Cell Tiss. Res. 163, 433-443 (1975) - 9 by Springer-Verlag 1975
Tanycyte Absorption Affected by the Hypothalamic Deafferentation in Japanese Quail, Coturnix coturnix japonica * Masumi Nozaki ** Misaki Marine Biological Station, University of Tokyo, Misaki, Kanagawa-ken, Japan
Summary. In Japanese quail, Coturnix coturnixjaponica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.
Key words: Hypothalamic deafferentation - Tanycyte absorption - Peroxidase reaction - Ovarian growth - Japanese quail (Coturnix coturnixjaponica).
Introduction In recent years, considerable evidence has been accumulated which suggests the possibility that tanycytes of the median eminence take up hormones from the ventricular fluid and transport them into the portal blood, thus, controlling adenohypophysial function. The facts supporting this idea are as follows: (1) pinocytotic vesicles are observed on the apical surface of the ependymal cells of the median eminence (Matsui and Kobayashi, 1968; Kobayashi etal., 1970; Sharp, 1972; Schechter and Weiner, 1972), (2)the fine structure of ependymal cells changes depending on different reproductive phases in the rhesus monkey (Knowles and Anand Kumar, 1969) and it also changes following castration Send offprint requests to: Masumi Nozaki, Misaki Marine Biological Station, University of Tokyo,
Miura-shi, Japan 238-02. * This work was supported by grants from the Ministry of Education, the Japan Society for Promotion of Science and the Ford Foundation. ** Acknowledgement. I wish to express my gratitude to Professor Hideshi Kobayashi for his valuable guidance and suggestion.
434
M. Nozaki
a n d estrogen a d m i n i s t r a t i o n in the rat ( K o b a y a s h i et al., 1970), (3) the tanycytes t r a n s p o r t peroxidase a n d labelled thyroxine which were injected i n t r a v e n t r i c u l a r l y from the third ventricle to capillaries of the p r i m a r y plexus (peroxidase, K o b a y a s h i et al., 1972; L b r a n t h a n d Schiebler, 1974; thyroxine, Silverman et al., 1973b). It has also been shown in vitro that the e p e n d y m a l c o m p o n e n t s of the m e d i a n e m i n e n c e actively a c c u m u l a t e a l p h a - a m i n o - i s o b u t y r i c acid a n d thyroxine (Silverm a n et al., 1973 a), (4) i n t r a v e n t r i c u l a r l y injected L R H a n d T R H reach the adenohypophysis, resulting in increases o f serum L H (Weiner et al., 1972; O n d o et al., 1973; U e m u r a et al., 1975) a n d of serum T S H ( G o r d o n et al., 1972; Knigge e t a l . , 1973), (5) n e u r o h y p o p h y s i a l h o r m o n e s (Heller, 1969), n o r a d r e n a l i n e ( D e n c k e r a n d Hfiggendal, 1969), T R H (Knigge a n d Joseph, 1974) a n d L R H ( C r a m e r a n d Barraclough, 1975 ; U e m u r a et al., 1975) are present in the ventricular fluid. O n the other h a n d , it has been d e m o n s t r a t e d electron microscopically that possible m o n o a m i n e r g i c n e u r o n s form s y n a p t o i d contacts with the tanycytes of the m e d i a n e m i n e n c e (Matsui, 1966a, b ; K o b a y a s h i et al., 1970; Gfildner, 1973). It seems that tanycyte a b s o r p t i o n is regulated by m o n o a m i n e r g i c n e u r o n s . In favor o f this hypothesis, in m a m m a l s a n d birds, i n n e r v a t i o n of the m e d i a n e m i n e n c e by c a t e c h o l a m i n e nerve fibers has been d e m o n s t r a t e d by m e a n s of fluorescence m i c r o s c o p y (Sharp a n d Follett, 1970; B j 6 r k l u n d et al., 1973; Soest et al., 1973), a l t h o u g h origins of these c a t e c h o l a m i n e fibers have n o t yet been clearly shown. C o n s i d e r i n g the o b s e r v a t i o n s m e n t i o n e d above, the a u t h o r has a t t e m p t e d to study whether the t a n y c y t e absorptive f u n c t i o n is affected by t r a n s e c t i o n of the c a t e c h o l a m i n e fibers. F o r this purpose, h y p o t h a l a m i c deafferentation was first attempted. A t the same time some e n d o c r i n e organs were observed after deafferentation.
