Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine

Brain Research, 60 (1973) 93-127 © Elsevier Scientific Pub"ushmg " Company, Amsterdam

93 Printed in The Netherlands

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN TO NERVE ENDINGS. I. RADIOAUTOGRAPHIC ANALYSIS OF THE RENEWAL OF PROTEIN IN NERVE ENDINGS OF CHICKEN CILIARY GANGLION AFTER INTRACEREBRAL INJECTION OF [aH]LYSINE

BERNARD DROZ, HERBERT L. KOENIG AND LUIGI DI GIAlVlBERARDINO

Ddpartement de Biologie, Commissariat d l'Energie Atomique, C.E.N. Saclay, 91190 Gif-sur-Yvette and Laboratoire de Cytologie, Universit~ Paris VI, 75005 Paris (France) (Accepted February 28th, 1973)

SUMMARY

The axonal transport of protein to nerve endings has been investigated in the giant presynaptic calices of the avian ciliary ganglion by means of high resolution radioautography. At early time intervals alter the intracerebral injection of [aH]lysine in the vicinity of the nerve cell bodies, labeled proteins, scattered along the axons, are conveyed at rates of about 288 ram/day to presynaptic calices, in which they spend about 17 h. One fraction of the rapidly transported proteins collects in presynaptic areas containing synaptic vesicles and presynaptic plasma membranes and, to a lesser extent, in mitochondria; another fraction is retained in axons, especially in the axolemmal region. When fast moving labeled proteins are arrested above a local compression, they accumulate in axons and appear to be associated with membranous profiles. Later on, a bulk of labeled proteins, distributed in the axoplasm and mitochondria, migrate along the axons at rates of 1.5-10 ram/day. The great majority of slowly moving labeled proteins is retained in the axons with a life-span of 22 days; a minor part reaches the presynaptic calices in which they turn over within 14.5 days. These labeled proteins are collected in regions of axoplasm devoid of synaptic vesicles and in mitochondria. When the synthesis of protein is inhibited after a local application of puromycin to a ciliary ganglion, very small amounts of label are found in the postsynaptic perikaryon. This fact would indicate that only a restricted number of protein or peptide molecules would be transferred from pre- to postsynaptic structures. In conclusion, fast moving proteins are mainly assigned to the renewal of various membrane components (synaptic vesicles, presynaptic and axolemmal plasma membranes, mitochondria, endoplasmic reticulum) whereas the slowly migrating proteins purvey mainly the replacement of the axoplasmic components and mitochondrial organelles.

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INTRODUCTION

The macromolecular components of nerve endings may originate from a triple source: a local synthesis in the axon terminal, an exchange of material with the surrounding microenvironment and an arrival of migratory proteins transported along the axon after their synthesis in the nerve cell body. Among these possible mechanisms, the last-mentioned ensures the renewal of the great majority of proteins present in various types of nerve ending14,1L Biochemical and radioautographic investigations performed in the central and peripheral nervous system have provided information dealing with various aspects of the axonal migration of proteins. According to these studies, the rates of axonal migration could be subdivided into two 21,3~,al,aS, three s,51 or four phases 29 and even into a wide spectrum of speeds 4. However, the heterogeneity of the rates of axonal migration could also reflect the heterogeneity of the axonal population composed of neuronal processes of different length and diameter and using chemical transmitter of possibly different nature. This alternative may be cleared up by examining a neuronal system possessing more homogeneous characteristics. Proteins transported at fast rate were found to be mainly associated with *particulate' fractions in homogenates of cerebral tissues, nerve fibers and syr ~tosomes3,S,16,'-'9,al,4L Electron microscope radioautographs have revealed that the fast moving protein molecules were generally located in peripheral regions of the axons '-'6,a6,5'-'and could be related to the presence of various axonal components such as the axolemma 5", axoplasma6, 55, a mitochondria-endoplasmic reticulum compartmer.t 25, the agranular endoplasmic reticulum 5'-' and amine storage vesicles of large diameterlg,sL Once arrived at nerve endings, the fast moving proteins have been detected in areas containing synaptic vesiclesla,'-'5,52,~L mitochondria la,'-'5,s2, microfilaments 52, preterminal membranesa'-', 5~ and peripheral axoplasm a6. The life span of fast moving proteins in nerve endings has been estimated to range between a few hours or daysS, '-'9 and several monthsXO,21. Proteins transported at slow rates were mainly recovered in 'soluble' and 'mitochondrial' fractionsa,S,27,~9,4~,aS. Electron microscope radioautographs of various axons have indicated that most of the slowly moving proteins were associated with the axoplasm x~,28, including bundles of microfilaments 11, the axolemma 11, the endoplasmic reticulum and mitochondrialX,2L The question has been recently raised whether slowly moving proteins enter nerve endings and take a significant part in the renewal of presynaptic components8,~3,29, a2 or whether they do not invade the synaptic terminals and remain within the axon2~, °-8 before being catabolized. Finally, the possibility that protein molecules move from presynaptic terminals to postsynaptic structures2,Zo,-~,'-'5,34,aa has been reported. However it is essential to make sure whether proteins have been transferred as macromolecules or whether free labeled amino acids, released from radioactive protein broken down in nerve endings, have been reutilized for the synthesis of new proteins in postsynaptic structures e2. We have thought that individual nerve endings of large size would possess advantageous features to study with radioautography the localization, kinetics and

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fate of fast and slowly migrating proteins. Furthermore, a comparison of the behavior of the tracer after inhibition or not of the synthesis of protein would provide a decisive argument for or against the transsynaptic passage of proteins. The preganglionic neurons forming giant nerve endings in the avian ciliary ganglion fulfil the conditions required for this study: (1) the nerve cell bodies are grouped in the Edinger-Westphal nucleus (accessory oculomotor nucleus) in the vicinity of the cerebral aqueduct and are therefore easily reached by the tracer deposited in the cerebral ventricle; (2) the preganglionic axons have a 10 mm length and 9 #m diameter; (3) each preganglionic axon terminates in the ciliary ganglion by forming one giant nerve ending, the presynaptic calyx, which encompasses each large ganglion cell body; (4) in the same animal, one of the ciliary ganglia submitted to experimental conditions (puromycin, local pressure) may be compared with the second one used as control; (5) the transmission of nerve impulses is known to be mediated by acetylcholine and by electrical coupling 4°. MATERIALS AND METHODS

(I) Main experhnent Two-week-old chickens (Leghorn) weighing 70 ± 10 g were lightly anesthetized with chloroform and held in a stereotaxic apparatus. 400 #Ci of L-[4-aH]lysine (specific activity: 30 Ci/mmole, Commissariat/t l'Energie Atomique, Saclay, France) diluted in

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Fig. I. Diagrammatic representation of the administration of labeled precursors to the preganglionic neurons of the ciliary ganglion in chickens. The radioactive tracer injected into the cerebral aqueduct reaches the nerve cell bodies of the preganglionic neurons located in the Edinger-Westphal nucleus

near the cerebral aqueduct. The preganglionic axons follow the pathway of the oculomotor nerve OlD and terminate in the ciliary ganglion by forming giant presynaptic calices.

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50 ,ul of saline were injected during 1 min into the cerebral aqueduct of the brain (Fig. 1). At each time interval, ranging from 1.5 h to 23 days after the injection, 3-5 chickens were sacrificed by decapitation. The periaqueductal region of the midbrain and the left ciliary ganglion were excised to measure the specific radioactivity of protein; the right ciliary ganglion was processed for radioautography. (A) Radioactivity assays. To determine the specific radioactivity of protein, the left ciliary ganglia of the chickens sacrificed at each time interval were pooled and then homogenized in 250 ~d of water. A volume of 250/~1 of a solution containing 20% PCA and 0.12 mM of L-lysine was added to the homogenate and after storage for 1 h at 0 °C, the mixture was centrifuged for 15 min at 4000 × g. The pellet was washed and resuspended in 0.5 ml of a solution containing 10 % PCA and 0.06 mM of L-lysine and recentrifuged. The pellet, dissolved in 0.5 ml of a 0.1 N NaOH solution, was referred to as the 'PCA-precipitable' fraction. The supernatants obtained after the two centrifugations were pooled and referred to as the 'PCA-soluble' fraction. Volumes of 250/~1 of the 'PCA-precipitable' and of the 'PCA-soluble' fractions were respectively mixed with 10 ml of Bray's scintillation mixture~; the vials containing the 'PCAsoluble' fractions were neutralized with 60/~1 of a 5 N NaOH solution. The counting efficiency was tested by spiking each vial with tritiated water. The amount of protein contained in the ~PCA-precipitable' fractions was measured by the method of Lowry et aL as and the radioactivity for both the 'PCA-precipitable' and the 'PCA-soluble' fractions was expressed as disint./min/mg of protein in the homogenate. In each animal, the radioactivity of the 'PCA-precipitable' and 'PCA-soluble' fractions of the midbrain was measured according to the same procedure. The percentage of radioactivity due to [aH]lysine in the 'PCA-soluble' fraction of the midbrain was determined as follows. Volumes of 250/~1 of each sample were evaporated at 100 °C in the vial. The residue was resuspended in 10 ml of Bray's scintillation mixture and the radioactivity counted referred to as the non-volatile fraction. The percentage of radioactivity due to [aH]lysine in the non-volatile fraction was determined by thin layer chromatography (Eastman Chromogram sheet 6040 cellulose; solvent pyridine-acetic acid-water, 50:35:15). The percentage of radioactivity due to [aH]lysine in the 'PCA-precipitable' fraction was determined after hydrolysis by 6 N HCI at 100 °C for 24 h in vials sealed under vacuum and after chromatography of the hydrolysate. (B) Light microscope radioautography. The ciliary ganglia were fixed by immersion in 4 % formaldehyde phosphate buffered to pH 7. I, postfixed in osmium tetroxide in the same buffer, dehydrated and embedded in Epon, care being taken to obtain coronal sections of the middle part of the ganglia. After polymerization (16 h at 60 °C), l-/zm thick sections were cut, deposited on glass slides and coated by dipping aa in llford K5 emulsion diluted 1:1. After 1 and 2 months of exposure, the radioautographs were processed in D-19 and poststained with toluidine blue. The radioactivity concentration was measured in various structures by counting the number of silver grains per 100 sq./~m with an ocular grid (Fig. 11). After a 2-month exposure, the radioautographs were photographed at the same magnification (Figs. 5-10). The relative volume of the different structures found in the ciliary ganglion (Table I) was

