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Axonal transport in growing and mature retinal ganglion cells
Brain Research, 62 (1973) 395-397
395
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
AXONAL TRANSFORT IN GROWING A N D MATURE RETINAL GANGLION CELLS
JOHAN SJ(~STRAND, J.-O. KARLSSON AND P. C. MARCHISIO Institute of Neurobiology, University of Giiteborg, GiJteborg (Sweden) and Department of Human Anatomy, University of Turin, Turin (Italy)
The transport of axonal constituents in mature neurons has been well established. In immature nerve cells the elongation of axons is assumed to involve the continuous transport of newly formed material toward the growing tips. Since our knowledge of axonal transport in growing neurons is sparse, our present work is aimed at obtaining information about the axonal transport in immature, embryonic neurons and at studying the changes of axonal transport in the optic pathway during development. We have studied the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos during the period when the formation of retinotectal connections occurs from the 7th day up to the 18th day 1. Labeled precursor ([ZH]leucine, [3H]proline and [ZH]fucose) was injected into the right eyeball of chick embryos of the white Leghorn strain. In chicks the chiasmal crossing is virtually complete and in the main experimental series the recovery of protein-bound radioactivity was followed in the contralateral optic tectum using a liquid scintillation technique 7. In another series the axonal transport of proteins from the retina to the contralateral tectum was studied by radioautography according to techniques previously described 6. Following intraocular injection the labeled precursor was incorporated into retinal proteins at all embryonic stages studied. The turnover in the retina of both proteins and glycoproteins was exceedingly high in the 7-day embryos compared to that of later stages. In the 10-day embryos, labeled proteins and glycoproteins reached the contralateral optic tectum at 2 h after intraocular injection of precursor. Since the length of the optic pathway is 5.3 mm at the 10-day stages a transport rate of at least 60 mm/ day can be calculated. Also at day 13 and day 18 a phase of rapidly transported protein was detected in the contralateral optic tectum at 2 h following intraocular injection. Since at these stages the length ot the optic pathway is 7.9 and 9.3 mm, respectively, transport rates of at least 95 and 110 mm/day can be estimated. If the changes in the rate of rapid axonal transport are considered as a function of the embryonic age, a continuous increase in the rate of rapid flow may be observed in the optic pathway. The velocity of rapid transport almost doubles between day 10
396
J. SJOSTRANDet al.
TABLE I COMPARISON BETWEEN RETINAL INCORPORATION AND AXONAL TRANSPORT TO THE TERMINALS OF CHICK EMBRYOS~ ADULT RABBITS AND ADULT RATS
Animal
Precursor
Macromolecule
Axonal transport phase
Transport ratio*
Reference
Chick embryo 10-18 days
Leucine
Protein
Rapid
7
Fucose
Glycoprotein
Rapid
Leucine
Protein
Fucose Uridine
Glycoprotein RNA
Rapid (1) Rapid (II) Slow (IV) Rapid (I + II) Slow (IV)
Leucine Fucose
Protein Glycoprotein
Rapid Rapid
0.5 (10 days) 1.2 (13 days) 1.1 (18 days) 1.3 (10 days) 1.5 (13 days) 6.4 (18 days) 1.4 2.0 7.8 4.7 0.03 (LGB only) 1.4 8.9
Adult rabbit
Adult rat
7 4 4 4 5 3 8 Sjtistrand and Hansson, in preparation.
* Maximal amount of protein-bound radioactivity transported to the terminal region in percent of maximal amount incorporated into retinal proteins. LGB ~ lateral geniculate body.
and day 18; at the latter stage, when the chick optic pathway has attained electrophysiological signs of maturity, the rate of rapid transport is very close to that recorded in the hatched chick (at least 120 mm/day; Gremo and Marchisio, in preparation), where the visual function is considered fully mature. The changes in the rate of rapid axonal transport reported in the present paper are closely comparable to those reported for the developing optic pathway of newborn rabbits z. In embryos injected at the 7th day of incubation the protein-bound radioactivity was only slightly elevated in the left tectum at 4 h for [3H]proline and at 12 h for [aH]fucose, and no significant difference between the left and the right tectum could be demonstrated with the present technique. Since the failure to detect axonal transport by liquid scintillation technique in 7-day embryos could be due to the high background radioactivity, at this stage radioautography was used to reveal small amounts of protein-bound radioactivity which may be conveyed by axonal transport in the still relatively few axons spreading to the surface of the tectum. Both rapid and slow axonal transport of protein and glycoprotein could be demonstrated by radioautography in the 7-day embryos (see ref. 6, and Marchisio and Sj6strand, in preparation). Our results therefore indicate that axonal transport is already working efficiently early in embryogenesis before synaptic contacts have been formed. In order to study the polypeptide constituents of the rapid transport phase SDS gel electrophoresis was carried out. The electrophoresis of leucine- or fucose-labeled tectal proteins revealed that a large number of relatively high mol. wt. polypeptides
AXONAL TRANSPORTIN RETINAL GANGLIONCELLS
397
reached the optic tectum by a rapid phase of axonal transport in 13-day and 18-day chick embryos. The labeling pattern of the polypeptides was similar in 13-day and 18day embryos and no obvious differences were seen when either fucose or leucine was used as precursor. In the 18-day embryo 6 h after precursor injection a large amount of proteinbound radioactivity transported by the rapid phase may be recovered in the tectum. At this time-interval the amount of radioactivity transported to the tectum compared to the level of retinal incorporation was approximately 1.1 ~ for proline and 6.4 ~ for fucose. When the variations of this 'transport index' tor fucose and proline 6 h after injection are considered as a function of embryonic age (Table I) it may be observed that (1) the relative proportion of fucose radioactivity transported from the retina is consistently higher throughout the development, and (2) a relatively larger proportion of retinal glycoproteins is destined for rapid axonal transport at 18 days when compared to earlier stages. A comparison of the amounts of material transported to the axon terminals following the injection of different labeled precursors is shown in Table I for the adult rabbit and rat. In comparison to retinal macromolecular synthesis, rapid axonal transport in mature neurons is characterized by a relatively high content o f fucosecontaining glycoproteins.
