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Developmental change of cholinergic enzymes during the song learning period of the zebra finch
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MAPIB: ITS M O L E C U L A R STRUCTURE, LOCALIZATION, A N D P H O S P H O R Y L A T I O N - D E P E N D E N T E X P R E S S I O N IN D E V E L O P I N G NEURONS. REIKO SATO-YOSHITAKE., HIROSHI MIYASAKA., Y O K O SHIOMURA., A N D N O B U T A K A HIROKAWA, Department of A n a t o m y a n d . C e l lB i o l o g y , School of Medicine, Universit~v of Tokyo, Hongo, Tokyo i 1 3 , J_aA?an. The neuronal cytoskeleton is c o m p o s e d of three major elements -- microtuble proteins, neurofilaments, and microfilaments. M A P I B (microtuhule-associated protein IB) is a major component of neuronal microtubule proteins, but little has been disclosed about its molecular structure and its p h o s p h o r y l a t e d levels in s i t u . We have raised two monoclonal antibodies against MAPIB, one detecting its entire population, the other reacting only to its p h o s p h o r y l a t e d form. M A P I B was purified on an immuno-affinity column, and its molecular structure was analyzed using the rotary-shadowing m e t h o d and the quick-freeze, deep-etch technique. It appeared as a long filamentous molecule (186 ± 38 nm) with a small spherical portion at one end, forming long cross-bridges between microtubules in v i fro. Together with i, ,~ivo d a t a f r o m i m m u n o g o l d m e t h o d s , i t i s c o n c l u d e d t h a t MAP1B i s a c o m p o n e n t o f c r o s s bridges between microtubules in neurons. By i m m u n o h i s t o c h e m i c a l analysis, phosphorylated forms were shown to exist mainly in axons, whereas dephosphorylated forms were limited to cell bodies and dendrites. Phosphorylated MAP1B w a s q u i t e a b u n d a n t in developing axons, suggesting its essential role in axonal elongation.
A 130 kD PHOSPHOPROTEIN IN ISOLATED GROWTH CONES. MICHIHIRO IGARASHI 1'2 AND YOSHIAKI KOMIYA 2, tDepartment of Biochemistry, lichi Medical School, Minamikawachi-machi, Tochigi 329-04, and 2Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi-shi, Gunma 371, Japan. Nerve growth cones are the growing tips of extending neurites, and play an important role for exact neural connection. To study their molecular mechanisms on nerve pathfinding, we analyzed protein phosphorylation in growth cones isolated biochemically. The growth cone particle ( GCP ) fraction was isolated from rat embryonic day 17 brains by Pfenninger's method with slight modifications, and then lysed with 6 mM Tris-HCl ( p8 8.1 ). 15 a g of lysed GCP protein was added to the reaction mixture. After the addition of [7-32P]ATP, the reaction mixture was incubated at 30 °C for 15 sec. The reaction mixture was subjected to SDS-PAGE and the phosphorylated proteins were analyzed by autoradiograms. In the reaction mixture containing 1 mM Ca ~+ and 5 mM Mg 2+. the four major phosphoproteins were 130 kD, 80 kD, 46 kD, and 40 kD. 20 ~ M of H-7 severely inhibited phosphorylation of the 80 kO, 46 kD. and 40 kD proteins, however, it had no effect on the phosphorylation of the 130 kD protein. As with tubulin and ppGO c-ere, the 130 kO protein was also phosphorylated under conditions which preferentially caused phosphorylation of Tyr residues. The 130 kD phosphoprotein reacted with anti-phosphotyrosine antibody, and was resistant to both 4N HCI and iN KOH. These data suggest that the 130 kD phosphoprotein is subjected to selective phosphorylation of its Tyr residues. We also detected that the 130 kD protein was endogenously phosphorylated more strikingly in isolated growth cones than in adult synaptosomes.
DEVELOPMENTAL CHANGE OF C H O L I N E R G I C ENZYMES DURING THE SONG LEARNING P E R I O D OF THE ZEBRA FINCH. HIRONOBU SAKAGUCHI AND NOZOMU S A I T O , Department of Physiology, Medical School, Dokkyo University, Mibu, Tochigi, 321-02. Song learning in the male zebra finch closely correlates with the development of song control nuclei in the forebrain. Acetylcholine (ACh) concentration increases transiently in the song control nuclei, RA, H V c , a n d MAN d u r i n g the critical period of song learning (Sakaguchi ~ Saito, Develop. Brain Res.,1989). The present report confirms this by measurement of cholinergic enzyme activity of the nuclei during the same period. Male birds were used at 30, 50, and 90 days of age. The enzyme activity of acetylcholine esterase (ACHE) o r c h o l i n e acetyltransferase (CHAT) in the song control nuclei, RA, H V c , a n d MAN w a s m e a s u r e d by the photometric or radioehemical method (gilman,1966; Fonnum, 1975). The three cholinergic markers, ACh concentration, AChE a n d ChAT a c t i v i t y showed similar developmental changes in the song control nuclei, This indicates that cholinergic neuronal substrates in the song control system should develop during the critical period. An a n t i b o d y to chick ChAT(from Dr.Epstein) was able to label cholinergic cells in the adult RA, HVc, a n d MAN b y t h e a v i d i n e - b i o t i n e complex method. No CHATpositive cell bodies, but ChAT-positive fibers and terminals w e r e f o u n d i n t h e RA, HVc and MAN, indicating that their cholinergie origin is in an unknown nuclei rather than in the cerebral song control nuclei including t h e RA, H V c , a n d MAN.