AXONAL MIGRATIONOF PROTEIN AND GLYCOPROTEIN. [

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TABLE 1 RELATIVE VOLUME OF THE VARIOUS STRUCTURES PRESENT IN CORONAL SECTIONS OF CILIARY GANGLIA

The relative volume of each structure is expressed as percent of hits~counted from radioautographed sections of ciliary ganglia removed 6 and l0 days after the intracerebrai injection of [aH]lysine. At those times, the preganglionicaxons are heavilylabeled and can therefore be easily distinguishedfrom the unlabeled postganglionicaxons. Six ciliary ganglia were used and 640 hits counted for each ganglion in this study.

Percent Preganglionic axons Presynaptic calices Ganglion cell bodies Postganglionic axons Schwann cells, myelin sheath, blood vessels, connective tissue

13.2 ± 1.1 7.2 '= 1.0 19.8 ! 1.7 15.8 ± 2.0 44.0 -4- 1.7

determined by the hit method of Chalkley 7. Spinal ganglia of the dorsal cord were also removed and radioautographed as control. (C) Electron microscope radioautography. Minute areas of selected blocks were then chosen for electron microscopic examination. The blocks were retrimmed accordingly, polymerized further (14-30 h at 60 °C) and cut on the LKB Ultrotome UM3. Ribbons of uniformly thin sections were deposited on glass slides previously coated with a celloidin film, stained with uranyl acetate and lead citrate, covered with a thin layer of vaporized carbon 49 then dipped into llford L4 emulsion diluted 1:42a. After a 2-month exposure at 4 °C, the radioautographs were processed in phenidon developer aT, collected on copper grids and examined with a Siemens Elmiskop I, after the celloidin membrane had been thinned in isoamyl-acetate. For quantitative evaluation, sections of similar thickness were prepared, exposed and developed together. At each time interval, all the silver grains found over the calices were counted. The grains located over one organelle were attributed to this organeUe as 'exclusive' whereas those overlying more than one type of structure were classed as 'shared'. These crude grain counts are recorded in Table II. The 'shared' grains were then assigned to each structure by dividing their count by the number of structures overlain 57, except in the following cases. First, owing to the very low frequency of grains observed over the postsynaptic perikarya and the glial cells, the shared grains associated with presynaptic plasma membranes and postsynaptic perikarya, on the one hand, and those associated with lateral plasma membranes covering the remainder of the calices and gila! cells, on the other, were assigned to presynaptic and lateral plasma membranes respectively. Second, in the regions of the calices containing synaptic vesicles, it was important to determine which part of the labeling was taken by the synaptic vesicles and by the embedding axoplasm 42,5s. Assuming that the concentration of the label is the same in the axoplasm embedding the synaptic vesicles as in the

n. DROZet al.

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axoplasm free of synaptic vesicles, an approximate value of the percent of grains Nv associated with synaptic vesicles may be derived: Nv = Na + v - - Na

Va'

Va

in which Na + v is the percent of grains counted over regions containing synaptic vesicles mixed with axoplasm; Na is the percent of grains counted over regions containing only axoplasm; Va' Va

is the ratio of the volumes of axoplasm containing synaptic vesicles and of axoplasm free of synaptic vesicles. The percent corrected grain counts are reported in Table Ill. Finally, the changes of label content of each presynaptic structure with time (Fig. 23) were obtained by multiplying the percent corrected grain counts (Table Ill) by the total grain concentration measured in calices with light microscopic radioautographs (Fig. 1l). The percent corrected grain counts (Table lid and the curves of the label content (Fig. 23) obtained by this method do not provide a measure of the true distribution and of the true content of radioactive proteins in each cell organelle but reflect the time sequence of the changes of labeling in subcellular structures of the presynaptic calices.

(H) Complenlentary experiments In 4 chickens, the synthesis of protein was inhibited in one ciliary ganglion by local application of puromycin. The right ciliary ganglion was wrapped in a thin piece of gelfoam, soaked with a 5 . 1 0 -a M puromycin solution, 30 min prior to the intracerebral injection of 400 #Ci of L-[4-aH]lysine diluted in 70 #1 of saline; the left ciliary ganglion was used as control (Table IV). Three and 48 h later, the ciliary ganglia were removed and prepared for light and electron microscopic radioautography. In one additional chicken, a slight compression was exerted on the right ciliary ganglion by applying a larger piece of gelfoam previously soaked with puromycin; under these conditions, the ciliary ganglion was progressively squeezed against the orbital wall. After 30 min, [aH]lysine was intracerebrally administered and the animal was sacrificed 3 h later. RESULTS

(I) Main experiment (A) Radioactivity assays The specific radioactivity (SRA) of the protein was calculated for the periaqueductal region of the midbrain and the ciliary ganglia at various time intervals (Fig. 2). Whereas a maximum was reached around 18 h in the midbrain (that is in a region close to the site of injection), the ciliary ganglia showed only a moderate and

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irregular increase of the SRA of protein. At 6 days after injection, when labeling of the midbrain had considerably diminished, a peak of SRA occurred in protein of ciliary ganglia. The decay rate of the specific radioactivity of protein in the midbrain and ciliary ganglia would correspond to a mean life of 14 days. The analysis of the hydrolysate of the 'PCA-precipitable' fractions of the midbrain indicated that all the radioactivity detectable in the chromatogram was bound to lysine. In contrast, the radioactivity of the 'PCA-soluble' fraction declined gradually with time in both structures and was about 20 times lower in the ciliary ganglia than in the midbrain. The analysis of the ?CA-soluble' fractions in midbrain homogenates (Fig. 3) showed that the [aH]lysine content decreased with time, whereas a relative increase was noted in the percentage of labeled volatile compounds (probably tritiated water) and in labeled non-volatile compounds (probably tritiated metabolites).

(B) Light microscopic radioa.tographs The preganglionic axons of 8-10/tm diameter displayed only scattered silver grains at 1.5 h after the intracerebral injection of [aH]lysine (Fig. 5); the concentration

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of radioactivity rose gradually, but at a slow rate until 24 h (Figs. 6 and 7), then was tremendously enhanced (Fig. 8) and reached a maximum at 6 days (Fig. 9); finally, the label content of the axons declined progressively (Figs. 10 and ! 1) at a rate corresponding to a turnover time of about 22 days. The preterminal segments of the preganglionic axons lose their myelin sheath before giving rise to the calyx; at each time interval, the preganglionic axons exhibited a stronger reaction in their preterminai segment than in regions located farther from the nerve terminals (Figs. 4, 7 and 11). The axon terminals of all ciliary ganglia prepared for radioautography between 1.5 h and 23 days after the intracerebral injection of [3H]lysine were the sites of a strong radioautographic reaction at all the time intervals examined. The ciliary ganglia of the chicken contain at least 2 types of axon terminals characterized by their size, their distribution and the diameter of the postsynaptic ganglion cell body30: minute nerve endings, appearing as 'boutons', are in synaptic junction with small ganglion ceils (25 pm) occupying a lateral and limited portion of the ciliary ganglion; huge nerve endings, forming a giant presynaptic calyx (Fig. 4), encompass each large ganglion cell body (45 pm) encountered throughout the major part of the ganglion. The intensity of the reaction appeared to be fairly similar in all giant presynaptic calices, whereas a weaker labeling was found in the minute nerve endings in contact

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Fig. 4. Light microscope radioautograph of a ciliary ganglion 18 h after the intracerebral injection of [aH]lysine. The radioautographic reaction, moderate over the large and myelinated preganglionic axons (Ax) is more pronounced over their unmyelinated preterminal segment (~,). An intense accumulation of silver grains occurs over the presynaptic calices (Ca) which encompass the postsynaptic perikarya (GC). The ganglion cell bodies (GC) display a slight reaction; only few silver grains are seen over the small postganglionic axons (upper right corner), the glial cells (S) and connective tissue cells (lower left corner).