Material and Methods Female Japanese quail (Coturnix coturnix japonica) at the age of three weeks were obtained from a commercial source and kept under a short daily photoperiod of 8L 16D (light from 0800 to 1600). After keeping them under short-day conditions for 3 to 7 weeks, they were divided into two groups : one group was subjected to the medial basal hypothalamic deafferentation and another underwent a sham-operation. After the operation, they were transferred to a long daily photoperiod of 16L 8D (light from 0800 to 2400). For hypothalamic deafferentation, a Hal'~sztype knife was made following the method of Wada (1974b), as shown in Fig. la. The knife assembly was attached to a stereotaxic instrument. The bird was anaesthetized with Nembutal and fixed on a stereotaxic instrument in such a position that the floor of the median eminence was nearly horizontal, according to the technique of Wada (1972). The knife was inserted into the brain through a hole (about 2 mm in diameter), which had been made with a dental drill in the frontal bone. The blade of the knife was on the median plane while being lowered. In order to place the knife at a desired position, an X-ray exposure was taken from the side of the head before the insertion, and a precise level of the knife was attained by referring to the radiograph. After the insertion, the knife was turned once 360 degrees to perform complete deafferentation of the hypothalamus. Then the knife assembly was raised slowly from the head. Histological examination on the deafferented hypothalamus has revealed that the isolated hypothalamic island includes the nucleus tuberis, nucleus hypothalamicus inferior, nucleus mamillaris medialis, a ventral part of the nucleus hypothalamicus posterior medialis, and a posterior
Tanycyte Absorption and Hypothalamic Deafferentation
435
part of the paraventricular organ (Figs. 1b and 2). Control birds were subjected to the same procedures without turning the knife. Two weeks after the operation, the birds received a single injection of peroxidase in the third ventricle under Nembutal anaesthesia. For the injection, the bird was fixed on a stereotaxic instrument and X-ray exposures were made from the side and the front. The tip of an injection needle was placed just above the median eminence by referring to the radiographs. Each animal received 0.4 to 0.6 mg horseradish peroxidase (60 U/mg Carl Roth), which was dissolved in 50 lal of physiological saline, within a period of one minute. Six minutes after the injection, the bird was killed by decapitation. The hypothalamic tissue was rapidly removed and fixed in ice-cold 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 3.5 hours. The tissue was rinsed in ice-cold 0.1 M Tris buffer (pH 7.6) for about 3 hours. Transverse sections were cut at 37.5 p.m with a freezing microtome. Special attention was paid to obtain serial sections of the tissue. Then, the sections were incubated at room temperature in a medium consisting of 50 mg of 3,'3-diaminobenzidine-tetrahydrochloride, 1 ml of 1% hydrogen peroxide and 100 ml of 0.05 M Tris buffer (pH 7.6). After incubation, the sections were rinsed in de-ionized water and dehydrated in a graded series of ethanol. The tissues were mounted on slides with Caedax (Merck) for light microscopy. Adenohypophysis, thyroid glands, adrenal glands and ovary were weighed with a torsion balance immediately after removal of the hypothalamic tissue. In the ovary the diameter of the largest follicle was also noted.
Statistical Methods After histological examination, only the birds with complete deafferentation were used for the study (Fig. 2a and b). All the tanycyte processes showing peroxidase reaction in the subependymal and fiber layers of the median eminence were counted in every section under a light microscope. It was necessary to count the processes particularly in these two layers, because they branch further into fine processes in the reticular and palisade layers. Peroxidase-positive tanycytes counted were usually distributed in about eight sections of both the anterior and posterior median eminence. The tanycyte processes located around the lateral recess of the median eminence were not counted. Counting was repeated three times in the same section and an average was calculated in each section. Numbers obtained from each section were added to give a total number in a bird. The total number was divided by numbers of sections examined in individual birds. Thus, the data are expressed in terms of number of processes per section in each bird. For comparison, the data of experimental birds and those of controls were subjected to the Student's t-test. The group means of ovarian weight and of the diameter of the largest follicle were treated by the randomization test. The weights of other organs were compared by the Student's t-test.
