Brain Research, 85 (1975) 273-280

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

273

RETROGRADE INTRAAXONAL TRANSPORT OF HORSERADISH P E R O X I D A S E IN R E T I N A L G A N G L I O N CELLS OF T H E C H I C K

MATTHEW M. LAVAIL AND JENNIFER H. LAVAIL Department of Neuropathology, Harvard Medical School and Department of Neuroscience, Children's Hospital Medical Center, Boston, Mass. 02115 (U.S.A.)

Since the 1920's several investigators have noted the bidirectional movement of vesicles within neurites in tissue culture26,28, 80. Other investigators have demonstrated the retrograde movement of materials within axons more clearly by showing the accumulation of axoplasm as well as radioactive and endogenous chemical markers in both the proximal and distal portions of constricted or severed peripheral nerves 2, 10,11,~2,27,z8. Recently Kristensson and coworkers 1s-21 have demonstrated morphologically that when the exogenous proteins, Evans blue-labeled albumin or horseradish peroxidase (HRP) are injected into the vicinity of neuromuscular junctions, they are taken up and subsequently found within the cell bodies whose axons project to the injection sites. We have extended these observations to include neurons of the central nervous system 2a-25. We previously considered the transport to be intraaxonal because we found vesicles containing H R P within chick retinal ganglion cell axons soon after tectal injection of the protein marker 23,24. A similar intracellular localization in rat optic nerve has been noted by Hansson is. This report deals with the cellular organelles and mechanisms involved in the uptake and retrograde transport of H R P in chick retinal ganglion cells. H R P (Type VI, Sigma Chemical) dissolved in saline (0.5 mg in 2-25/~l) was injected into the right optic tectum of thirteen 1-7-day-old chicks. After survival times of 0.5-26 h the chicks were perfused with formaldehyde-glutaraldehyde fixative in phosphate buffer (pH 7.2). Following overnight fixation, dissection and an overnight wash in phosphate buffer (pH 7.2) with 5 ~ sucrose (w/v) added, 40-#m frozen sections were taken in a plane which included the injection site, optic tracts and optic nerves. After cytochemical reaction of the sections and unfrozen slices of retina to demonstrate the presence o f peroxidase, reaction product could be seen in the lateral surface of the tectum at the injection site (Fig. 1). The frozen sections and retinal slices were then post-fixed in osmium tetroxide and embedded in an Epon-Araldite mixture, and different regions of the retino-tectal pathway were examined either in cross-section or longitudinal section by electron microscopy. Further details of the histochemical and cytological procedures have been presented elsewhere ~4. Retinal ganglion cell axons terminate in the superficial one-third of the contralateral optic tectum 8. In axon terminals of this region, 30 min to 2 h after injection,

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Fig. 1. Light micrograph of a 40-ttm frozen section illustrating the distribution of H RP after injectior~ into the left optic tectum. The small dark spots throughout the tissue are red blood cells which show endogenous peroxidase activity, ot, optic tract; oc, optic chiasm; on, optic nerve. Section not counterstained. × 5. (From LaVail and LaVail2~.) Figs. 2-4. Electron micrographs from the superficial third of the optic tectum in the region of retinal ganglion cell terminals 1.5 h after tectal injection of HRP. (From LaVail and LaVai124.) Fig. 2. HRP is present in tubules (t) and small vesicles within axon terminals (arrows). In the terminal at the right some of the HRP-filled vesicles are apposed to the presynaptic membrane. ::. 38,780. Fig. 3. A tubular multivesicular body containing HRP is present in an axon terminal, and it is fused with one HRP-positive synaptic vesicle (arrow). :~', 48,800. Fig. 4. A large pinocytotic vesicle is present in an unmyelinated axon (arrow). , 38,780.

H R P reaction p r o d u c t was f o u n d in a small percentage o f the 4 5 - 5 0 - n m p r e s u m e d synaptic vesicles (Figs. 2 and 3), in tubules (Fig. 2) a n d in t u b u l a r multivesicular bodies (Fig. 3). L a r g e r 100-125-nm vesicles c o n t a i n i n g H R P were u n c o m m o n in the terminals, b u t they were often seen in dendrites, axons (Fig. 4) and n e u r o n a l cell bodies a n d a p p e a r e d to be f o r m e d by p i n o c y t o s i s as c o a t e d vesicles. In the s t r a t u m o p t i c u m near the injection site, 30 min after injection, H R P reaction p r o d u c t was f o u n d in the extracellular spaces a r o u n d a l m o s t all o f the u n m y e l i n a t e d axons, in the external a n d internal m e s a x o n s a n d in the p a r a a x o n a l spaces o f m y e l i n a t e d axons. T h e enzyme was t a k e n up in large vesicles by superficial glial cells, a n d it was also present diffusely in some m y e l i n a t e d a n d u n m y e l i n a t e d axons. A t this time H R P was also evident within some 100-125-nm m e m b r a n e - b o u n d vesicles a n d c u p - s h a p e d organelles within the axons. A l t h o u g h a x o n a l p i n o c y t o s i s in the s t r a t u m

