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1204 Basic FGF determines the anterior-posterior axis during early neural development in Xenopus
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1203
CHARACTERIZATION OF RETINAL CHOLINERGIC N E U R O N - N E U R O T R O P H I C FACTOR DERIVED FROM CULTURED HIPPOCAMPAL CELLS TOHRU YASUKIJRA, KYOKO OMORI, TOSHIKO MIKAMI, MITSUYOSHI HARA AND CHIYOKO INAGAKI. _Department of Pharmacolog,v. Kansal Medical University, Fumizono-eho l,Meriguti, Osaka 570. lnpan.
A neurotrophie factor which supports the development of eholinergic retinal neuron was isolated from media-conditioned with embryonic hlppocampal neurons in culture. We purified the factor by collecting fractions active in promoting neurite-outgrowth and Choline acetyltransferase (CHAT) activity of cultured retinal cells from 1 7 - d a y - o l d rat fetus. After Sephadex-G15 column chromatography and two steps of r e v e r s e - p h a s e (Cls) HPLC, the final peak fraction of the neurotrophie activity corresponded to the absorlmnee peak. G a s - p h a s e peptide sequencing of the active substance suggested that the neurotrophic factor is a peptide composed of 10 amino acids. A synthetic peptide with the same sequence dose-dependently stimulated ChAT activity in retinal cell culture. The peptide also showed similar ChAT-stimulating activity in the cell culture of septal nucleus. These findings demonstrated that hippocampal neurons produce neurotrophie peptide which stimulates the development of cholinergie neurons.
1204
BASIC FGF DETERMINES THE ANTERIOR-POSTER/OR AXIS DURING EARLY NEURAL
DEVELOPMENT IN XENOPUS. MINEKQ KENGAKU AND HARUMASA QKAMQTQ, Department of Neurobioloa'v. Institute of Bgain Research. School of Me41i~ine. Univcr~i|y of Tokyo. Hgn~'o7-3-I, Tokyo 113. ]~pan The anterior-posterior (A-P) axis of the vermbrate nervous system is established during gastrulation as the dorsal mesoderm induces the overlying ectoderm towards neural development in a region specific manner. We have previously shown that bFGF at concentrations in the picomolar range specifically mimics the actions of dorsal mesoderm to induce both neural tube-derived neurons and neural crest-derived melanophores from cultured gastrula ectoderm cells of Xenopus. This raises the possibility that bFGF elicits multiple responses of ectoderm cells to give rise to a complete set of the nervous systems along the A-P axis. Quantitative RT-PCR analysis revealed that bFGF induced the expression of three positionspecific neural markers (homeobox genes NK2, En-2 and X1Hbox6) in a dose-dependent fashion. The sequence of the concentration thresholds of bFGF required for inducing these markers was collinear with the sequence of A-P locations of their expression. NK-2, the gene expressed at the anterior end, was induced at the lowest dose, En-2 next, and XIHbox6 at the highest dose. These data suggest that bFGF is the natural neural inducer secreted from the dorsal mesoderm cells in a graded manner along the A-P axis, and plays a crucial role in determination of the A-P axis in the developing nervous system.
1205
DEPOLARIZATION-EVOKED ACETYLCHOLINE RELEASE FROM CULTURED RAT SEPTAL NEURONS IS INDUCED BY NERVE GROWTH FACTOR BUT NOT BY DIBUTYRYL CYCLIC AMP TREATMENT. TAKESHI SUZUKI and KOICHIRO KAWASHIMA. Deoarlment of Pharmacology, Kvoritsu College of Pharmacy. Minato-ku, Tokyo 105.'Japan.
In the present study, we investigated the effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) on the inductiori of choline acetyltransferase (CHAT) and spontaneous and stimulation-evoked acetylcholine (ACh) release in the cultured embryonic (EIT) rat septal neurons. Both compounds increased ChAT activity, cellular ACh content and spontaneous ACh release. Total protein content was not changed significantly by these corn'pounds, thus, these effects was specific for Cholinergic neurons. A23187, a Ca2+ionophore, evoked extmcellular C.a2+-dependent ACh release in all cells tested (including control group). Therefore, the process of ACh secretion triggered by increase in intraceilular Ca 2+ level is revealed spontaneously in all developing cholinergic neurons. On the other hand, high-K+ stimulation-evoked Ca2+dependent ACh release was observed in NGF treated group but not observed in control and dbcAMP treated groups. These results indicate that NGF can induce not only ChAT but also the'pathway of excitation-secretion coupling.
