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Dynamics of neuronal intermediate filaments in vivo
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COLUMNAR DISTRIBUTION OF AXON COLLATERALS OF SINGLE PYRAMIDAL NEURONS 1N THE CAT PRII%L~kRY AUDITORY CORTEX(AI), HISAYUKI OJIMA) TSUTOMU HASHIKAWA AND EDWARD G. JONES*, Neural Systems Laboratory, Frontier Research Pro m'am, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-01 Japan. In the ectosylvian gyrus of barbiturate-anesthetized, paralyzed cats, layer 2/3 ceils were penetrated using glass micropippets filled with 2% biocytin (Sigma). After checking the response frequency range (the frequency range in which action potentials or EPSPs were evoked at a low intensity of pure tone ranging 2-20 kHz in frequency), the cell was injected ientophoretically. The animal was peffused 6-24 hours after injection. Thin collaterals given off from main axon formed a cluster of fine terminal branches in the vicinity of the ceII body. As a whole, they displayed a columnar distribution with the high density in layers 1, 2 and 3 and in layer 5. From horizontally coursing, thick collaterals, terminal branches were given off. They were distributed in a columnar fashion at a distance from the cell body. The distance ranged flora 500 um to several mm. Their laminax distribution retained the features found in the column situated in the vicinity of the cell body, and was rich in layers 1, 2, 3 and 5. Some collaterals, which branched from the main axon in the white matter, contributed to the columnar formation in the vicinity of the cell body. Some of layer 3 cells projected to extra-AI fields with the main axon passing through the white matter. Their laminar distribution was, again, identical to that of other columns formed within the intrinsic area.
5. Axoplasmic transport M E C H A N I S M OF THE B I D I R E C T I O N A L A X O N A L TRANSPORT. L O C A L I Z A T I O N OF BRAIN D Y N E I N (MAPIC) A N D K I N E S I N IN VIVO. N O B U T A K A HIROKAWA, R E I K O S A T O - y O S H I T A K E & NAOTO KOBAYASHI. D e p a r t m e n t of A n a t o m y and Cell Biology, F a c u l t y of Medi_cine r U n i v e r s i t y of Tokyo, Honqo, Tokyo, 113 Japan. K i n e s i n and brain d y n e i n are m i c r o t u b u l e - a c t i v a t e d A T P a s e c o n s i d e r e d to be candidates to function as m o l e c u l a r motors to t r a n s p o r t m e m b r a n o u s o r g a n e l l e s a n t e r o g r a d e l y and r e t r o g r a d e l y in the axon b a s e d on in v i t r o experiments. However there are no in vivo e v i d e n c e s indicating that they are really m o l e c u l a r motors for each tansports. To e l u c i d a t e this q u e s t i o n we studied the l o c a l i z a t i o n of k i n e s i n and brain dynein in axons after the ligation of peripheral nerves by light and e l e c t r o n m i c r o s c o p i c i m m u n o c y t o c h e m i s t r y using a n t i - b r a i n dynein a n t i b o d i e s and anti kinesin antibodies. Different classes of o r g a n e l l e s p r e f e r e n t i a l l y a c c u m u l a t e d at the r e g i o n s proximal and distal to the ligated part. K i n e s i n was a s s o c i a t e d p r i m a r i l y w i t h a n t e r o g r a d e l y t r a n s p o r t e d m e m b r a n o u s organelles, while b r a i n d y n e i n l o c a l i z e d not only on r e t r o g r a d e l y t r a n s p o r t e d m e m b r a n o u s o r g a n e l l e s but also on a n t e r g r a d e l y t r a n s p o r t e d ones. This is the first in vivo evidence to s h o w that k i n e s i n p r i m a r i l y associates with a n t e r o g r a d e l y - t r a n s p o r t e d m e m b r a n o u s organelles in vivo and that brain dynein a s s o c i a t e s with r e t r o g r a d e l y t r a n s p o r t e d o r g a n e l l e s in vivo and that brain dynein is t r a n s p o r t e d to the n e r v e terminal by fast flow. This also suggests that there may be some m e c h a n i s m that a c t i v a t e d brain dynein only for r e t r o g r a d e transport.