Results 2 7 . 3 % o f t h e d e a f f e r e n t e d b i r d s d i d n o t s u r v i v e t h e o p e r a t i o n , w h e r e a s all t h e sham-operated birds survived. One of the most conspicuous features observed in the experimental birds was their large consumption of water and subsequent p r o d u c t i o n o f d i l u t e u r i n e as c o m p a r e d t o t h e c o n t r o l s .
Effects of Medial Basal Hypothalamic Deafferentation on Tanycyte Absorption of Peroxidase Peroxidase injected intraventricularly diffused into brain parenchyma around the ventricle. Brown staining indicating peroxidase activity became weaker with a n i n c r e a s e in d i s t a n c e f r o m t h e v e n t r i c u l a r s u r f a c e ( F i g . 2 a a n d b). T h e t a n y c y t e cell b o d i e s a n d t h e i r p r o c e s s e s i n t h e m e d i a n e m i n e n c e s h o w e d s t r o n g e r p e r o x i d a s e
436
M. Nozaki
E
~D LD
E
lE la 1mm
lb
Tanycyte Absorption and Hypothalamic Deafferentation
437
Table 1. Effect of hypothalamic deafferentation on tanycyte uptake of intra-
ventricularly injected peroxidase in Japanese quail Number of birds
Number of tanycyte processes showing peroxidase reaction anterior median eminence
Control (sham) Deafferented
14 7
,
posterior median eminence
a
110_ 13 b ; ; - - -
18 5- 2
18.9_+2.5 /
40.4+4.3"
b
c
* Expressed in terms of number of processes (Mean + SE) per histological section. a P
Tanycyte Absorption Affected by the Hypothalamic Deafferentation in Japanese Quail, Coturnix coturnix japonica * Masumi Nozaki ** Misaki Marine Biological Station, University of Tokyo, Misaki, Kanagawa-ken, Japan
Summary. In Japanese quail, Coturnix coturnixjaponica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.
Key words: Hypothalamic deafferentation - Tanycyte absorption - Peroxidase reaction - Ovarian growth - Japanese quail (Coturnix coturnixjaponica).
Introduction In recent years, considerable evidence has been accumulated which suggests the possibility that tanycytes of the median eminence take up hormones from the ventricular fluid and transport them into the portal blood, thus, controlling adenohypophysial function. The facts supporting this idea are as follows: (1) pinocytotic vesicles are observed on the apical surface of the ependymal cells of the median eminence (Matsui and Kobayashi, 1968; Kobayashi etal., 1970; Sharp, 1972; Schechter and Weiner, 1972), (2)the fine structure of ependymal cells changes depending on different reproductive phases in the rhesus monkey (Knowles and Anand Kumar, 1969) and it also changes following castration Send offprint requests to: Masumi Nozaki, Misaki Marine Biological Station, University of Tokyo,
Miura-shi, Japan 238-02. * This work was supported by grants from the Ministry of Education, the Japan Society for Promotion of Science and the Ford Foundation. ** Acknowledgement. I wish to express my gratitude to Professor Hideshi Kobayashi for his valuable guidance and suggestion.