with small perikarya. Our radioautographic study was therefore restricted to the giant presynaptic calices. From 1.5 h (Fig. 5) to 6 h (Fig. 6) after the intracerebral injection, silver grains were most heavily accumulated over the calices and their counts rose steeply to reach a maximal concentration of the label by 18 h (Figs. 7 and 11). By then, the radioactivity had attained levels unexceeded by labeling in any other structure at any time interval. From 18 to 48 h, labeling dropped off precipitously in axon terminals (Figs. 8 and i 1). The decay rate of the label obtained after subtracting the values corresponding to the later increase of radioactivity indicates that one part of the labeled material would stay in calices for a mean time of sojourn of 17 h (turnover

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Figs. 5-10. Light microscope radioatltographs of ciliary ganglia at various time intervals after the intracerebral injection of [3H]lysine. Fig. 5. At 1.5 h, the reaction is already intense over the presynaptic calices (Ca) but rather weak over the preganglionic axons (Ax). Fig. 6. Six hours later, the accumulation of label in presynaptic calices is intensified. Fig. 7. By 18 h, a very strong reaction is noted over the oresynaptic calices and the preterminal segment (~) of the preganglionic axons. Large myelinated t 'ganglionic axons (Ax) contain also more label than at earlier time intervals. Fig. 8. After 36 h, the reaction increases over the preganglionic axons but diminishes over the presynaptic calices. Fig. 9. At 6 days, the calices display again a strong reaction; each preganglionic axon is covered with numerous silver grains. Fig. 10. By 16 days, the intensity of the reaction decreases over the calices and to a lesser extent over the preganglionic axons. At each time interval, note the weak but constant labeling of the ganglion cell bodies (GC) and the very small number of silver grains over glia! cells, postgang!ionic axons and connective tissue.

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Figs. 12 and 13. Electron microscope radioautographs of myelinated preganglionic axons 1.5 h and 2 days after the intracerebral injection of [aH]iysine, Fig. 12. At 1.5 h, only occasional axons show some label, in this case silver grains are located over or near axolemma (axl) and profiles of smooth endoplasmic reticulum (ser). Fig. 13. By 2 days, the axons are heavily labeled. N~merous silver grains are distributed over the axoplasm (ax) containing microfilaments; some grains appear related to mitochondria (mi). Fig. 14. Part of a caliciform nerve ending after fixation with glutaraldehyde. Among the numerous synaptic vesicles, coated vesicles (~), dense core vesiclesof various types (~), tubular profiles ( ~" ), and large vesicles ( ",~,) may be seen. Microtubules (mr) and microfilaments (mr) are also embedded in the axoplasmic matrix of the calyx (Ca). Postsynaptic perikarya: GC.

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rate: 0.1 • 10-2 min-1). This prominent but short-lived increase of radioactivity was followed by a later and slower rise culminating at 6 days after injection (Fig. 9). Finally, the concentration of the label declined gradually (Figs. 10 and 11) at a rate indicating a mean time of sojourn of 14.5 days (turnover rate: 7 • 10-2 day-X). The postsynaptic perikarya (ciliary ganglion cell bodies) were the sites of a weak but constant labeling (Figs. 5-11), slightly more pronounced than in the spinal ganglion cells used as control. In both ganglion cells, the dim perikaryal reaction diminished slowly with time. The postganglionic nerve fibers remained mostly unreactive at any time intervals. Only occasional silver grains were found over glial cells, myelin sheath, connective tissue cells and blood vessels.

(C) Electron microscopic radioautographs The preganglionic axons examined at 1.5 h after the intracerebral injection of [3H]lysine were poorly labeled (Fig. 5). The grain counts were too low to undertake a quantitative study of their distribution among the subcellular components of the axons. However, a few labeled axons showed that silver grains were closely related to profiles of the smooth endoplasmic reticulum mainly in the vicinity of the axolemmal region (Fig. 12). At 3 and 18 h, labeled axons were more frequently found; silver grains were located over the axolemmal region, mitochondria, endoplasmic reticulum profiles and to a lesser extent over the axoplasm. At 2 and 6 days, all the preganglionic axons were labeled; the reaction was no more prominent over the axolemmal region and the smooth endoplasmic reticulum, while most of the silver grains were seen over the axoplasm embedding numerous microfilaments and relatively scattered microtubules and, to a lesser extent, over the axonal mitochondria (Figs. 13 and 20). The preterminal segments of the preganglionic axons contained an increased concentration of mitochondria and membranous profiles of the smooth endoplasmic reticulum; contrary to the regions of the axons located farther from the terminal, numerous vesicular profiles, possessing the ultrastructural characteristics of the synaptie vesicles seen in the calices, were frequently found (Fig. 17). The preterminal segments were overlain by a greater number of silver grains than in their myelinated portion (Fig. 16). Most of the silver grains were shared between several organelles and could therefore hardly be ascribed to a definite structure. Nevertheless, the reactions observed early after the injection appeared to be relatively prominent over the areas containing membranous and vesicular profiles (Fig. 17). The axon terminals or calices were more heavily labeled than any other structures in the ciliary ganglion during the period 1.5 h-16 days after the intracerebral injection of [aH]lysine. From 1.5 h to 18 h, most label was located in the vicinity of the presynaptic plasma membrane (Figs. 15-17); the silver grains indeed predominated over areas containing synaptic vesicles (Tables I1 and 11I) and their counts rose rapidly to reach a peak at 18 h (Fig. 22), whereas they remained at a much lower level in areas occupied by axoplasm containing microfilaments. Simultaneously, an increase of the number of 'shared' silver grains was recorded over the areas containing presynaptic plasma membranes and their adjacent structures (Table lI and Fig. 22). At

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TABLE I1 CRUDE COUNTS OVER THE PRESYNAPTIC CALICES IN CILIARY G A N G L I A AT VARIOUS TIME INTERVALS AFTER INTRACEREBRAL INJECTION OF [8H]LYSINE

1.5 h Exclusive grains Synaptic vesicles 0 Presynaptic plasma membranes 12 Lateral plasma membranes 0 Mitochondria 11 Axoplasm 8 Shared grains Synaptic vesicles + axoplasm 132 Presynaptic plasma membrane, s + synaptic vesicles -~axoplasm 41 Presynaptic plasma membranes + postsynaptic perikaryon 46 Lateral plasma membranes -taxoplasm 13 Lateral plasma membranes + glial cells 1 Mitochondria + synaptic vesicles + axoplasm 48 Mitochondria ÷ axoplasm 5 Total

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these early time intervals, mitochondria accounted only for a moderate part of the total labeling in calices (Tables 11 and Ill; Fig. 22). Between 18 h and 48 h, the grain counts performed over the areas containing synaptic vesicles dropped off rather steeply (Tables II and Ill; Fig. 22), whereas the radioactivity started to rise in mitochondria and in areas occupied by axoplasm containing microfilaments (Fig. 18). At 6 days, most silver grains were located at a distance from the presynaptic plasma membrane. Clusters of silver grains were frequently found over mitochondria.

Figs. 15 and 16. Electron microscope radioautographs of presynaptic calices 1.5 h and 18 h after the intracerebral injection of [3H]lysine. Fig. 15. At 1.5 h, numerous silver grains are seen over the calyx (Ca), especially over presynaptic plasma membranes (pro), including whorls of membranes (w). Regions containing synaptic vesicles (sv) are also radioactive (see inset). Preganglionic axons (Ax) are devoid of reaction. Fig. 16. At 18 h, the preterminal segment (PS) of the axon and the presynaptic calyx (Ca) are heavily labeled. Most of the silver grains are located over presynaptic membranes (pro) and areas rich in synaptic vesicles (sv) (see inset). Silver grains are seen over mitochondria (mi) and to a lesser extent over regions of axoplasm free of synaptic vesicles (ax).

B. OROZ et al.

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Fig. 17. Electron microscope radioautograph of a presynaptic calyx ! 8 h after the intracerebral injection of [:~H]lysine. In the preterminal segment (PS) of the preganglionic axons, silver grains are located over lateral plasma membranes (Ira) and mitochondria (mi). Clusters of synaptic vesicles (sv) and numerous membranous profiles (er) are also frequently found, in the medial part of the calyx as well as in more lateral regions (see inset), presynaptic plasma membranes (pro) and areas rich in synaptic vesicles (sv) near the synaptic junction are labeled. The axoplasm (ax) devoid of synaptic vesicles is unlabeled. The postsynaptic perikaryon (OC) exhibits only few silver grains.

A X O N A L M I G R A T I O N OF P R O T E I N A N D G L Y C O P R O T E I N .

