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Labelling of spinal neuron by E. coli enterotoxin subunit B.
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LABELLING OF SPINAL NEURON BY E. COLI ENTEROTOXIN SUBUNIT B. OUBO.N OKADO . I, IHIDEO . HAYASH~..2 , YASUHIKO . HOSOYAI, AND NUTSUNI NATSUKAWA* I , Departments o f Anatomy and B a c t e r i o l o g y , ~ n n i v e r s i t y ~f ~ , I n s t i t u t e of Basic Medical S c i e n c e s , Tsukuba, I b a r a k i 305, Japan. Subunit B o f E s c h e r i c h i a c o l i h e a t - l a b i l e e n t e r o t o x i n (LTB) was be was i n j e c t e d i n t o t h e limb muscle o f t h e c h i c k , or i n t o t h e s u p e r i o r c e r v i c a l ganglion of the rat. Sections were p r o c e s s e d with an immunohistochemical technique using an a n t i b o d y against LTB. Following LTB in~eetions into the muscle, r e t r o g r a d e l l y labelled m o t o n e u r o n s were found: the entire extent of dendritic a r b o r i z a t i o n s a p p e a r e d labelled, because many d e n d r i t e s reached to the pial surface of the lateral funiculus. Primary afferent fibers were
l a b e l l e d , and t h e y were l o c a t e d mainly on d e n d r i t i c p r o f i l e s . Primary a f f e r e n t f i b e r s were c h a r a c t e r i z e d by v a r i c o s i t i e s , t h a t were n o t o b s e r v e d on d e n d r i t i c profiles. After the LTB injection into the ganglion, v i r t u a l l y all of the preganglionic neurons were r e t r o g r a d e l l y labelled in the rat spinal cord. Sensitivity and labelling c a p a b i l i t y with LTB a p p e a r e d the same with those with c h o l e r a t o x i n subunit B. The present study d e m o n s t r a t e d that LTB can be utilized as a powerful tracer in n e u r o a n a t o m i e a l studies.
FLUORESCENT LATEX MICROSPHERES AS A NEW MARKER FOR EMBRYONIC CHICK MOTONEURONS IN CULTURE. YOSHIHIRO KIMATAI~ KIYONORI HARIII~ KAZUKO KEINO2~ JUN FUKUDA2T Department of Iplastic and Reconstructive Surgeryr and Department of Zphysiology~ Faculty of Medicine~ University of Tokyo~ 7-3-I~ Hongot Bunkyo-kur Tokyo~ l13t Japan. We have developed a new cell-labeling technique using fluorescent latex microspheres, and tested the technique on embryonicchick motoneuronsin culture. Fluorescentlatex microspheres(0.125 um diameter, 0.1 g/ml, dissolvedin 0.01 mM PBS), 3 ul was injected into the thigh and shank of 5 days old chick embryos. Wheat germ agglutinin-fluoresceinisothiocyanate (WGA-FITC) was also injected into other groups of embryo for comparison. After 16 hrs of incubation, the lumbar spinal cord was dissected, and mincedinto pieces of 0.5mm in diameter. The pieces were digested with trypsin, and collected by centrifugation. Neurons were then dissociated by pipetting, and grown on glass cover-sl]p coated with poly-L-ornithine for two weeks. In comparison with labeling the motoneuronsby WGA-FITC, the present microspheretechnique had the following advantages : I) The intensity of fluorescenceof motoneuronsby latex microsphereswas approximately 1,00010,000 fold of that by WGA-FITG. 2) The location of the fluorescent microspheres was more sharply discriminated in the cytoplasm than that by WGA. 3) Fluorescent motoneuronscould be easily differentiated from other types of neurons and from non-neuronalcells, even after culturing for 2 weeks in serum-containing medium, while WGA-labeledmotoneuronsbecame hard to be differentiated from other cells within a week. The technique using fluorescentlatex mierospheresmay be a useful method for studyingmotoneurons.