1 COWAN,W. M., Studies on the development of the avian visual system. In D. C. PgASE(Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Univ. Press, Berkeley, Calif., 1971, pp. 177-222. 2 HENDRICKSON,A. E., AND COWAN,W. M., Changes in the rate of axoplasmic transport during postnatal development of the rabbit's optic nerve and tract, Exp. Neurol., 30 (1971) 403-422. 3 JARLSTEDT,J., ANDKARLSSON,J.-O., Evidence for axonal transport of RNA in mammalian neurons, Exp. Brain Res., 16 (1973) 501-506. 4 KARL~ON,J.-O., ANDSJSSTRAND,J., Synthesis, migration amd turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749-767. 5 KARLSSON,J.-O., AND SJOSTRAND,J., Rapid intracellular transport of fucose-containing glycoproteins in retinal ganglion cells, J. Neurochem., 18 (1971) 2209-2216. 6 MARCH~SIO,P. C., AND SJOSTRAND, J., Radioautographic evidence for protein transport along the optic pathway of early chick embryos, J. Neurocytol., 1 (1972) 101-108. 7 MARCHISIO,P. C., SJ()STRAND,J., AGLIETTA,M., ANDKARLSSON,J.-O., The development of axonal transport of proteins and glycoproteins in the optic pathway of chick embryos, Brain Research, 63 (1973) 273-284. 8 SJOSTRAND,J., ANDHANSSON,H.-A., Effect of colchicine on the transport of axonal protein in the retinal ganglion cells of the rat, Exp. Eye Res., 12 (1972) 261-269.
395
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
AXONAL TRANSFORT IN GROWING A N D MATURE RETINAL GANGLION CELLS
JOHAN SJ(~STRAND, J.-O. KARLSSON AND P. C. MARCHISIO Institute of Neurobiology, University of Giiteborg, GiJteborg (Sweden) and Department of Human Anatomy, University of Turin, Turin (Italy)
The transport of axonal constituents in mature neurons has been well established. In immature nerve cells the elongation of axons is assumed to involve the continuous transport of newly formed material toward the growing tips. Since our knowledge of axonal transport in growing neurons is sparse, our present work is aimed at obtaining information about the axonal transport in immature, embryonic neurons and at studying the changes of axonal transport in the optic pathway during development. We have studied the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos during the period when the formation of retinotectal connections occurs from the 7th day up to the 18th day 1. Labeled precursor ([ZH]leucine, [3H]proline and [ZH]fucose) was injected into the right eyeball of chick embryos of the white Leghorn strain. In chicks the chiasmal crossing is virtually complete and in the main experimental series the recovery of protein-bound radioactivity was followed in the contralateral optic tectum using a liquid scintillation technique 7. In another series the axonal transport of proteins from the retina to the contralateral tectum was studied by radioautography according to techniques previously described 6. Following intraocular injection the labeled precursor was incorporated into retinal proteins at all embryonic stages studied. The turnover in the retina of both proteins and glycoproteins was exceedingly high in the 7-day embryos compared to that of later stages. In the 10-day embryos, labeled proteins and glycoproteins reached the contralateral optic tectum at 2 h after intraocular injection of precursor. Since the length of the optic pathway is 5.3 mm at the 10-day stages a transport rate of at least 60 mm/ day can be calculated. Also at day 13 and day 18 a phase of rapidly transported protein was detected in the contralateral optic tectum at 2 h following intraocular injection. Since at these stages the length ot the optic pathway is 7.9 and 9.3 mm, respectively, transport rates of at least 95 and 110 mm/day can be estimated. If the changes in the rate of rapid axonal transport are considered as a function of the embryonic age, a continuous increase in the rate of rapid flow may be observed in the optic pathway. The velocity of rapid transport almost doubles between day 10
396
J. SJOSTRANDet al.
TABLE I COMPARISON BETWEEN RETINAL INCORPORATION AND AXONAL TRANSPORT TO THE TERMINALS OF CHICK EMBRYOS~ ADULT RABBITS AND ADULT RATS
Animal
Precursor
Macromolecule
Axonal transport phase
Transport ratio*
Reference
Chick embryo 10-18 days
Leucine
Protein
Rapid
7
Fucose
Glycoprotein
Rapid
Leucine
Protein
Fucose Uridine
Glycoprotein RNA
Rapid (1) Rapid (II) Slow (IV) Rapid (I + II) Slow (IV)
Leucine Fucose
Protein Glycoprotein
Rapid Rapid
0.5 (10 days) 1.2 (13 days) 1.1 (18 days) 1.3 (10 days) 1.5 (13 days) 6.4 (18 days) 1.4 2.0 7.8 4.7 0.03 (LGB only) 1.4 8.9
Adult rabbit
Adult rat
7 4 4 4 5 3 8 Sjtistrand and Hansson, in preparation.