434
M. Nozaki
a n d estrogen a d m i n i s t r a t i o n in the rat ( K o b a y a s h i et al., 1970), (3) the tanycytes t r a n s p o r t peroxidase a n d labelled thyroxine which were injected i n t r a v e n t r i c u l a r l y from the third ventricle to capillaries of the p r i m a r y plexus (peroxidase, K o b a y a s h i et al., 1972; L b r a n t h a n d Schiebler, 1974; thyroxine, Silverman et al., 1973b). It has also been shown in vitro that the e p e n d y m a l c o m p o n e n t s of the m e d i a n e m i n e n c e actively a c c u m u l a t e a l p h a - a m i n o - i s o b u t y r i c acid a n d thyroxine (Silverm a n et al., 1973 a), (4) i n t r a v e n t r i c u l a r l y injected L R H a n d T R H reach the adenohypophysis, resulting in increases o f serum L H (Weiner et al., 1972; O n d o et al., 1973; U e m u r a et al., 1975) a n d of serum T S H ( G o r d o n et al., 1972; Knigge e t a l . , 1973), (5) n e u r o h y p o p h y s i a l h o r m o n e s (Heller, 1969), n o r a d r e n a l i n e ( D e n c k e r a n d Hfiggendal, 1969), T R H (Knigge a n d Joseph, 1974) a n d L R H ( C r a m e r a n d Barraclough, 1975 ; U e m u r a et al., 1975) are present in the ventricular fluid. O n the other h a n d , it has been d e m o n s t r a t e d electron microscopically that possible m o n o a m i n e r g i c n e u r o n s form s y n a p t o i d contacts with the tanycytes of the m e d i a n e m i n e n c e (Matsui, 1966a, b ; K o b a y a s h i et al., 1970; Gfildner, 1973). It seems that tanycyte a b s o r p t i o n is regulated by m o n o a m i n e r g i c n e u r o n s . In favor o f this hypothesis, in m a m m a l s a n d birds, i n n e r v a t i o n of the m e d i a n e m i n e n c e by c a t e c h o l a m i n e nerve fibers has been d e m o n s t r a t e d by m e a n s of fluorescence m i c r o s c o p y (Sharp a n d Follett, 1970; B j 6 r k l u n d et al., 1973; Soest et al., 1973), a l t h o u g h origins of these c a t e c h o l a m i n e fibers have n o t yet been clearly shown. C o n s i d e r i n g the o b s e r v a t i o n s m e n t i o n e d above, the a u t h o r has a t t e m p t e d to study whether the t a n y c y t e absorptive f u n c t i o n is affected by t r a n s e c t i o n of the c a t e c h o l a m i n e fibers. F o r this purpose, h y p o t h a l a m i c deafferentation was first attempted. A t the same time some e n d o c r i n e organs were observed after deafferentation.
Material and Methods Female Japanese quail (Coturnix coturnix japonica) at the age of three weeks were obtained from a commercial source and kept under a short daily photoperiod of 8L 16D (light from 0800 to 1600). After keeping them under short-day conditions for 3 to 7 weeks, they were divided into two groups : one group was subjected to the medial basal hypothalamic deafferentation and another underwent a sham-operation. After the operation, they were transferred to a long daily photoperiod of 16L 8D (light from 0800 to 2400). For hypothalamic deafferentation, a Hal'~sztype knife was made following the method of Wada (1974b), as shown in Fig. la. The knife assembly was attached to a stereotaxic instrument. The bird was anaesthetized with Nembutal and fixed on a stereotaxic instrument in such a position that the floor of the median eminence was nearly horizontal, according to the technique of Wada (1972). The knife was inserted into the brain through a hole (about 2 mm in diameter), which had been made with a dental drill in the frontal bone. The blade of the knife was on the median plane while being lowered. In order to place the knife at a desired position, an X-ray exposure was taken from the side of the head before the insertion, and a precise level of the knife was attained by referring to the radiograph. After the insertion, the knife was turned once 360 degrees to perform complete deafferentation of the hypothalamus. Then the knife assembly was raised slowly from the head. Histological examination on the deafferented hypothalamus has revealed that the isolated hypothalamic island includes the nucleus tuberis, nucleus hypothalamicus inferior, nucleus mamillaris medialis, a ventral part of the nucleus hypothalamicus posterior medialis, and a posterior
Tanycyte Absorption and Hypothalamic Deafferentation
435