I

109

TABLE III RELATIVE VOLUME (PERCENT HITS) AND DISTRIBUTION OF THE LABEL (PERCENT CORRECTED GRAIN COUNTS) IN SUBCELLULAR STRUCTURE OF PRESYNAPTIC CALICES IN CILIARY GANGLIA AT VARIOUS TIME INTERVALS AFTER THE INTRACEREBRAL INJECTION OF I~tH]LYSlNE

Percent hits

Synaptic vesicles ÷ axoplasm* 44 ~ Synaptic vesicles 4-20 Axoplasm 24--40 Presynaptic plasma membranes !. Lateral plasma membranes , 13 ~ Mitochondria 21 ~ Axoplasm 22 ~

Percent corrected grain counts 1.5h

3

58 52 6 i 25 2 I 2 3 11 4 4

3h

18h

2days

6days

16 days

63 58 5 21 3 10 3

62 54 8 17 2 14 5

34 1 33 15 2 27 22

39 9 20 15 3 23 20

42 24 18 17 3 26 12

* The relative volume occupied by regions containing synaptic vesicles is fairly constant but the density of the synaptic vesicle population varies greatly within the calyx and also from one calyx to another. The approximate values for the relative distribution of the label in synaptic vesicles and in their embedding axoplasm have been calculated for a relative volume ratio synaptic vesicle: axoplasm of 1:3.

The regions of axoplasm devoid of synaptic vesicles attained at that time their maximal labeling (Figs. 19 and 21). Even the few silver grains seen over areas containing synaptic vesicles were mostly found far from the synaptic junction. After 6 days, a decrease of the label appeared in each organelle. The postsynaptic perikarya displayed only a small number of silver grains localized over the perinuclear ergastoplasm, Goigi apparatus and mitochondria, as well as over the cytoplasm near the synaptic junction. (I!) Complementary experhnents

The right ciliary ganglia locally treated with puromycin in geifoam were compared with the left ciliary ganglia of the same chickens. At 3 h after the intracerebral injection of [aH]lysine, the concentration of radioactivity in presynaptic calices was found to be identical in both right and !eft ganglia (Ca in Figs. 23 and 24;

TABLE IV CONCENTRATION OF RADIOACTIVITY (NUMBER OF SILVER GRAINS PER | 0 0 sq.pm) IN PRESYNAPTIC CALICES AND POSTSYNAPTIC PERIKARYA IN CILIARY GANGLIA PRETREATED OR UNTREATED WITH PUROMYCIN, 3 h AFTER THE INTRACEREBRAL INJECTION OF p H ] L Y S I N E

Presynaptic calices Postsynaptic perikarya

Control

Pttromycin-treated

Percent inhibition

70.9 + 2.3 9.6 ~ 0.5

68.8 ± 2.6 1.0 -4-0.1

89

0

110

B. DROZ et d.

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. 1

111

Table IV); however, the slight reaction detected in postsynaptic perikarya was depressed by 89 ~ in puromycin-treated ganglia (GC in Figs. 23 and 24; Table IV). At the ultrastructural level, the distribution of silver grains over the presynaptic calices was similar in both puromycin-treated and non-treated ciliary ganglia, that is over areas containing synaptic vesicles and presynaptic plasma membranes. In puromycintreated ganglia, the label concentration in postsynaptic perikarya was hardly any higher than the background level, whereas a slight reaction was observed in the control. When a gentle pressure was applied to a puromycin-treated ciliary ganglion, the radioautographs performed 3 h after the intracerebral injection of [aH]lysine showed that several preganglionic axons displayed an intense accumulation of label (Fig. 25), whereas several presynaptic calices were free of reaction. Such an axonal retention of the tracer was never observed in unsqueezed ganglia, whether or not treated with puromycin (compare the labeling of the axons in Figs. 23-25). These intensely labeled axons contained numerous vesicular and tubular profiles enclosing a moderately electron dense material; most silver grains were accumulated over the areas containing these membranous profiles whereas the regions of axoplasm rich in microfilaments were almost free of label (Figs. 26 and 27). DISCUSSION

After injection of [aH]lysine into the cerebral ventricle, the fate of the labeled molecules is followed as a function of time in the midbrain and the ciliary ganglia. In the midbrain, free [aH]lysine eventually declines. One part, taken up by neurons and glial cells, is incorporated for the synthesis of new protein molecules. All the measurable label present in ,the PCA-precipitable fraction of the midbrain is indeed due to [aH]lysyl residues; consequently, most of the label detected by radioautography does correspond to protein synthesized from the radioactive precursor. Another part of the injected [aH]lysine leaks into the bloodstream through the numerous capillaries irrigating the brain tissues and is then diluted in the lysine pool of the chickens. As reflected by the low concentration of free labeled lysine measured in the ciliary ganglia (Fig. 2), these small amounts of blood-borne [aH]lysine are probably responsible for the slight degree of synthesis of radioactive proteins observed in non-neuronal structures of the ciliary ganglia and in spinal ganglion cells.

Figs. 18 and 19. Electron microscope radioautographs of presynaptic calices 2 and 6 days after the intracerebrai injection of [aH]lysine. Fig. 18. By 2 days, the label is still present in areas near the synaptic junction (sv) but extends in regions of axoplasm (ax) devoid of synaptic vesicles; labeled mitochondria covered with several silver grains (mi) are frequently found. Fig. 19. At 6 days, the amount of label present in areas near the synaptic junction (pm and sv) decreases; the silver grains are located farther from the presynaptic plasma membrane (pro) over regions containing synaptic xesicles or over the axoplasm (ax) free of synaptic vesicles. Heavily labeled mitochondria (mi) covered with clusters of silver grains are found among unlabeled mitochondria.

i~

•

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_,,

Figs. 20 and 21. Electron microscope radioautographs of preganglionic axons (Fig. 20) and presynaptic calices (Fig. 21) 6 days after the intracerebral injection of [aH]lysine. Fig. 20. Two preganglionic axons (Ax) exhibit numgrous silver grains distributed over the axoplasm (ax) and mitochondria (mi). Few silver grains are also seen over profiles of the endoplasmic reticulum (er), the axolemmal membrane (axl) and the myelin she~lth. Three unlabeled postganglionic axons are visible in the upper part of the figure. Fig. 21. The presynaptic calyx contain numerous silver grains mainly distributed in a region distant from the presynaptic plasma membrane (pm). Mitochondria (mi) and areas containing synaptic vesicles (sv) (see inset) are labeled whereas presynaptic plasma membranes and whorls of membranes (w) are poorly labeled or unlabeled (compare with Figs. 15, 16 and 17).

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Fig. 22. Time curves of label content in various components of presynaptic calices, after the intracerebral injection of [aH]lysine. The percent corrected grain count obtained from electron microscope radioautographs (Table i !1) has been multiplied by the total grain concentration obtained from light microscope radioautographs (Fig. 1 !). in the upper curves, the solid black line indicates the evolution of the label in areas ofaxoplasm rich in synaptic vesicles. The dashed line has been obtained by subtracting the radioactivity associated with axoplasm and therefore corresponds to the radioactivity probably associated with synaptic vesicles.

puromy¢in •

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Figs. 23 and 24. Light microscope radioautographs of ciliary ganglia locally treated (Fig. 24) or untreated (Fig. 23) with puromycin in gelfoam prior to the intracerebral injection of [aH]lysine and removed 3 h later. Fig. 23. In non-treated ganglia, a strong reaction is seen over the presynaptic calyx (Ca). The perikaryai reaction (GC) is relatively more intense than in the main experiment, probably as a consequence of the greater volume injected into the cerebral ventricle (70 pl v e r s u s 50 ,,I). Fig. 24. In puromycin-treated ganglia, the strong reaction persists over the calyx (Ca), but the number of silver grains present over the postsynaptic perikaryon (GC) has considerably decreased. In both ganglia, the preganglionic axons (Ax) contain only small amounts of label.

114

n. DROZ et al.

