic nuclei and lower brain stem catecholaminergic cell groups of the rat, Brain Research, 117 (1976) 351-356.
400 16 Saito, M., Hirano, M., Uchimura, l-L, Nakahara, T. and Ito, M., Tyrosine hydroxylase activity in the catecholamine nerve terminals and cell bodies of the rat brain, J. Neurochem., 29 (1977) 161-165. 17 Wiesel, F.-A., Fuxe, K., H6ckfelt, T. and Agnati, L. F., Studies on dopamine turnover in ovariectomized or hypophysectomized female rats. Effects of 17fl-estradiol benzoate, ethynodioldiacetate and ovine prolactin, Brain Research, 148 (1978) 399-4l 1.