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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

MECHANISM OF THE BIDIRECTIONAL AXONAL TRANSPORT. LOCALIZATION OF BRAIN DYNEIN (MAPIC) AND KINESIN IN VIVO. NOBUTAKA HIROKAWA, REIKO SATO-yOSHITAKE & NAOTO KOBAYASHI. Department of Anatomy and Cell Biology, Faculty of Medi_cine r University of Tokyo, Honqo, Tokyo, 113 Japan.

Kinesin and brain dynein are microtubule-activated ATPase considered to be candidates to function as molecular motors to transport membranous organelles anterogradely and retrogradely in the axon based on in vitro experiments. However there are no in vivo evidences indicating that they are really molecular motors for each tansports. To elucidate this question we studied the localization of kinesin and brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using anti-brain dynein antibodies and anti kinesin antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Kinesin was associated primarily with anterogradely transported membranous organelles, while brain dynein localized not only on retrogradely transported membranous organelles but also on antergradely transported ones. This is the first in vivo evidence to show that kinesin primarily associates with anterogradely-transported membranous organelles in vivo and that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activated brain dynein only for retrograde transport.

DYNAMICS OF NEURONAL INTERMEDIATE FILAMENTS IN VIVO. SHIGEO OKABE AND NOBUTAKA HIROKAWArDepartment of Anatomy and Cell Biology r School of Medicine t University of Tokyo~ 7-3-I Hongo~ Bunkyo-ku~ Tokyo 113~ Japan.

Neurofilaments and microtubules, together with various kinds of associated proteins, form highly organized structures of the axonal cytoskeleton. Previous studies have shown that axonal microtubules and actin filaments are dynamic and are not translocated as a stable complex. To clarify the mechanism of intermediate filament turnover in vivo, we labeled neurofilament L (NF-L) protein with the fluorescent dye iodoacetoamide fluorescein. Labeled NF-L was soluble in a low ionic strength buffer and under this condition it was possible to introduce this probe into freshly plated dorsal root ganglion (DRG) neurons by microinjection. The injected cells were incubated for 12 to 20 hours for axon regeneration. The growing fluorescent axons were bleached with a laser beam and the recovery of fluorescence was analyzed. The recovery half-time of fluorescence was about 35 min and no movement of the bleached zone was observed. These results suggest that neurofilaments are dynamic structure which are mainly transported as free molecules and turnover ]ocally within growing axons.