DYNAMICS OF N E U R O N A L I N T E R M E D I A T E F I L A M E N T S IN VIVO. S H I G E O OKABE AND N O B U T A K A H I R O K A W A r D e p a r t m e n t of A n a t o m y and Cell B i o l o g y r School of M e d i c i n e t U n i v e r s i t y of Tokyo~ 7-3-I Hongo~ Bunkyo-ku~ T o k y o 113~ Japan. N e u r o f i l a m e n t s and m i c r o t u b u l e s , together with v a r i o u s kinds of a s s o c i a t e d proteins, form h i g h l y o r g a n i z e d structures of the axonal cytoskeleton. Previous studies have shown that axonal m i c r o t u b u l e s and actin filaments are d y n a m i c and are not translocated as a stable complex. To clarify the mechanism of intermediate filament t u r n o v e r in vivo, we labeled neurofilament L (NF-L) protein with the f l u o r e s c e n t dye i o d o a c e t o a m i d e fluorescein. Labeled NF-L was soluble in a low ionic strength buffer and under this c o n d i t i o n it was possible to i n t r o d u c e this probe into freshly plated dorsal root g a n g l i o n (DRG) neurons by microinjection. The injected cells were i n c u b a t e d for 12 to 20 hours for axon regeneration. The g r o w i n g f l u o r e s c e n t axons w e r e b l e a c h e d with a laser beam and the r e c o v e r y of f l u o r e s c e n c e was analyzed. The recovery h a l f - t i m e of fluorescence was about 35 min and no m o v e m e n t of the bleached zone was observed. These results suggest that n e u r o f i l a m e n t s are d y n a m i c s t r u c t u r e w h i c h are m a i n l y t r a n s p o r t e d as free m o l e c u l e s and t u r n o v e r ]ocally within growing axons.
COLUMNAR DISTRIBUTION OF AXON COLLATERALS OF SINGLE PYRAMIDAL NEURONS 1N THE CAT PRII%L~kRY AUDITORY CORTEX(AI), HISAYUKI OJIMA) TSUTOMU HASHIKAWA AND EDWARD G. JONES*, Neural Systems Laboratory, Frontier Research Pro m'am, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-01 Japan. In the ectosylvian gyrus of barbiturate-anesthetized, paralyzed cats, layer 2/3 ceils were penetrated using glass micropippets filled with 2% biocytin (Sigma). After checking the response frequency range (the frequency range in which action potentials or EPSPs were evoked at a low intensity of pure tone ranging 2-20 kHz in frequency), the cell was injected ientophoretically. The animal was peffused 6-24 hours after injection. Thin collaterals given off from main axon formed a cluster of fine terminal branches in the vicinity of the ceII body. As a whole, they displayed a columnar distribution with the high density in layers 1, 2 and 3 and in layer 5. From horizontally coursing, thick collaterals, terminal branches were given off. They were distributed in a columnar fashion at a distance from the cell body. The distance ranged flora 500 um to several mm. Their laminax distribution retained the features found in the column situated in the vicinity of the cell body, and was rich in layers 1, 2, 3 and 5. Some collaterals, which branched from the main axon in the white matter, contributed to the columnar formation in the vicinity of the cell body. Some of layer 3 cells projected to extra-AI fields with the main axon passing through the white matter. Their laminar distribution was, again, identical to that of other columns formed within the intrinsic area.