275 opticum, optic tract and optic nerve occurred, it was a rare finding compared to pinocytotic activity in the region of axon terminals. In 60 fields of stratum opticum which were examined, each containing cross-sections of about 400-600 axons, only two apparent pinocytotic profiles in axons were observed. In contrast, several pinocytotic configurations were seen in almost every micrograph from the region of axon terminals. HRP was found within 4 types of membrane-bound organelles in the myelinated and unmyelinated axons of the ipsilateral stratum opticum and optic tract and contralateral optic nerve and optic fiber layer of the retina. These were (1) vesicles approximately 100 nm in diameter (Figs. 5-7 and 12), (2) multivesicular bodies (Figs. 8 and 9), (3) cup-shaped organelles (Fig. 10), which are thought to be precursors of multivesicular bodiesl,12,1s, 17, and (4) tubules of the agranular reticulum (Fig. 11). Seventy randomly chosen vesicles in the optic nerves and optic tracts had an average crosssectional diameter of 99.1 nm, ranging from 44.5 to 220 nm. Although some of the smaller, less frequently observed vesicles may have been cross-sections of tubules or synaptic vesicles, they did not have the smooth, circular profiles usually observed in synaptic vesicles in axon terminals (Figs. 2 and 3). In longitudinal sections of the optic tracts and nerves, most of the HRP-filled vesicles were ovoid or somewhat elongate, and no spherical, 50-nm synaptic vesicles were observed. Most of the HRP-containing vesicles within axons of the optic tract and optic nerve were in close proximity to one or more microtubules. Almost half had 3 or more microtubules partially surrounding them (Fig. 6), and in one instance a regular array of microtubules completely surrounded a vesicle in transit (Fig. 7). The vesicles sometimes touched the microtubules (Fig. 7, large arrow), and on occasion possible cross-bridges 35 extended between the vesicle and microtubules. Vesicles lacking reaction product also sometimes contacted microtubules (Fig. 7, small arrow). Some vesicles containing HRP were also in close proximity to neurofilaments (Fig. 6), but less frequently than to microtubules. The HRP-containing organelles (or the HRP within organelles) apparently move within axons toward the cell body, since no extracellular HRP was found in the optic nerves of a chick fixed 7 h after tectal injection, yet numerous intraaxonal, HRP-containing organelles were present in the contralateral nerve. As in all of the experiments, no HRP-filled organelle was found within axons of the ipsilateral optic nerve. Furthermore, in no instance were axons in either optic nerve or optic tract diffusely filled with HRP. In a few experiments involving either large doses of HRP injected into the tectum or long survival times, extracellular HRP diffused from the injection site into the optic nerves, but in no instance did it diffuse into either retina. Within the retina contralateral to the injection site, 5 h or longer after HRP injection, HRP-containing vesicles were found in ganglion cell axons in the optic fiber layer, ganglion cell bodies in the ganglion cell layer (Fig. 13) and in displaced ganglion cell bodies in the inner nuclear layer. (Displaced ganglion cells have recently been demonstrated in the rat retina by the use of retrograde axonal transport of HRPS.) In the ganglion ceils located within the ganglion cell layer, many of the vesicles were observed coalescing with one another (Fig. 14) to form structures large enough to be readily visible with the light microscope. By 24 h many vesicles were at

