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1233 Estrogen affects the cytoskeleton of the cultured hypothalamic neuron
S129
1233
ESTROGEN AFFECTS THE CYTOSKELETON OF THE CULTURED HYPOTHALAMIC NEURON. KAZUNARI YURI AND MITSUHIRO KAWATA, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Estrogen plays a pivotal role in growth and differentiation of neurons in the sexually dimorphic area. In the present study, the effect of estrogen on the neurite growth was investigated by a dissociated culture of embryonic day 17 and posmatal day 1 female rat hypothalami. Phenol red free Eagle's minimum essential medium was supplemented by dextran-coated charcoal treated 10% fetal calf serum and experimental cultures were treated with estradiol-17t3 (100ng/ml). Cells were plated onto polyethylenimine-coated glass coverslips at densities ranging from lxl04 to 5x104. They were fixed at 1,3, 5 and 7days in vitro, and stained immunohistochemically using anti-MAP2 and anti-tau antisera. The length of neurites was measured by an image analysis system. It was found that the number of MAP2- and tau-immunoreactive neurons increased, and MAP2- and tau-immunoreactive neurites were elongated by es.trogen treatment. The outgrowth of neurites in specific type of neurons has been promoted, suggesting that these neurons may be sensitive to estrogen treatment.
1234
ANALYSIS
OF N E U R I T E
GROWTH
OF PC12h-R
USING F L O W S Y S T E M
TOMO0 HOMMAYASUSHI KAZUNO. KAZUAKI YOSHIOKA AND HIDEAKI MATSUOKA, Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16, Nakamaehi, Koganei, Tokyo 184, Japan. The flow type culture system was developed for analysis of neurite growth. The system could exchange the medium gently and sequentially, which reduced effects of coexisting cells on the neufite growth. In this study, N G F and db-cAMP were applied repeatedly with this system and their action on neurite growth were examined by continual observation of neurite behaviour. The multiplication medium and the differentiation medium containing NGF or db-cAMP were alternately flowed for every 1 hr. Neurite growth was evaluated by the relative number of neurite bearing cells (RN) and neurite length (L,~). In PC12h-R, R,~was changed after the supply and the removal of factors. However, RN did not show tl{e change after 5 times applications of NGF. In contrast, db-cAMP supply to the same cells indicated the neurite projection. On the other hand, in the case of db-cAMP application, PC12h-R showed the repeated response until 8 times applications. L.,4 changed with a vibrating pattern, that is, neurites elongated and retracted repeatedly. However, this pattern was not necessarily synchronized with addition and removal of factors exactly. These factors seemed to affect the initial stage of neurite growth (neurite projection) even rather than the successive stage (neurite elongation).
1235
CALCIUM CHANNEL PROPERTIES IN RAT SENSORY FIBERS DURING GROWTH IN C O L L A G E N - G E L JUN FUKUDA, KAZUKO KEINO-MASU, SHINGO TSUKADA, TAKU IWAMOTO AND KEIICHI TORIMITSU*, Department of Physiology, National Defense Medical School, Tokorozawa, Department of Physiology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, and *NTT Basic Research Laboratories, Musashino, Tokyo.
In order to examine properties of Ca channels of sensory fibers during regeneration both in normal Ca medium (2.5 raM) and in Ca-free medium (0.01 nM), we n~easured intracellular Ca concentration ([Ca2+]i) of neurites with flue-3 and a confocal laser microscope, and Ca channel activities were measured by the change of [Ca2+]i that was induced by depolarization with a high K-medium. Increment in [Ca2+]i was blocked by nicardipine (100 IaM), nitrendipine (100 pM), Co2+ (3 mM), Ni2+ (2 mM) and Cd2+ (2 mM) for the neurites grown in standard culture medium. While that increment was not blocked ill the presence of 20 IJM t0-c6notoxin (N-type channel blocker) and 200 nM t0-agatoxin (P-type channel blocker), indicating that the Ca-channels in neurites are mostly L-type. [Ca2÷]i of neurites that were grown in Ca-free medium was 1/2 of that of normal nettrites. Change of the Ca-free medium to the normal one induced increment in [Ca2+]i. This increment was not blocked either by'Co2+, Ni2÷, Cd2+ or verapamil, suggesting that the elevation of [Ca2+]i was not due to activities of Ca channels. Further exposure of the neurites with high K-solution induced elevation of [Ca2+]i, that wa~ blocked by Co2+, Ni2+ or Cd2+, but not by nicardipine, t0-conotoxin or to-agatoxin. It is likely that a new type of Ca-channels appears in neurites during growth in the Ca-free medium.