1203
CHARACTERIZATION OF RETINAL CHOLINERGIC N E U R O N - N E U R O T R O P H I C FACTOR DERIVED FROM CULTURED HIPPOCAMPAL CELLS TOHRU YASUKIJRA, KYOKO OMORI, TOSHIKO MIKAMI, MITSUYOSHI HARA AND CHIYOKO INAGAKI. _Department of Pharmacolog,v. Kansal Medical University, Fumizono-eho l,Meriguti, Osaka 570. lnpan.
A neurotrophie factor which supports the development of eholinergic retinal neuron was isolated from media-conditioned with embryonic hlppocampal neurons in culture. We purified the factor by collecting fractions active in promoting neurite-outgrowth and Choline acetyltransferase (CHAT) activity of cultured retinal cells from 1 7 - d a y - o l d rat fetus. After Sephadex-G15 column chromatography and two steps of r e v e r s e - p h a s e (Cls) HPLC, the final peak fraction of the neurotrophie activity corresponded to the absorlmnee peak. G a s - p h a s e peptide sequencing of the active substance suggested that the neurotrophic factor is a peptide composed of 10 amino acids. A synthetic peptide with the same sequence dose-dependently stimulated ChAT activity in retinal cell culture. The peptide also showed similar ChAT-stimulating activity in the cell culture of septal nucleus. These findings demonstrated that hippocampal neurons produce neurotrophie peptide which stimulates the development of cholinergie neurons.
1204
BASIC FGF DETERMINES THE ANTERIOR-POSTER/OR AXIS DURING EARLY NEURAL
DEVELOPMENT IN XENOPUS. MINEKQ KENGAKU AND HARUMASA QKAMQTQ, Department of Neurobioloa'v. Institute of Bgain Research. School of Me41i~ine. Univcr~i|y of Tokyo. Hgn~'o7-3-I, Tokyo 113. ]~pan The anterior-posterior (A-P) axis of the vermbrate nervous system is established during gastrulation as the dorsal mesoderm induces the overlying ectoderm towards neural development in a region specific manner. We have previously shown that bFGF at concentrations in the picomolar range specifically mimics the actions of dorsal mesoderm to induce both neural tube-derived neurons and neural crest-derived melanophores from cultured gastrula ectoderm cells of Xenopus. This raises the possibility that bFGF elicits multiple responses of ectoderm cells to give rise to a complete set of the nervous systems along the A-P axis. Quantitative RT-PCR analysis revealed that bFGF induced the expression of three positionspecific neural markers (homeobox genes NK2, En-2 and X1Hbox6) in a dose-dependent fashion. The sequence of the concentration thresholds of bFGF required for inducing these markers was collinear with the sequence of A-P locations of their expression. NK-2, the gene expressed at the anterior end, was induced at the lowest dose, En-2 next, and XIHbox6 at the highest dose. These data suggest that bFGF is the natural neural inducer secreted from the dorsal mesoderm cells in a graded manner along the A-P axis, and plays a crucial role in determination of the A-P axis in the developing nervous system.
1205
DEPOLARIZATION-EVOKED ACETYLCHOLINE RELEASE FROM CULTURED RAT SEPTAL NEURONS IS INDUCED BY NERVE GROWTH FACTOR BUT NOT BY DIBUTYRYL CYCLIC AMP TREATMENT. TAKESHI SUZUKI and KOICHIRO KAWASHIMA. Deoarlment of Pharmacology, Kvoritsu College of Pharmacy. Minato-ku, Tokyo 105.'Japan.
In the present study, we investigated the effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) on the inductiori of choline acetyltransferase (CHAT) and spontaneous and stimulation-evoked acetylcholine (ACh) release in the cultured embryonic (EIT) rat septal neurons. Both compounds increased ChAT activity, cellular ACh content and spontaneous ACh release. Total protein content was not changed significantly by these corn'pounds, thus, these effects was specific for Cholinergic neurons. A23187, a Ca2+ionophore, evoked extmcellular C.a2+-dependent ACh release in all cells tested (including control group). Therefore, the process of ACh secretion triggered by increase in intraceilular Ca 2+ level is revealed spontaneously in all developing cholinergic neurons. On the other hand, high-K+ stimulation-evoked Ca2+dependent ACh release was observed in NGF treated group but not observed in control and dbcAMP treated groups. These results indicate that NGF can induce not only ChAT but also the'pathway of excitation-secretion coupling.