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277 least 0.5 # m with some almost 1 # m in diameter. N o diffuse H R P was found in any ganglion cell. Many of the vesicles were found near the inner aspect of the Golgi complex in the supranuclear (outer) portion of the ganglion cells (Fig. 15). Presumably after fusion o f lysosomes with the vesicles z3 the peroxidase is degraded or inactivated. Little or no activity was found by light microscopy in retinal ganglion cells of a number of chicks with survival times longer than 4 days 24. What we have demonstrated is that when the extracellular tracer, HRP, is injected into the vicinity of axon terminals, it is taken up by pinocytosis by axon terminals and unmyelinated portions of axons in the region of the terminals. The enzyme is then localized in membrane-bound vesicles, tubules of the agranular reticulum, multivesicular bodies and their precursors, and either these organelles or the H R P within them are transported back to the parent cell body at a rate we had previously shown to be at least 72 mm/day 23. Organelle transformations may occur with the axons 1,t7,36 (e.g., from tubule to vesicle), so that we cannot say that the H R P found in the agranular reticulum is in a continuous 'transport channel' running from the terminal to the cell body. N o HRP-containing synaptic vesicles appeared to be transported back to the cell body, since none were found within axons; however, they may fuse with one another in the terminals to form larger vesicles or with multivesicular bodies which are then transported to the cell body. Alternatively, membrane which is recycled locally in the terminals3,6,14,16, 32 may be independent of that which is transported back to the cell body. The hypothesis that microtubules may be involved in the retrograde axonal transport mechanism is supported by the frequent observation of a morphological association between microtubules and vesicles and by the fact that colchicine, when injected into the chick eye, blocks retrograde transport of H R P from the contralateral tectum 24. Vinblastine has a similar effect in retinal ganglion cells of the rat 4, and Kristensson and Sj6strand 21 have found that retrograde transport in peripheral nerves is blocked by application of colchicine. These studies do not provide definitive p r o o f of

Figs. 5-11. Electron micrographs of the ipsilateral optic tract (Fig. 8) or contralateral optic nerve (others) either 7.3 h (Fig. 10) or l0 h (others) after a tectal injection of HRP. (Figs. 6, 7, 11 and 12 from LaVail and LaVail24.) Fig. 5. A large HRP-filled vesicle is present (arrow) within a myelinated axon. Fig. 6. An HRP-containing vesicle is shown in an unmyelinated axon, partially surrounded by microtubules (between arrows) and neurofilaments (n). × 76,300. Fig, 7. One HRP-positive vesicle in an unmyelinated axon is surrounded by a regular array of microtubules and contacts one of them (large arrow). A smaller vesicle free of HRP contacts another microtubule (small arrow). × 48,690. Fig. 8. A multivesicular body is present in an unmyelinated axon. × 61,620. Fig. 9. A tubular multivesicular body is shown in longitudinal section. × 46,250. Fig. 10. A cup-shaped organelle is present in an unmyelinated axon. × 79,050. Fig. 11. Longitudinal section of axons showing an agranular tubule partially filled with HRP (arrow) and one free of HRP (t). × 79,040. Fig. 12. Optic fiber layer of a retina contralateral to tectal injection of HRP, 10 h after injection. HRP-positive structures of various sizes and shapes are present within ganglion cell axons, but no HRP is evident extracellularly or in M~ller cell processes (rap). ilm, inner limiting membrane. × 17,185.

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Figs. 13-15, Electron micrographs of the retina contralateral to a tectal injection of HRP, 10 h after injection. (From LaVail and LaVail 2 L) Fig. 13. The ganglion cell layer is shown with many of the larger, HRP-containing vesicles (large arrows) located in the supranuclear (outer) region of the ganglion cell somas. Smaller HRP-positive vesicles are found in the ganglion cell axons within the optic fiber layer (small arrows). ;,: 3700. Fig. 14, HRP-containing vesicles appear to have coalesced into larger, more irregularly shaped structures (arrows). u 27,200. Fig. 15. Vesicles containing HRP accumulate near the Golgi membranes (g) in the supranuc[ear region of a ganglion cell. n, nucleus. >: 21,300,

279 microtubule involvement, however, since we do not yet know whether these alkaloids are affecting uptake, transport or some other metabolic system of the cells 37 (for review see ref. 29). Furthermore, in chick ganglion cell axons the frequent physical association of microtubules and HRP-filled vesicles may be an artifact of the small caliber of the axons. A number of regulatory reactions such as chromatolysis after axon injury a, retrograde transsynaptic changes 7, acute glial reactions 34, and growth regulation according to the size of the peripheral field 31 imply the existence of intracellular communication from the axons (and/or terminals) to the cell nucleus. Retrograde intraaxonal transport may provide the means for such communication. The transport mechanisms also may have other functions such as membrane reutilization 17. At present the role(s) of retrograde intraaxonal transport remains speculative. N o t e added in proof. P. T. Turner and A. B. Harris (Brain Research, 74 (1974) 305-326) have recently reported a similar time course and similar organelles involved in the uptake of H R P by cells in the rabbit, cat and monkey cerebral cortex as we have found in the chick optic tectum.

This work has been supported in part by United States Public Health Service Research Grants EY 01202 (M.M.L.) and NS 11237 (R. L. Sidman), Special Fellowship NS 02702 (J.H.L.) and The Children's Hospital Medical Center Mental Retardation and H u m a n Development Research Program Grant H D 03773.

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Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick.

Brain Research, 85 (1975) 273-280 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands 273 RETROGRADE INTRAAXONAL TRANS...
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