1233
ESTROGEN AFFECTS THE CYTOSKELETON OF THE CULTURED HYPOTHALAMIC NEURON. KAZUNARI YURI AND MITSUHIRO KAWATA, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Estrogen plays a pivotal role in growth and differentiation of neurons in the sexually dimorphic area. In the present study, the effect of estrogen on the neurite growth was investigated by a dissociated culture of embryonic day 17 and posmatal day 1 female rat hypothalami. Phenol red free Eagle's minimum essential medium was supplemented by dextran-coated charcoal treated 10% fetal calf serum and experimental cultures were treated with estradiol-17t3 (100ng/ml). Cells were plated onto polyethylenimine-coated glass coverslips at densities ranging from lxl04 to 5x104. They were fixed at 1,3, 5 and 7days in vitro, and stained immunohistochemically using anti-MAP2 and anti-tau antisera. The length of neurites was measured by an image analysis system. It was found that the number of MAP2- and tau-immunoreactive neurons increased, and MAP2- and tau-immunoreactive neurites were elongated by es.trogen treatment. The outgrowth of neurites in specific type of neurons has been promoted, suggesting that these neurons may be sensitive to estrogen treatment.
1234
ANALYSIS
OF N E U R I T E
GROWTH
OF PC12h-R
USING F L O W S Y S T E M
TOMO0 HOMMAYASUSHI KAZUNO. KAZUAKI YOSHIOKA AND HIDEAKI MATSUOKA, Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16, Nakamaehi, Koganei, Tokyo 184, Japan. The flow type culture system was developed for analysis of neurite growth. The system could exchange the medium gently and sequentially, which reduced effects of coexisting cells on the neufite growth. In this study, N G F and db-cAMP were applied repeatedly with this system and their action on neurite growth were examined by continual observation of neurite behaviour. The multiplication medium and the differentiation medium containing NGF or db-cAMP were alternately flowed for every 1 hr. Neurite growth was evaluated by the relative number of neurite bearing cells (RN) and neurite length (L,~). In PC12h-R, R,~was changed after the supply and the removal of factors. However, RN did not show tl{e change after 5 times applications of NGF. In contrast, db-cAMP supply to the same cells indicated the neurite projection. On the other hand, in the case of db-cAMP application, PC12h-R showed the repeated response until 8 times applications. L.,4 changed with a vibrating pattern, that is, neurites elongated and retracted repeatedly. However, this pattern was not necessarily synchronized with addition and removal of factors exactly. These factors seemed to affect the initial stage of neurite growth (neurite projection) even rather than the successive stage (neurite elongation).
1235
CALCIUM CHANNEL PROPERTIES IN RAT SENSORY FIBERS DURING GROWTH IN C O L L A G E N - G E L JUN FUKUDA, KAZUKO KEINO-MASU, SHINGO TSUKADA, TAKU IWAMOTO AND KEIICHI TORIMITSU*, Department of Physiology, National Defense Medical School, Tokorozawa, Department of Physiology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, and *NTT Basic Research Laboratories, Musashino, Tokyo.
In order to examine properties of Ca channels of sensory fibers during regeneration both in normal Ca medium (2.5 raM) and in Ca-free medium (0.01 nM), we n~easured intracellular Ca concentration ([Ca2+]i) of neurites with flue-3 and a confocal laser microscope, and Ca channel activities were measured by the change of [Ca2+]i that was induced by depolarization with a high K-medium. Increment in [Ca2+]i was blocked by nicardipine (100 IaM), nitrendipine (100 pM), Co2+ (3 mM), Ni2+ (2 mM) and Cd2+ (2 mM) for the neurites grown in standard culture medium. While that increment was not blocked ill the presence of 20 IJM t0-c6notoxin (N-type channel blocker) and 200 nM t0-agatoxin (P-type channel blocker), indicating that the Ca-channels in neurites are mostly L-type. [Ca2÷]i of neurites that were grown in Ca-free medium was 1/2 of that of normal nettrites. Change of the Ca-free medium to the normal one induced increment in [Ca2+]i. This increment was not blocked either by'Co2+, Ni2÷, Cd2+ or verapamil, suggesting that the elevation of [Ca2+]i was not due to activities of Ca channels. Further exposure of the neurites with high K-solution induced elevation of [Ca2+]i, that wa~ blocked by Co2+, Ni2+ or Cd2+, but not by nicardipine, t0-conotoxin or to-agatoxin. It is likely that a new type of Ca-channels appears in neurites during growth in the Ca-free medium.