STRUCTURE OF T R I G E M I N A L GANGLION NEURONS. S H I N - I C H I TERASHIMA, P E N G J I A JIANG*, A N D Y U N - F E I LIANG*, D e p a r t m e n t of Ph[siolo~[, U n i v e r s i t y of the Ryuk[us School of Medicine, N i s h i h a r a - c h o , O k i n a w a 9 0 3 - 0 1 , Japan. H o r s e r a d i s h p e r o x i d a s e (HRP) was i n t r a s o m a l l y i n j e c t e d into s e n s o r y neurons of the trigeminal g a n g l i o n of c r o t a l i n e snakes, T r i m e r e s u r u s flavoviridis, w i t h one i n j e c t i o n per ganglion. By light and electron m l c r o s c o p y , we o b s e r v e d d e g e n e r a t e d m y e l i n a t e d and n o n m y e l i n a t e d axons, some of w h i c h w e r e HRP-labeled. The glomerular structure reported in the cat was not o b s e r v e d a r o u n d the H R P - l a b e l e d s e n s o r y neuron; however, twisting axons of other neurons w e r e found. The n a k e d part of the node of Ranvier v a r i e d in length and showed different manifestations of b i f u r c a t i o n of the stem axon. Thus the s t r u c t u r a l d i f f e r e n c e of the node of Ranvier at the b i f u r c a t i o n seemed not to be closely r e l a t e d to its modality, a l t h o u g h the d i a m e t e r of 3 axons (stem, peripheral, and c e n t r a l axons) m a y be related to the modality. Various lengths of the internodal d i s t a n c e were found. A few unmyelinated axons (66/1612 =3.8%) w e r e o b s e r v e d in the p e r i p h e r a l nerve trunk and in the root of the ganglion. This finding is q u i t e d i f f e r e n t from that in the rat r e p o r t e d by others in w h i c h the n u m b e r of u n m y e l i n a t e d fibers was more than twice as m a n y as that of myelinated. This m i g h t be due to the species difference. No b r a n c h i n g was d e t e c t e d by H R P - i n j e c t i o n into p e r i p h e r a l and central axons in the v i c i n i t y of the ganglion.
LABELLING OF SPINAL NEURON BY E. COLI ENTEROTOXIN SUBUNIT B. OUBO.N OKADO . I, IHIDEO . HAYASH~..2 , YASUHIKO . HOSOYAI, AND NUTSUNI NATSUKAWA* I , Departments o f Anatomy and B a c t e r i o l o g y , ~ n n i v e r s i t y ~f ~ , I n s t i t u t e of Basic Medical S c i e n c e s , Tsukuba, I b a r a k i 305, Japan. Subunit B o f E s c h e r i c h i a c o l i h e a t - l a b i l e e n t e r o t o x i n (LTB) was be was i n j e c t e d i n t o t h e limb muscle o f t h e c h i c k , or i n t o t h e s u p e r i o r c e r v i c a l ganglion of the rat. Sections were p r o c e s s e d with an immunohistochemical technique using an a n t i b o d y against LTB. Following LTB in~eetions into the muscle, r e t r o g r a d e l l y labelled m o t o n e u r o n s were found: the entire extent of dendritic a r b o r i z a t i o n s a p p e a r e d labelled, because many d e n d r i t e s reached to the pial surface of the lateral funiculus. Primary afferent fibers were
l a b e l l e d , and t h e y were l o c a t e d mainly on d e n d r i t i c p r o f i l e s . Primary a f f e r e n t f i b e r s were c h a r a c t e r i z e d by v a r i c o s i t i e s , t h a t were n o t o b s e r v e d on d e n d r i t i c profiles. After the LTB injection into the ganglion, v i r t u a l l y all of the preganglionic neurons were r e t r o g r a d e l l y labelled in the rat spinal cord. Sensitivity and labelling c a p a b i l i t y with LTB a p p e a r e d the same with those with c h o l e r a t o x i n subunit B. The present study d e m o n s t r a t e d that LTB can be utilized as a powerful tracer in n e u r o a n a t o m i e a l studies.