Figs. 25-27. Light (Fig. 25) and electron (Figs. 26 Pnd 27) microscope radioautographs of preganglion. ic axons 3 h after a slight compression of the ciliary ganglion locally treated with puromycin and after an intracerebral injection of [:3H]lysine. Fig. 25. Several axons ( = ) contain large amounts of label (compare with the preganglionic axons of Figs. 23 and 24). Figs. 26 and 27, These sections of the same preganglionic axon at two different levels show an intense accumulation of label, mainly in areas containing vesicular and tubular profiles (~,) enclosing an electron dense material. The regions of axoplasm (ax) rich in microfilaments are almost devoid of label

(I) Preganglionic axons and their pretermhlal segments Radioautographs of the preganglionic axons examined in the ciliary ganglia 1.5-18 h after the intracerebral injection of [aH]lysine show first a very low concentration of intraaxonal labeled proteins which increases about 7 times within this time interval (Fig. 11); this fact contrasts with the large amounts of labeled proteins accumulated in nerve endings. A similar behavior of the axonal labeling has been noted by Schonbach and Cu6nod 5~ in the regions containing retino-tectal fibers while, in other studies2a,29,as, 45, a sharp peak or a crest of radioactivity corresponding to a wave of labeled proteins has been observed to move rapidly toward the nerve endings. Such an event does not seem to occur in the preganglionic axons of the ciliary ganglion. The subcellular localization of the label is rendered difficult by the small number of silver grains recorded over the preganglionic axons at i.5 and 3 h. In intact axons, the

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. 1

I 15

rare silver grains are generally associated with membranous profiles of the endoplasmic reticulum found in the outer regions of the axons (Fig. 12). However, when the axonal traffic is interrupted by a slight pressure applied to the ciliary ganglion, numerous membranous profiles are overlain by clumps of silver grains (Figs. 26 and 27). This finding indicates that rapidly transported proteins move along the axons either as ready-made components of the endoplasmic reticulumS0 or as macromolecular subunits capable of self-assembly to build up membranous profiles when the axonal traffic is jammed. The size, the shar~e and the electron dense content of the membranous profiles found in the axons do not fit the characteristics of the synaptic vesicles present in the axon terminals. Thus, in these cholinergic axons, as well as in catecholaminergic axonslS,ta,~, there is no evidence for an axonal migration of ready-made synaptic vesicles; the labeled proteins associated with these membranous profiles and their electron dense content are probably used as precursors of synaptic vesicles and plasma membranes in nerve terminals. This hypothesis would account for the fact that the labeled proteins arriving early in nerve terminals are mainly found in areas of axoplasm rich in synaptic vesicles and close to the presynaptic plasma membrane. On the other hand, the increasing labeling of other axonal structures such as the axolemmal region and mitochondria, observed in preganglionic axons between 3 and 18 h, raises the question whether some fast moving protein contributes also to the renewal of axonal components and in young chickens to the growth of the axon in length and diameter. This possibility is discussed in the Appendix. At later time intervals, 2-16 days after the intracerebral injection of [aH]iysine, the axons are congested with labeled proteins. The dramatic increase of the label observed between l and 6 days in axons at about i0 mm from their origin indicates a mean rate of transport of 1.5-10 ram/day similar to that recorded in various peripheral nerves of young growing animalst2,2~, aS. The labeled proteins transported at a slow rate are mainly associated with the axoplasm, including the numerous microfilaments and the scarce microtubules, and with the axonal mitochondria. These data confirm the results of previous studies carried out in various types of nerve 3,s,tl,17'eS-27'41'82'Se. As already shown in other nervesll,29, St, a progressive disappearance of the radioactivity is observed in the preganglionic axons after a maximum has been reached. The renewal of the labeled proteins in the axons would correspond to a turnover time of about 22 days and seems mainly due to a local breakdown of proteins probably ensured by the intraaxonal i~ydrolytic enzymes44, 4~. The necessary material for the replacement of worn-out axonal proteins and also for the growth of the axons in young animals is indeed supplied by the axonal migration of slowly transported proteins. The question then arises to determine to what extent the slow flow components do or do not invade the axon terminalsS,21,°-5,29. In the case of the preganglionic axons of young growing chickens, the kinetic analysis of our results (see Appendix) indicates that more than 95 % of the slowly migrating proteins do not reach the calices. A special behavior of the labeled proteins is noted in the preterminal segments of the axons (Fig. l l): at all the time intervals studied, the concentration of the radioactive proteins is always higher in preterminal segments than in regions located farther from the calices. Several factors may influence this higher concentration of the label.

116

B. DROZ et ai.

The organelles normally found in the preganglionic axons such as endoplasmic reticulum profiles and mitochondria are more numerous in the preterminal segment; in addition, clusters of vesicular profiles possessing the ultrastructural characteristics of the synaptic vesicles are frequently observed (Fig. 17). Thus, the preterminal segment of the axons would be a special site for storing and dispatching new molecules to nerve endings. (II) Nerve endings

The rapid appearance of labeled proteins at nerve endings may result from various processes. The presynaptic calices of the ciliary ganglion have been shown to incorporate hi vitro labeled amino acids into proteins 14,1~. After an intracerebral injection, small amounts of free [3H]lysine may reach the ciliary ganglia either by leakage into the circulating blood or by rapid transport along the nerves 4~. Various experiments have been performed to test this latter possibility but have failed to demonstrate that a significant amount of free labeled amino acids is intraaxonaily transported to nerve endings 10,4t. The comparison of the distrib~tion of the label in the ciliary ganglion after various ways of administration provides a more decisive argument to determine the part taken by a local incorporation of labeled amino acids. When ciliary ganglia are incubated 01 vitro in a medium containing a labeled amino acid 15, or when labeled amino acids are introduced hl vivo into the circulating blood 14, the concentration of the radioactivity observed at very early time intervals is 3-4 times higher in the ganglion cell bodies than in the presynaptic calices; in both structures, the incorporation of the label is severely impaired by puromycin. When the labeled amino acid is intracerebrally injected, the distribution of the radioactivity is reversed: labeled proteins are 8 times more concentrated in the presynaptic calices than in the ganglion cell bodies (Figs. 5 and !1). Furthermore the strong reaction observed over the calices is no more impeded by puromycin (Fig. 24; Table IV). Thus, the accumulation of radioactive proteins observed in nerve endings after the intracerebral injection of [aH]lysine does not correspond by any means to a local incorporation of the label. The glial cells which closely encompass the preganglionic axons and their calices are able to synthesize proteinsl~; according to Singer and Salpeter 5a, labeled proteins manufactured in glial cells would be delivered to the axon even through the myelin sheath. After an intracerebral injection of [aH]lysine, cells adjacent to preganglionic axons and their calices are poorly labeled at any time (Figs. 4-10) and therefore cannot account for the high content of label present in nerve endings. Finally, when the axonal traffic is interrupted by means of a slight pressure, several calices are devoid of reaction whereas the accumulation of large amounts of label occurs in several preganglionic axons (Figs. 25-27). Thus it can be concluded that most of the labeled proteins collected in nerve endings must result from the arrival of proteins migrating within the axons of the preganglionic neurons. According to former experiments performed after intracerebral injection of [3H]leucine, a significant amount of label starts to appear in calices only after 1 h 82. The relatively high concentration of radioactivity measured in the calices at 1.5 h

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. I

117

indicates that the first protein molecules reach the nerve endipgs between i and i.5 h after the intracerebral injection. Thus they would migrate along the axons at a rate of 288 mm/day. The peak observed at 18 h (Fig. 11) signals the accumulation of labeled proteins either transported at a slower rate or synthesized at a later time (see Appendix). Once arrived at nerve endings, one part of the fast moving proteins is rapidly replaced within about 17 h, whereas glycoproteins seem to stay for a longer period of 10 days (see the following paper). The late increase of radioactivity observed at 6 days in both preganglionic axons and their terminal calices corresponds to the arrival of proteins transported at a rate of 1.5-10 ram/day. The kinetic analysis of our results (see Appendix) shows however that only a minor part of the slowly migrating proteins - - probably less than 5 ~ enters the nerve endings in which they spend about 14.5 days. Alter 6 days of transport along the axons, the life-span of these proteins averages 21 days. (a) Synaptic resicles. In regions containing synaptic vesicles, the volume occupied by these organeiles embedded in the axoplasmic matrix shows great variation (Table Ill). Among the population of synaptic vesicles, large dense core vesicles, coated vesicles and tubular profiles of the endoplasmic reticulum can also be identified (Fig. 14). Between 1.5 h and 18 h the areas rich in synaptic vesicles contain definitely more label than the regions of axoplasm devoid of these organelles (Fig. 22 and Table ll): thus it can be assumed that most of the radioactive proteins rapidly transported to these areas are associated with synaptic vesicles themselves. This assumption is reinforced by the finding of Cu6nod et al. 9 who have shown that, after blo. king the fast flow with colchicine, the ultrastructural features of the synaptic vesi ties are deeply altered. Consequently both observations indicate that some macromolecular material rapidly transported along the axons is required for the maintenance ofsynaptic vesicles in nerve endings. The rapid fall of the label after the 18 hpeak in calices (Fig. I !) and more specifically in areas rich in synaptic vesicles (Fig. 23) would correspond to a high turnover rate (0. I • 10-'-' min -1) of some protein subunits associated with synaptic vesicles. At later time intervals, most of the label found in areas rich in synaptic vesicles probably corresponds to slowly transported axoplasmic proteins distributed in the embedding axoplasmic matrix and to a lesser extent to vesicular components (Table Iii), generally located at a farther distance from the presynaptic plasma membrane than at earlier time intervals. A reutilization of labeled protein, such as an exchange between plasma membranes and pinocytotic vesicles6, could also account for a part of the persisting labeling in areas containing synaptic vesicles. (b) Plasma membranes. Arriving in nerve endings mainly with the fast flow components, a relatively large amount of labeled proteins is related to areas occupied by presynaptic plasma membranes, rather than by lateral membranes covering the remainder of the calices. The lack of resolution of the electron microscope radioautography does not permit one to ascertain whether the higher content of label found in the areas occupied by the presynaptic plasma membranes is due to a faster turnover of the membrane subunits, to the renewal of the special molecular differentiation of the presynaptic membrane1, 24, to a deposit of protein and glycoprotein in the