MAPIB: ITS M O L E C U L A R STRUCTURE, LOCALIZATION, A N D P H O S P H O R Y L A T I O N - D E P E N D E N T E X P R E S S I O N IN D E V E L O P I N G NEURONS. REIKO SATO-YOSHITAKE., HIROSHI MIYASAKA., Y O K O SHIOMURA., A N D N O B U T A K A HIROKAWA, Department of A n a t o m y a n d . C e l lB i o l o g y , School of Medicine, Universit~v of Tokyo, Hongo, Tokyo i 1 3 , J_aA?an. The neuronal cytoskeleton is c o m p o s e d of three major elements -- microtuble proteins, neurofilaments, and microfilaments. M A P I B (microtuhule-associated protein IB) is a major component of neuronal microtubule proteins, but little has been disclosed about its molecular structure and its p h o s p h o r y l a t e d levels in s i t u . We have raised two monoclonal antibodies against MAPIB, one detecting its entire population, the other reacting only to its p h o s p h o r y l a t e d form. M A P I B was purified on an immuno-affinity column, and its molecular structure was analyzed using the rotary-shadowing m e t h o d and the quick-freeze, deep-etch technique. It appeared as a long filamentous molecule (186 ± 38 nm) with a small spherical portion at one end, forming long cross-bridges between microtubules in v i fro. Together with i, ,~ivo d a t a f r o m i m m u n o g o l d m e t h o d s , i t i s c o n c l u d e d t h a t MAP1B i s a c o m p o n e n t o f c r o s s bridges between microtubules in neurons. By i m m u n o h i s t o c h e m i c a l analysis, phosphorylated forms were shown to exist mainly in axons, whereas dephosphorylated forms were limited to cell bodies and dendrites. Phosphorylated MAP1B w a s q u i t e a b u n d a n t in developing axons, suggesting its essential role in axonal elongation.
A 130 kD PHOSPHOPROTEIN IN ISOLATED GROWTH CONES. MICHIHIRO IGARASHI 1'2 AND YOSHIAKI KOMIYA 2, tDepartment of Biochemistry, lichi Medical School, Minamikawachi-machi, Tochigi 329-04, and 2Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi-shi, Gunma 371, Japan. Nerve growth cones are the growing tips of extending neurites, and play an important role for exact neural connection. To study their molecular mechanisms on nerve pathfinding, we analyzed protein phosphorylation in growth cones isolated biochemically. The growth cone particle ( GCP ) fraction was isolated from rat embryonic day 17 brains by Pfenninger's method with slight modifications, and then lysed with 6 mM Tris-HCl ( p8 8.1 ). 15 a g of lysed GCP protein was added to the reaction mixture. After the addition of [7-32P]ATP, the reaction mixture was incubated at 30 °C for 15 sec. The reaction mixture was subjected to SDS-PAGE and the phosphorylated proteins were analyzed by autoradiograms. In the reaction mixture containing 1 mM Ca ~+ and 5 mM Mg 2+. the four major phosphoproteins were 130 kD, 80 kD, 46 kD, and 40 kD. 20 ~ M of H-7 severely inhibited phosphorylation of the 80 kO, 46 kD. and 40 kD proteins, however, it had no effect on the phosphorylation of the 130 kD protein. As with tubulin and ppGO c-ere, the 130 kO protein was also phosphorylated under conditions which preferentially caused phosphorylation of Tyr residues. The 130 kD phosphoprotein reacted with anti-phosphotyrosine antibody, and was resistant to both 4N HCI and iN KOH. These data suggest that the 130 kD phosphoprotein is subjected to selective phosphorylation of its Tyr residues. We also detected that the 130 kD protein was endogenously phosphorylated more strikingly in isolated growth cones than in adult synaptosomes.
DEVELOPMENTAL CHANGE OF C H O L I N E R G I C ENZYMES DURING THE SONG LEARNING P E R I O D OF THE ZEBRA FINCH. HIRONOBU SAKAGUCHI AND NOZOMU S A I T O , Department of Physiology, Medical School, Dokkyo University, Mibu, Tochigi, 321-02. Song learning in the male zebra finch closely correlates with the development of song control nuclei in the forebrain. Acetylcholine (ACh) concentration increases transiently in the song control nuclei, RA, H V c , a n d MAN d u r i n g the critical period of song learning (Sakaguchi ~ Saito, Develop. Brain Res.,1989). The present report confirms this by measurement of cholinergic enzyme activity of the nuclei during the same period. Male birds were used at 30, 50, and 90 days of age. The enzyme activity of acetylcholine esterase (ACHE) o r c h o l i n e acetyltransferase (CHAT) in the song control nuclei, RA, H V c , a n d MAN w a s m e a s u r e d by the photometric or radioehemical method (gilman,1966; Fonnum, 1975). The three cholinergic markers, ACh concentration, AChE a n d ChAT a c t i v i t y showed similar developmental changes in the song control nuclei, This indicates that cholinergic neuronal substrates in the song control system should develop during the critical period. An a n t i b o d y to chick ChAT(from Dr.Epstein) was able to label cholinergic cells in the adult RA, HVc, a n d MAN b y t h e a v i d i n e - b i o t i n e complex method. No CHATpositive cell bodies, but ChAT-positive fibers and terminals w e r e f o u n d i n t h e RA, HVc and MAN, indicating that their cholinergie origin is in an unknown nuclei rather than in the cerebral song control nuclei including t h e RA, H V c , a n d MAN.