part of the paraventricular organ (Figs. 1b and 2). Control birds were subjected to the same procedures without turning the knife. Two weeks after the operation, the birds received a single injection of peroxidase in the third ventricle under Nembutal anaesthesia. For the injection, the bird was fixed on a stereotaxic instrument and X-ray exposures were made from the side and the front. The tip of an injection needle was placed just above the median eminence by referring to the radiographs. Each animal received 0.4 to 0.6 mg horseradish peroxidase (60 U/mg Carl Roth), which was dissolved in 50 lal of physiological saline, within a period of one minute. Six minutes after the injection, the bird was killed by decapitation. The hypothalamic tissue was rapidly removed and fixed in ice-cold 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 3.5 hours. The tissue was rinsed in ice-cold 0.1 M Tris buffer (pH 7.6) for about 3 hours. Transverse sections were cut at 37.5 p.m with a freezing microtome. Special attention was paid to obtain serial sections of the tissue. Then, the sections were incubated at room temperature in a medium consisting of 50 mg of 3,'3-diaminobenzidine-tetrahydrochloride, 1 ml of 1% hydrogen peroxide and 100 ml of 0.05 M Tris buffer (pH 7.6). After incubation, the sections were rinsed in de-ionized water and dehydrated in a graded series of ethanol. The tissues were mounted on slides with Caedax (Merck) for light microscopy. Adenohypophysis, thyroid glands, adrenal glands and ovary were weighed with a torsion balance immediately after removal of the hypothalamic tissue. In the ovary the diameter of the largest follicle was also noted.
Statistical Methods After histological examination, only the birds with complete deafferentation were used for the study (Fig. 2a and b). All the tanycyte processes showing peroxidase reaction in the subependymal and fiber layers of the median eminence were counted in every section under a light microscope. It was necessary to count the processes particularly in these two layers, because they branch further into fine processes in the reticular and palisade layers. Peroxidase-positive tanycytes counted were usually distributed in about eight sections of both the anterior and posterior median eminence. The tanycyte processes located around the lateral recess of the median eminence were not counted. Counting was repeated three times in the same section and an average was calculated in each section. Numbers obtained from each section were added to give a total number in a bird. The total number was divided by numbers of sections examined in individual birds. Thus, the data are expressed in terms of number of processes per section in each bird. For comparison, the data of experimental birds and those of controls were subjected to the Student's t-test. The group means of ovarian weight and of the diameter of the largest follicle were treated by the randomization test. The weights of other organs were compared by the Student's t-test.
Results 2 7 . 3 % o f t h e d e a f f e r e n t e d b i r d s d i d n o t s u r v i v e t h e o p e r a t i o n , w h e r e a s all t h e sham-operated birds survived. One of the most conspicuous features observed in the experimental birds was their large consumption of water and subsequent p r o d u c t i o n o f d i l u t e u r i n e as c o m p a r e d t o t h e c o n t r o l s .
Effects of Medial Basal Hypothalamic Deafferentation on Tanycyte Absorption of Peroxidase Peroxidase injected intraventricularly diffused into brain parenchyma around the ventricle. Brown staining indicating peroxidase activity became weaker with a n i n c r e a s e in d i s t a n c e f r o m t h e v e n t r i c u l a r s u r f a c e ( F i g . 2 a a n d b). T h e t a n y c y t e cell b o d i e s a n d t h e i r p r o c e s s e s i n t h e m e d i a n e m i n e n c e s h o w e d s t r o n g e r p e r o x i d a s e
436
M. Nozaki
E
~D LD
E
lE la 1mm
lb
Tanycyte Absorption and Hypothalamic Deafferentation
437
Table 1. Effect of hypothalamic deafferentation on tanycyte uptake of intra-
ventricularly injected peroxidase in Japanese quail Number of birds
Number of tanycyte processes showing peroxidase reaction anterior median eminence
Control (sham) Deafferented
14 7
,
posterior median eminence
a
110_ 13 b ; ; - - -
18 5- 2
18.9_+2.5 /
40.4+4.3"
b
c
* Expressed in terms of number of processes (Mean + SE) per histological section. a P