118

B. DROZ et aL

synaptic cleft 4s or to a stronger labeling of the synaptic vesicles closely apposed to presynaptic membranes. Nevertheless a supplementary argument in favor of the fast transport of membrane subunits to nerve endings is provided by the fact that whorls of membranes, frequently found in calices, are definitely more heavily iab~led at very early time intervals (Fig. 15) than at 6 days (Fig. 21). As visualized in Fig. 22, the turnover of proteins in regions containing plasma membranes is slower than in areas occupied by synaptic vesicles. (c) Mitochondria. At early time intervals, a moderate but significant labeling is observed in mitochondria of all the calices. This finding indicates that some fast moving protein contributes to the renewal of mitochondrial components in nerve endings. Since the net movem,~nt of mitochondria observed within axons 27,47 does not seem to fit the rates of fast moving components, the small amount of radioactive proteins present in mitochondria at 1.5 h and 3 h correspond more probably to the arrival of fast moving subunits eventually added to these organelles than to a fast transport of the mitochondria themselves. Most mitochondrial proteins are indeed synthesized in cytoplasmic ribosomes and subsequently transferred to mitochondria'-'8; when located in axons or nerve endings, these organelles do receive new proteins by transfer from the neuronal perikaryon. At 2 and 6 days, the increase of radioactivity in the mitochondrial population of calices coincides with a similar increase of the amount of label in mitochondria of the preganglionic axons. At these late time intervals, corresponding to the arrival of the slowly migrating proteins in nerve endings, strongly labeled mitochondria are seen among unlabeled ones {Fig. 19); thus, most of the strongly labeled mitochondria present in calices woold be the consequence of the axonal transport of the organdies themselves rather than of mitochondriai protein. The disappearance of labeled mitochondria in nerve endings has been given no explanation by this study. (d) Axoplasm. Reaping profit from the stratified distribution of the organelles in calices, the label content in the axoplasm of nerve terminals is referred to as the radioactivity registered in areas devoid of synaptic vesicles. Nevertheless, the axoplasm of axon terminals contains various components: microfilaments mainly distributed under the lateral plasma membranes, microtubules and tubular membranous profiles of the endoplasmic reticulum are indeed embedded in the axoplasmic matrix proper. At early time intervals, only very few labeled proteins are detected in the axoplasm of the calices; later on, the amount of labeled proteins increases dramatically and reaches a maximum at 6 days (Figs. 18 and 19). The late arrival of labeled axoplasmic proteins in both the preganglionic axons and their nerve endings probably accounts for most of the radioactivity simultaneously recovered in soluble fractions of homogenized ciliary ganglia (see the third paper in this series) and in other materialsS,29,41.

(III) The postsynaptic perikarya At any time the concentration of the radioactivity is always slightly higher in the ciliary than in the spinal ganglion cells. This supplementary labeling may be the consequence of either a transsynaptic delivery of proteins, or of amino acids released

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. I

119

after breakdown of proteins in the nerve endings. Nerve endings contain a sufficient amount of hydrolytic enzymes to ensure a complete breakdown of most proteins to polypeptides or amino acidsaL The local catabolism of radioactive proteins in calices would produce a release of free labeled peptides or lysine in the synaptic cleft, that is in the immediate vicinity of the postsynaptic cell surface, and would therefore facilitate the reutilization of the label for the synthesis of radioactive proteins in the postsynaptic perikarya. If this interpretation were correct, the local application of high doses of puromycin to the ciliary ganglion would completely inhibit the appearance of the label in the postsynaptic perikarya. The results (Fig. 24 and Table IV) indicate that about 90 O//oof the radioactivity found in the ganglion cells is due to a local synthesis of labeled proteins; the other 10% could result from a transneuronal passage of protein or polypeptide. Several reports mention that labeled axonal material could be massively transferred from pre- to postsynaptic structures 2,z°,22,25,aq,43, but no special care has been taken in the exFerimental procedure to inhibit the synthesis of protein in the postsynaptic cells. In the present study, it could be surmised that the gap junctions occasionally found in the caliciform synapses al would facilitate a massive interneuronal exchange of protein. Surprisingly, the small amount of label present in the postsynaptic perikarya, when the synthesis of protein is inhibited, indicate that only a restricted number of protein or peptide molecules would be transferred from the presynaptic calices. This fact, however, does not necessarily mean that a small number of specific protein or peptide molecules cannot play an important role in the exchange of information taking place between a neuron and its effector cell. ACKNOWLEDGEMENTS The authors are especially indebted to Mrs. J. Boyenval for her efficient assistance in preparing radioautographs and to Mrs. R. Hiissig for her appreciated help throughout this work.

REFERENCES

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9 CU[NOD, M., SANDRI, C., AND AKERT, K., Enlarged synaptic vesicles in optic nerve terminals induced by intraocular injection of colchicine, Brain Research, 39 (1972) 285-296. 10 DI GIAMnERARDiNO, L., Independence of the rapid axonal transport of protein from the flow of free amino acids, Acta Neuropath. (Berl.), Suppl. 5 (1971) 132-135. 1 ! DROZ, B., Synth6se et transfert des prot6ines cellulaires dans les neurones ganglionnaires. Etude radioautographique quantitative en microscopie 61ectronique, J. Microscopie, 6 (1967) 201-228. 12 DROZ, B., Protein metabolism in nerve cells, bit. Rev. Cytol., 25 (1969) 363-390. 13 DROZ, B., AND BARONDES,S. H., Nerve endings: rapid appearance of labeled protein shown by electron microscope radioautography, Science, 165 (1969) 1131-1133. 14 DROZ, B., AND KOENm, H. L., The turnover of proteins in axons and nerve endings. In S. H. BARONDES(Ed.), Celhdar Dynamics of the Neuron, Academic Press, New York, 1969, pp. 35-50. 15 DROZ, B., AND KOENm, H. L., Dynamic condition of protein in axons and axon terminals, Acta Neuropath. (Berl.), Suppl. 5 (1971) 109-118. 16 ELAM,J. S., AND AGRANOFF,B. W., Rapid transport of protein in the optic system of the goldfish, J. Neurochem., 18 (1971) 375-387. 17 FELT,H., DUTrON, G. R., BARONDES,S. H., AND SHELANSKI,M. L., Microtubule protein. Identification in and transport to nerve endings, J. Cell Biol., 51 (1971) 138-147. 18 GEFFEN, L. B., AND LlVET'r, B. G., Synaptic vesicles in sympathetic neurons, Physiol. Re,,., 51 (1971) 98-157. 19 GEFFEN, L. B., DESCARRIES, L., AND DROZ, B., Intraaxonal migration of aH-norepinephrine injected into the coeliac ganglion of cats: radioautographic study of the proximal segments of constricted splenic nerves, Brain Research, 35 (1971) 315-318. 20 GLonvs, A., Lux, H. D., ANO SCHUaERT, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1968) 440-445. 21 GRAFSTEIN,B., Axonal transport: communication between soma and synapse, Advanc. Biochem. P~vchopharmacol., 1 (1969)11-25. 22 GRAFSTEIN,B., Transneuronal transfer of radioactivity in the central nervous system, Science, 172 (1971) 177-179. 23 GRANaOULAN, P., Comparison of emulsions and techniques in electron microscope radioauto graphy. In C. P. LEaLONDAN,'~K. B. WnRRF.N, (Eds.) The Use o.f Radioautography in bwestigating Protein SynthesLs', Academic P~ess, New York, 1965, pp. 43-63. 24 GRAY, E. G., Round and fiat syl~aptic vesicles in the fish central nervous system, in S. H. BARONt)LS (Ed.), C(:'l/uh; Dynamics of the Neuron, Academic Press, New York, 1969, pp. 211-227. 25 HF.NDaK'KSON, A. E., Electron microscopic distribution of a×oplasmic transport, J. comp. Neurol., 144 (1972) 381-397. 26 H~NDRK'gSON, A. E., ANrJ 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. 27 ,IF.FFR~Y,P. L., JA~,tFS, K. A. C., KmMAN, A. D., RICHAROS,A. M., AND AUSTIN, L., The flow of mitochondria in chicken sciatic nerve, J. Neurobiol., 3 (1972) 199-208. 28 KADENnA(,H, B., A quantitative study of the biosynthesis of cytochrome C, Europ. J. Biochem., 10 (1969) 312-317. 29 KARLSSON,J. O., AND S~6STRANO,J., Synthesis, migration and turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749-767. 30 KOENIG, H. L., Relations entre la distribution de l'activit~ ac6tylcholinest~rasique et celle de rergastoplasme dans les neurones du ganglion ciliaire du poulet, Arch. Anat. micr. Morph. exp., 54 (1965) 937-964. 31 KOENm, H. L., Quelques particularit~s ultrastructurales des zones synaptiques darts le ganglion ciliaire du poulet, Bull. Ass. Anat. (Nancy), 52 (1967) 711-719. 32 KOENm, H. L., V.T DROZ, B., Transports axonaux de prot~ines aux terminaisons nerveuses du ganglion ciliaire du poulet, apr~s injection intraventriculaire c~r~brale de leucine-aH, C.R. Aead. Sci. (Paris), 272 (1971) 2812-2815. 33 KOPmWA,B. M., A.~DLEnLOND,C. P., Improvements in the coating technique of radioautography. J. Histochem. Cytochem., 10 O961)269-284. 34 KORR, I. M., WILKrNSON,P. N., A~D CHORNOCK, F. W., Axonal delivery of neuroplasmic components to muscle cells, Science, 155 (1967) 342-345. 35 LASEK,R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 36 LEN'rZ,T. L., Distribution of !eucine-aH during axoplasmic transport within regenerating neurons as determined by electron-microscope radioautography, J. Cell Biol., 52 (1972) 719-732.