FLUORESCENT LATEX MICROSPHERES AS A NEW MARKER FOR EMBRYONIC CHICK MOTONEURONS IN CULTURE. YOSHIHIRO KIMATAI~ KIYONORI HARIII~ KAZUKO KEINO2~ JUN FUKUDA2T Department of Iplastic and Reconstructive Surgeryr and Department of Zphysiology~ Faculty of Medicine~ University of Tokyo~ 7-3-I~ Hongot Bunkyo-kur Tokyo~ l13t Japan. We have developed a new cell-labeling technique using fluorescent latex microspheres, and tested the technique on embryonicchick motoneuronsin culture. Fluorescentlatex microspheres(0.125 um diameter, 0.1 g/ml, dissolvedin 0.01 mM PBS), 3 ul was injected into the thigh and shank of 5 days old chick embryos. Wheat germ agglutinin-fluoresceinisothiocyanate (WGA-FITC) was also injected into other groups of embryo for comparison. After 16 hrs of incubation, the lumbar spinal cord was dissected, and mincedinto pieces of 0.5mm in diameter. The pieces were digested with trypsin, and collected by centrifugation. Neurons were then dissociated by pipetting, and grown on glass cover-sl]p coated with poly-L-ornithine for two weeks. In comparison with labeling the motoneuronsby WGA-FITC, the present microspheretechnique had the following advantages : I) The intensity of fluorescenceof motoneuronsby latex microsphereswas approximately 1,00010,000 fold of that by WGA-FITG. 2) The location of the fluorescent microspheres was more sharply discriminated in the cytoplasm than that by WGA. 3) Fluorescent motoneuronscould be easily differentiated from other types of neurons and from non-neuronalcells, even after culturing for 2 weeks in serum-containing medium, while WGA-labeledmotoneuronsbecame hard to be differentiated from other cells within a week. The technique using fluorescentlatex mierospheresmay be a useful method for studyingmotoneurons.
STRUCTURE OF T R I G E M I N A L GANGLION NEURONS. S H I N - I C H I TERASHIMA, P E N G J I A JIANG*, A N D Y U N - F E I LIANG*, D e p a r t m e n t of Ph[siolo~[, U n i v e r s i t y of the Ryuk[us School of Medicine, N i s h i h a r a - c h o , O k i n a w a 9 0 3 - 0 1 , Japan. H o r s e r a d i s h p e r o x i d a s e (HRP) was i n t r a s o m a l l y i n j e c t e d into s e n s o r y neurons of the trigeminal g a n g l i o n of c r o t a l i n e snakes, T r i m e r e s u r u s flavoviridis, w i t h one i n j e c t i o n per ganglion. By light and electron m l c r o s c o p y , we o b s e r v e d d e g e n e r a t e d m y e l i n a t e d and n o n m y e l i n a t e d axons, some of w h i c h w e r e HRP-labeled. The glomerular structure reported in the cat was not o b s e r v e d a r o u n d the H R P - l a b e l e d s e n s o r y neuron; however, twisting axons of other neurons w e r e found. The n a k e d part of the node of Ranvier v a r i e d in length and showed different manifestations of b i f u r c a t i o n of the stem axon. Thus the s t r u c t u r a l d i f f e r e n c e of the node of Ranvier at the b i f u r c a t i o n seemed not to be closely r e l a t e d to its modality, a l t h o u g h the d i a m e t e r of 3 axons (stem, peripheral, and c e n t r a l axons) m a y be related to the modality. Various lengths of the internodal d i s t a n c e were found. A few unmyelinated axons (66/1612 =3.8%) w e r e o b s e r v e d in the p e r i p h e r a l nerve trunk and in the root of the ganglion. This finding is q u i t e d i f f e r e n t from that in the rat r e p o r t e d by others in w h i c h the n u m b e r of u n m y e l i n a t e d fibers was more than twice as m a n y as that of myelinated. This m i g h t be due to the species difference. No b r a n c h i n g was d e t e c t e d by H R P - i n j e c t i o n into p e r i p h e r a l and central axons in the v i c i n i t y of the ganglion.