5. Axoplasmic transport M E C H A N I S M OF THE B I D I R E C T I O N A L A X O N A L TRANSPORT. L O C A L I Z A T I O N OF BRAIN D Y N E I N (MAPIC) A N D K I N E S I N IN VIVO. N O B U T A K A HIROKAWA, R E I K O S A T O - y O S H I T A K E & NAOTO KOBAYASHI. D e p a r t m e n t of A n a t o m y and Cell Biology, F a c u l t y of Medi_cine r U n i v e r s i t y of Tokyo, Honqo, Tokyo, 113 Japan. K i n e s i n and brain d y n e i n are m i c r o t u b u l e - a c t i v a t e d A T P a s e c o n s i d e r e d to be candidates to function as m o l e c u l a r motors to t r a n s p o r t m e m b r a n o u s o r g a n e l l e s a n t e r o g r a d e l y and r e t r o g r a d e l y in the axon b a s e d on in v i t r o experiments. However there are no in vivo e v i d e n c e s indicating that they are really m o l e c u l a r motors for each tansports. To e l u c i d a t e this q u e s t i o n we studied the l o c a l i z a t i o n of k i n e s i n and brain dynein in axons after the ligation of peripheral nerves by light and e l e c t r o n m i c r o s c o p i c i m m u n o c y t o c h e m i s t r y using a n t i - b r a i n dynein a n t i b o d i e s and anti kinesin antibodies. Different classes of o r g a n e l l e s p r e f e r e n t i a l l y a c c u m u l a t e d at the r e g i o n s proximal and distal to the ligated part. K i n e s i n was a s s o c i a t e d p r i m a r i l y w i t h a n t e r o g r a d e l y t r a n s p o r t e d m e m b r a n o u s organelles, while b r a i n d y n e i n l o c a l i z e d not only on r e t r o g r a d e l y t r a n s p o r t e d m e m b r a n o u s o r g a n e l l e s but also on a n t e r g r a d e l y t r a n s p o r t e d ones. This is the first in vivo evidence to s h o w that k i n e s i n p r i m a r i l y associates with a n t e r o g r a d e l y - t r a n s p o r t e d m e m b r a n o u s organelles in vivo and that brain dynein a s s o c i a t e s with r e t r o g r a d e l y t r a n s p o r t e d o r g a n e l l e s in vivo and that brain dynein is t r a n s p o r t e d to the n e r v e terminal by fast flow. This also suggests that there may be some m e c h a n i s m that a c t i v a t e d brain dynein only for r e t r o g r a d e transport.
DYNAMICS OF N E U R O N A L I N T E R M E D I A T E F I L A M E N T S IN VIVO. S H I G E O OKABE AND N O B U T A K A H I R O K A W A r D e p a r t m e n t of A n a t o m y and Cell B i o l o g y r School of M e d i c i n e t U n i v e r s i t y of Tokyo~ 7-3-I Hongo~ Bunkyo-ku~ T o k y o 113~ Japan. N e u r o f i l a m e n t s and m i c r o t u b u l e s , together with v a r i o u s kinds of a s s o c i a t e d proteins, form h i g h l y o r g a n i z e d structures of the axonal cytoskeleton. Previous studies have shown that axonal m i c r o t u b u l e s and actin filaments are d y n a m i c and are not translocated as a stable complex. To clarify the mechanism of intermediate filament t u r n o v e r in vivo, we labeled neurofilament L (NF-L) protein with the f l u o r e s c e n t dye i o d o a c e t o a m i d e fluorescein. Labeled NF-L was soluble in a low ionic strength buffer and under this c o n d i t i o n it was possible to i n t r o d u c e this probe into freshly plated dorsal root g a n g l i o n (DRG) neurons by microinjection. The injected cells were i n c u b a t e d for 12 to 20 hours for axon regeneration. The g r o w i n g f l u o r e s c e n t axons w e r e b l e a c h e d with a laser beam and the r e c o v e r y of f l u o r e s c e n c e was analyzed. The recovery h a l f - t i m e of fluorescence was about 35 min and no m o v e m e n t of the bleached zone was observed. These results suggest that n e u r o f i l a m e n t s are d y n a m i c s t r u c t u r e w h i c h are m a i n l y t r a n s p o r t e d as free m o l e c u l e s and t u r n o v e r ]ocally within growing axons.