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121

37 LETTRE, H., UND PAWELETZ, N., Probleme der elektronenmikroskopischen Autoradiographie, Naturwissenschaften, 53 (I 966) 268-27 I. 38 LowRy, O. H., ROSEaROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. bh~l. Chem., 193 (1951) 265-275. 39 MARKS,N., AND LAJTHA,A., Protein and polypeptide breakdown. In A. LAJTHA(Ed.), Handbook ofNeurochemistry, Vol. 5A, Plenum Press, New York, 1971, pp. 49-139. 40 MARWiTT, R., PILAR, G., AND WEAKLY,J. N., Characterization of two ganglion cell populations in avian ciliary ganglia, Brain Research, 25 (1971) 317-334. 41 McEwEN, B. S., FORMAN, D. S., AND GRAFSTEtN, B., Components of fast and slow axonal transport in the goldfish optic nerve, J. NeurobioL, 2 0971) 361-377. 42 NADLER, N. J., The interpretation of grain counts in electron microscope radioautography, J. Cell Biol., 49 (1971) 877-882. 43 NEALE, J. H., NEALE, E. A., AND AGRANOFF, B. W., Radioautography of the optic rectum of the goldfish after intraocular injection of aH-proline, Science, 176 (1972) 407-410. 44 NOVIKOFF,A. B., Lysosomes in nerve cells. In H. HVD~N(Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 319-377. 45 OCHS, S., Fast transport of materials in mammalian nerve fibers, Science, 176 (1972) 252-260. 46 ORREGO, F., Protein degradation in squid giant axons, J. Neurochem., 18 (1971) 2249-2254. 47 POMERAT,C. M., HENDELMAN,W. J., RamOrN, C. W., AND MASSEY,J. F., Dynamic activities of nervous tissue in vitro. In H. HYD[N (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 119-178. 48 RAMaOURG,A., Morphological and histochemical aspects of glycoproieins at the surface of animal cells, hit. Rev. Cytol., 31 (1971) 57-114. 49 SALPETER, M. M., AND BACHMANN,L., Assessment of technical steps in electron microscope autoradiography, in C. P. LEnLONDAND K. B. WARREN(Ed~.), The Use of Radioautography in hwestigating Protein Synthesis, Academic Press, New York, 1965, pp. 23-41. 50 SCHMffT, F. O., Fibrous proteins and neuronal dynamics, in S. H. BARONDES(Ed.), Celhdar Dynamk's of the Neuron, Academic Press, New York, 1969, pp. 95-111. 51 SCH6NBAC'H,J., AND CUi~NOD, M., Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retino-tectai pathway, Exp. Brain Res., 12 (1971) 275-282. 52 SCH/JNBACH,J., SCH/)NnACH,C., AND CUI~NOD, M., Rapid phase of axoplasmic flow and synaptic proteins: an electron microscopical autoradiographic study, J. comp. NeuroL, 141 (1971 ) 485-498. 53 SINGEr, M., AND SALPETrr, M. M., The transport of aH-t-histidine through the Schwann and myelin sheath into the axon, including a reevaluation of myelin function, J. MorphoL, 120 (1966} 281 a16. 54 SJ/JSTRAND, J., Rapid axoplasmic transport of labeled proteins in the vagus and hypoglossal nerves of the rabbit, Exp. Brain Res., 8 (1969) 105-112. 55 Taxi, J., et SOTELO,C., Le probl6me de la migration des cat6cholamines dans les neurones sympathiques, Rev. neuroL, 127 (1972) 23-36. 56 Weiss, P. A., Neuronal dynamics and neuroplasmic ("axonal") flow. In S. H. BARONDES(Ed.), Cellular Dynamics of the Neuron, Academic Press, New York, 1969, pp. 3-34. 57 WHUR, P., HERSCOVlCS,A., AND LenLOND, C. P., Radioautographic visualization of the incorporation of galactose-aH and mannose-aH by rat thyroid in vitro in relation to the stages of thyroglobulin synthesis, J. Cell BioL, 43 (1969) 289-311. 58 WILUAMS, M. A., The assessment of electron microscopic autoradiographs, Adv. Optic. Electr. Microsc., 3 (1969) 219-272. 59 YOUNG, R. W., AND DROZ, B., The renewal of protein in retinal rods and cones, J. Cell Biol., 39 (1968) 169-184.

B. DROZ et al.

122 APPENDIX

CRITICAL ANALYSIS OF THE RATES ESTIMATED FROM RADIOAUTOGRAPHS

OF

AXONAL

MIGRATION

B. D R O Z A N D L. DI G I A M B E R A R D I N O

The rate of migration of a tracer along an axon may be expressed as v = L/t where L is the distance between the nerve cell body and the axon terminal and t the time interval required by the tracer to cover this distance. It is the aim of this Appendix to discuss the influence of several factors on the determination of the rate of axonal transport observed in the preganglionic neurons of the ciliary ganglion in young chickens.

(!) Determhzation of the.last rates of migration (1) Measure of the length of the preganglionic axons. The ner,~e cell bodies, closely packed in the Edinger-Westphal nucleus (Fig. 1 of the accompanying paper), possess axons which first cross the ventral part of the midbrain (2.5-3 mm), then run within the oculomotor nerve !!I (6.5-7 mm) and terminate in the ciliary ganglion after an intraganglionic pathway of 0.3-0.9 mm. Thus the whole axonal length ranges between 9.3 and 10.9 mm.

C

.o 6C (11

g

i

so

U

~o

/ -

/

e/

0 •

0

,

I

1

,

I

2 Hours

I

I~

3"-

'1 0

.,Jjl

L 1

I i 2 Days

I 3-'L

Fig. 28. Time of arrival of the first labeled molecules transported by fast and slow flow. The percent of maximal label concentration recorded in presynaptic calices is plotted on ordinate at various time intervals after administration of pH]leucine (C,), [3H]lysine ( 0 ) and [3H]fucose (A). The data are collected from previous experiments-°,r, and from the accompanying papers I and II. According to this graph, the first labeled macromolecules moving with the fast flow arrive in nerve endings at about 1 11; the first labeled proteins migrating with the slow flow reach the calices after 1 day.

123

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. |

(2) Measure o.I"the time of arriral of the first fast moring components in cafices. After the injection of a [aH] labeled amino acid into the cerebral ventricle, the radioactive precursor diffuses rapidly through the adjacent brain tissue arid can therefore be readily taken up by the nerve cell bodies located in the vicinity of the cerebral aqueduct. One part of the labeled proteins synthesized in the Nissl substance is transferred to the Golgi apparatus and packaged with membranes °. These probably correspond to the fast moving proteins found associated with membrane profiles within the axons (Figs. 12, 26 and 27 of the accompanying paper). According to previous studies 2, the m:an time spent by new proteins in the Nissl substance and the Golgi apparatus is 1-2 min and 6-10 min respectively. Thus, fast moving proteins would spend 6-12 min in the nerve cell bodies bffore they enter the axons. This intraperikaryai sojourn is probably shorter after injection of [aH]fucose. The time interval between the intracerebral injection of the tracer and the arrival of the first labeled molecules in calices may be obtained by extrapolating to zero the curves of radioactivity concentration in nerve endings and corresponds to about 1 h (Fig. 28). After subtraction of the 6-12 min spent in the nerve cell bodies, the time of transport is 48-54 min and the rate of migration ranges between 248 and 329 mm/day, that is, a mean rate of migration of 288 mm/day. (3) Measure of the flux of label through ttle axon. The rate of change of the label content in presynaptic calices dQp/dt results from two processes: (1) the influx of label dQa/dt moving within the axon toward the nerve endings; (2) the efflux of label KQp released from nerve endings.