* Maximal amount of protein-bound radioactivity transported to the terminal region in percent of maximal amount incorporated into retinal proteins. LGB ~ lateral geniculate body.
and day 18; at the latter stage, when the chick optic pathway has attained electrophysiological signs of maturity, the rate of rapid transport is very close to that recorded in the hatched chick (at least 120 mm/day; Gremo and Marchisio, in preparation), where the visual function is considered fully mature. The changes in the rate of rapid axonal transport reported in the present paper are closely comparable to those reported for the developing optic pathway of newborn rabbits z. In embryos injected at the 7th day of incubation the protein-bound radioactivity was only slightly elevated in the left tectum at 4 h for [3H]proline and at 12 h for [aH]fucose, and no significant difference between the left and the right tectum could be demonstrated with the present technique. Since the failure to detect axonal transport by liquid scintillation technique in 7-day embryos could be due to the high background radioactivity, at this stage radioautography was used to reveal small amounts of protein-bound radioactivity which may be conveyed by axonal transport in the still relatively few axons spreading to the surface of the tectum. Both rapid and slow axonal transport of protein and glycoprotein could be demonstrated by radioautography in the 7-day embryos (see ref. 6, and Marchisio and Sj6strand, in preparation). Our results therefore indicate that axonal transport is already working efficiently early in embryogenesis before synaptic contacts have been formed. In order to study the polypeptide constituents of the rapid transport phase SDS gel electrophoresis was carried out. The electrophoresis of leucine- or fucose-labeled tectal proteins revealed that a large number of relatively high mol. wt. polypeptides
AXONAL TRANSPORTIN RETINAL GANGLIONCELLS
397
reached the optic tectum by a rapid phase of axonal transport in 13-day and 18-day chick embryos. The labeling pattern of the polypeptides was similar in 13-day and 18day embryos and no obvious differences were seen when either fucose or leucine was used as precursor. In the 18-day embryo 6 h after precursor injection a large amount of proteinbound radioactivity transported by the rapid phase may be recovered in the tectum. At this time-interval the amount of radioactivity transported to the tectum compared to the level of retinal incorporation was approximately 1.1 ~ for proline and 6.4 ~ for fucose. When the variations of this 'transport index' tor fucose and proline 6 h after injection are considered as a function of embryonic age (Table I) it may be observed that (1) the relative proportion of fucose radioactivity transported from the retina is consistently higher throughout the development, and (2) a relatively larger proportion of retinal glycoproteins is destined for rapid axonal transport at 18 days when compared to earlier stages. A comparison of the amounts of material transported to the axon terminals following the injection of different labeled precursors is shown in Table I for the adult rabbit and rat. In comparison to retinal macromolecular synthesis, rapid axonal transport in mature neurons is characterized by a relatively high content o f fucosecontaining glycoproteins.
1 COWAN,W. M., Studies on the development of the avian visual system. In D. C. PgASE(Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Univ. Press, Berkeley, Calif., 1971, pp. 177-222. 2 HENDRICKSON,A. E., AND COWAN,W. M., Changes in the rate of axoplasmic transport during postnatal development of the rabbit's optic nerve and tract, Exp. Neurol., 30 (1971) 403-422. 3 JARLSTEDT,J., ANDKARLSSON,J.-O., Evidence for axonal transport of RNA in mammalian neurons, Exp. Brain Res., 16 (1973) 501-506. 4 KARL~ON,J.-O., ANDSJSSTRAND,J., Synthesis, migration amd turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749-767. 5 KARLSSON,J.-O., AND SJOSTRAND,J., Rapid intracellular transport of fucose-containing glycoproteins in retinal ganglion cells, J. Neurochem., 18 (1971) 2209-2216. 6 MARCH~SIO,P. C., AND SJOSTRAND, J., Radioautographic evidence for protein transport along the optic pathway of early chick embryos, J. Neurocytol., 1 (1972) 101-108. 7 MARCHISIO,P. C., SJ()STRAND,J., AGLIETTA,M., ANDKARLSSON,J.-O., The development of axonal transport of proteins and glycoproteins in the optic pathway of chick embryos, Brain Research, 63 (1973) 273-284. 8 SJOSTRAND,J., ANDHANSSON,H.-A., Effect of colchicine on the transport of axonal protein in the retinal ganglion cells of the rat, Exp. Eye Res., 12 (1972) 261-269.