Since

dQp

dQa

dt

dt

dQp dt

- -

- - KQp

dCp dt

Vp

and

(1) dQa dt

-- Ca~Ra2

dx dt

equation (!) becomes: Vp

dCp dt

= C~rRa ~

dx dt

V,,KCp

(2)

in which: volume of the presynaptic calyx Cp -- concentration of the label in presynaptic calyx Ca = concentration of the label 'in transit' in the preganglionic axon radius of the section of the axon Ra dx = length of the axonal cylinder containing an amount of label equal to dQa K = decay rate of the label expressed as the percent of label replaced per unit of time From equation (2), the rate of migration of the label from the axon to the nerve ending is: =

dx dt

1 Ca

Vp ~Ra 2

I

dCp

~

dt

-~- KCD

(3)

B. DROZ et ai.

124

At the earliest time interval studied after the injection of [aH]lysine (I.5 h) Ca --- 0.9 grain/100 sq./~m Co --- 47.9 grains/100 sq4~m dCp

55 ~ 35 -

-

dt

40

K

-- 0.5 grain/min

---- 0.1 • 10-"/min (this turnover rate was obtained graphically from the decay curve drawn after 18 h) = 19,300-14,700 cu./~m (from Table I of the accompanying paper, the ratio

V~

volume of the presynaptic calyx

7.2 :~ 1.0

volume of the ganglion cell body

19.8 ~ !.7

and the volume of a ganglion cell body of 45 ,urn diameter corresponds to 47,700 cu. pro) RII, -- 4.5 :~ 0.5 ,urn From equation 13),

or

dx

1

19,300

dt

0.9

3.14 i~.~4"-'

I 0.9

:.,"

14,700 3.14 ~:.: 4.5 z

~.: (0.5 + 47.9 ::,:0.1 × 10-2) x 1440 = 338 mm/day

:~< (0.5 + 47.9 × 0.1 ;.: 10-2) x 1440 ---- 202 mm/day

The mean rate of migration of the first arriving protein, determined from the flux of the label (270 mm/day), fits the mean rate of migration obtained by measuring the arrival ot" the first labeled molecules in calices (288 mm/day). (4) hlterpretation oJ' the late arrival o['fast nlov#ig components. The speeds calculated by these two independent methods account tbr the axonal transport of proteins collected in nerve endings at very early time intervals. Later, between 1.5 h and 18 h, the rate of accumulation of the tracer in calices might result from two pos',:ble phenomena: either labeled proteins are transported at different speeds 1, those moving at lower rates arriving later in nerve endings, or labeled proteins are transported at the same rate after being synthesized over a long period of time. To clarify this issue, several assumptions must be examined and confronted with the radioautographic data. It is widely accepted that fast moving proteins are in totality or at least in great majority conveyed to and collected in nerve endings. According to this assumption, the concentration of label actually 'in transit' to nerve endings would correspond to the concentration of label measured in the preganglionic axons. Under these conditions the rate of migration dx/dt, calculated at each time interval from equation (3), would decline from 270 to 14 mm/day, between 1.5 h and 18 h. This result would apparently support the evidence for a wide spectrum of velocities in an homogeneous population of axonsL However the validity of this assumption may be questioned" the behavior of the concentration of the label in preganglionic axons and especially in their preterminal segment is somewhat suspect when other parameters are considered. After an intracerebral injection, free labeled amino acids last for several hours in the midbrain and

AXONAL MIGRATIONOF PROTEINAND GLYCOPROTEIN. |

125

% 100 -

99:

97

89 /// /// /// ///

/I (I

75

//

/// ///

//

68

[ /// ///

, A n ~ ,

/// ///

50

~\~E

/// /// ///

E~ 25

/// /// /// ///

v v v ~ l

A~

luU

..... AA~ VVU

.,/i

~L^.L ,

//,, /// /// iJl

0-

3h.

IV/2 h.

17"/71SH-Lysiae

6h. ~

---

12 h.

3H.Fucose

Fig. 29. Percent of axonal label retained in preganglionic axons at early time intervals after intracerebral injection of [:~H]lysineand 13H]fucose. thereby the synthesis of radioactive proteins is going on until the free labeled amino acids have declined to a low level. Since the concentration of free labeled amino acids diminishes with time, a decreasing number of radioactive protein molecules is synthesized per unit of time and consequently a'reduced number of migratory proteins is dispatched to nerve endings per unit of time. If we assume that all the tracer molecules present in the axon are actually 'in transit' to nerve endings and do not stay in the axon, we would expect to find a gradual decrease of the concentration of the label in the preganglionic axons and their preterminal segments. Such an event does not occur: the concentration of the label in preganglionic axons and their preterminal segments increases gradually until 18 h (Fig. 11 of the accompanying paper). This peculiar behavior of the axonal labeling suggests that only one part of the label present in the axons is actually 'in transit' to nerve endings, while the other part is probably 'retained' in the preganglionic axons (Fig. 29) and their preterminal segments. The percent of axonal concentration of the label retained in the axon may be calculated from equation (3) at various time intervals: C~ =

1 v

Vp(dCp Ra ~ dt

+ KCp

)

(4)

where v -- 288 mm/day. Thus the percent of axonal label rapidly transported to calices drops off with time, whereas the relative amount of label 'retained' in the axons is increased and more pronounced after injection of [aH]fucose than after injection of pH]lysine (Fig. 29). The question then arises to determine which axonal organelles are associated with macromolecules 'in transit' to nerve endings or with macromolecules 'retained' in

126

B. DROZ et aL

preganglionic axons. At early time intervals, most of the labeled proteins 'in transit" along the preganglionic axons are associated with membranous profiles of the endoplasmic reticulum (Fig. 12 of the accompanying paper). It is likely that these membranous profiles or assembling subunits migrate along the axon at such a speed that, between 1 and 3 h after the injection of [aH]lysine, they account for more than 50 % of the total amount of protein conveyed by the fast flow into calices. Between 3 and 18 h, the rate of accumulation of fast moving components in calices decreases gradually as the rate of synthesis decreases in the midbrain; simultaneously, in the preganglionic axons and their preterminal segments, axonal organeiles other than the endoplasmic reticulum become progressively labeled: axolemma, mitochondria and to a lesser extent axoplasm. A similar distribution of the label occurs after [aH]fucose injection but persists for a longer time than after [3H]lysine injection. Thus, one fraction of fast moving proteins and glycoproteins, probably associated with the endoplasmic reticulum profiles frequently found in the vicinity of the axolemma, would be incorporated into the plasma membranes of the preganglionic axons, and of their preterminal segments as well as of their presynaptic calices. In the course of their rapid migration toward nerve endings, labeled proteins could also hit axonal mitochondria and be transferred to these organelles as they probably do in terminals. Such an exchange of enzyme protein from microsome to mitochondr,.'a has been shown to occur in other cell types 4. Labeled protein transferred to mitochondria would continue to migrate slowly with these organelles, whereas labeled molecules incorporated into the axolemma are probably immobilized in the axon. Under these conditions, the velocity dx/dt, calculated from equation (3) would not reflect the 'true' flux of label but rather a mean value resulting from the relative amounts of different molecules migrating at rates ranging from 0 to 288 ram/day.

(11) Determination of the slow rates of migration (1) Measure of the time of arrival of the first slowly moving components. The arrival time of the first labeled molecules transported with the slow flow may be obtained by subtracting the decay curve of the fast moving components (turnover rate of 0.1 • 10-2/min) from the experimental curve: the time required for the transport of slowly migrating protein is provided by extrapolating to zero the curve of radioactivity concentration (Fig. 11 of the accompanying paper); a delay of 24-36 h is found (Fig. 28). Another piece of information is obtained by examining the concentration of the radioactivity in preganglionic axons (Fig. 11 of the accompanying paper): between 1.5 h and 24 h, a gradual increase is noted, followed by a sudden rise starting around 24 h. Thus the first slowly moving components arriving in preganglionic axons and in nerve endings migrate at a rate of 6-10.9 mm/day. (2) The radioactive wave of the slowly moving proteins. After the arrival of the first slowly moving proteins between 24 and 36 h the influx of labeled components persists for several days and reaches a maximum by 6 days. The 5-day delay observed between the front and the crest of the radioactive wave greatly exceeds the period of synthesis of labeled proteins; thus this spreading wave is probably due to molecules moving at different speeds, with a value of 1.5-1.8 mm/day for most ef them 3.

AXONAL MIGRATION OF PROTEIN AND GLYCOPROTEIN. I

127

At late time intervals, only a very small fraction of the label contained in the axons is collected in nerve endings as estimated from the flux of label equation (4): about 3 % at 2 days and only 0.6 % at 6 days.

REFERENCES

1 BRADLEY,W. G., MURCHISON, D., AND DAY, M. J., The range of velocities of axoplasmic flow. A new approach and its application to mice with genetically inherited spinal muscular atrophy, Brain Research, 35 (1971) 185-197. 2 DROZ, B., AND KOENIG, H. L., Localization of protein metabolism in neurons. In A. LA.ITHA (Ed.), Protein Metabolism of the Nervous System, Plenum Press, New York, 1970, pp. 93-108. 3 DROZ, B., AND LEBLOND,C. R., Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography, J. comp. Neurol., 121 (1963) 325-346. 4 KADENBACH,B., A quantitative study of the biosynthesis ofcytochrome C, Europ. J. Biochem., 10 (1969) 312-317. 5 KOENIG, H. L., ET DROZ, B., Transports axonaux de ptot6ines aux terminaisons ner~euses du ganglion ciliaire de poulet, apr6s injection intraventriculaire c6r6brale de leucine-aH, C.R. Acad. Sci. (Paris), 272 O971) 7.812-2815.