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Synthesis and transport of newly formed proteins in dendrites of rat hippocampal pyramid cells. An electron microscope autoradiographic study
Brain Research, 124 (1977) 237-250
237
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
SYNTHESIS AND TRANSPORT OF NEWLY FORMED PROTEINS IN DENDRITES OF RAT HIPPOCAMPAL PYRAMID CELLS. AN ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY
J.
KISS
Second Department of Anatomy, Histology and Embryology, Semmelweis University Medical School Budapest (Hungary)
(Accepted July 20th, 1976)
SUMMARY The synthesis and transport of newly synthesized proteins in dendrites of rat hippocampal pyramid cells were investigated. Labelled leucine was injected into the left lateral ventricle and the hippocampal region was processed for light and electron microscopic autoradiography. To differentiate between the silver grains originating from 'sedentary' or 'migratory' proteins, the radioactivity in dendritic areas free of ribosomes and rich in ribosomes was determined separately. Several conclusions were reached. (1) Protein synthesis in dendrites takes place mainly in the proximal parts although a slight synthetic activity can be observed along the whole dendritic tree as well. (2) Newly synthesized proteins are transported toward the distal dendritic region; the data obtained suggest that cisterns of the smooth endoplasmic reticulum and the microtubular system may be involved in this transport. (3) Two phases of dendritic transport may be distinguished; a fast phase with a rate of 100-200 mm/day and a slow phase with a rate of 2.8-10 mm/day. It also seems probable that the majority of the proteins newly synthesized in dendrites are transported by the slow phase.
INTRODUCTION There is a continuous and very intensive protein synthesis in the perikaryon of neurons. The majority of the synthesized proteins flow continuously toward the most distal parts of the cell processes, primarily in axons. Since the first evidence for axonal protein flow has been reported by Weiss and Hiscoe~6, much effort has been directed towards the clarification of the mechanism underlying this process1,4-s,11,15, 18,27. It is very likely that the microtubules and the smooth endoplasmic reticulum present in the axon play an important role in the mechanism of axonal protein
238 transport. This is suggested by the finding that colchicine inhibits axonal protein transportle,la,16, 2a. The numerous, predominantly electron microscopic autoradiographic investigations demonstrating that in the fast and the slow phase of the axonal flow the labelling occurs primarily around the microtubules, neurofilaments and smooth endoplasmic reticulum cisterns4, 5,s also support this view. Soon after the publication of the axon-flux theory of Weiss the assumption arose that there may also be a protein flow in the dendrites. Autoradiographic examinations have revealed that intracellularly injected [aH]glycine is incorporated into proteins and distributed throughout not only the perikaryon but the whole dendritic tree as well 9. On the basis of light 24,25 and electron microscopic autoradiographic 14 investigations, the appearance of labelled proteins in dendrites was attributed to protein transport from the perikaryon. It was proposed that dendritic transport is either due to simple diffusion or to a mechanism similar to that responsible for axonal flow. In this respect it has been shown that colchicine inhibits not only axonal protein transport but also reduces dendritic transport 25, which suggests that similar or identical mechanisms are operative in both cases. In the present study, dendritic protein transport was examined by electron microscopic autoradiography. The main purpose of the investigation was to provide further characterization of this process, especially by seeking information on the structures involved and on the speed of the transport. MATERIAL AND METHODS Male Wistar rats, weighing 220 zk 5 g, were used. Under Nembutal anaesthesia 1.0 mCi of labelled leucine (OL-4,5-ZH, spec. act. 1.4 Ci/mmole; Radioisotope Institute of the Hungarian Academy of Sciences, Budapest), diluted in 70 #1 of saline was injected under constant pressure into the left lateral ventricle, over a period of 2 min. The animals were perfused 3 with a 4 % paraformaldehyde solution containing phosphate buffer (pH 7.1) and 1.4 mmole of cold leucine, 5 and 30 min, 6 and 24 h and 4 days following [aH]leucine injection. After perfusion the brain was removed and cut into 2 mm thick frontal sections. Four sections containing the hippocampal regions were immersed for 2 h in the same paraformaldehyde solution, postfixed for 2 h in osmium tetroxide, dehydrated, embedded in Epon and polymerized at 60 °C for 16 h. After this polymerization, 1/~m thick sections were cut for light microscopic autoradiography 17. Light microscopic autoradiography was used to select hippocampal areas which contained the Ammon pyramid cell layer, which shaved a longitudinal orientation of the pyramid cell dendrite arborizations and which exhibited a number of silver grains sufficient for electron microscopic autoradiography. The blocks were retrimmed accordingly, and polymerized for a further 20 h at 60 °C. Then, ribbons corresponding to pale gold interference colour were cut and processed for electron microscopic autoradiography 10. After 3 months of exposure the autoradiographs were processed in Kodak Microsol X and subsequently studied under a JEM 6C and JEM 100 B elec-
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Fig. 1. Diagrammatic representation of the hippocampal area examined. The squales show the excised specimens used for light microscopic autoradiography, from which minute areas were retrimmed for electron microscopic autoradiography. The examined areas include the stratum pyramidale (SP); stratum radiatum (SR); stratum lacunosum (SL); stratum moleculare (SM); stratum oriens (SO) and the alveus (a). CA1 and CA~, hippocampal regions (in terminology of Lorente de N6); GD, gyrus dendatus; VL, lateral ventricle; Vm, third ventricle; I, II, III, IV, dendrite categories.
tron microscope. Direct magnifications of 4000 × , 10,000 ×, and final magnifications of 10,000 x and 25,000 × were used in all experiments. The area chosen for examination had the advantage of regular dendrite orientation and relative accessibility to the labelled precursor injected in the lateral ventricle. It contained the alveus hippocampi, the stratum oriens, the stratum pyramidale, the stratum radiatum, the stratum lacunosum and the stratum moleculare. In some cases the layer of the gyrus dentatus was also examined (Fig. 1). The stratum radiatum includes the thick trunks and the branches of the dendrites of the pyramidal cells; the ramification of the dendrites into thin terminal branches occurs in the molecular layer 21. The area extending from the pyramidal layer to the molecular layer, approximately 700-1000 # m wide, was divided into 4 zones and was investigated in both the quantitative and qualitative studies. Zone I contained the pyramidal layer and the initial part of the stratum radiatum, which is mainly composed of the most proximal parts of dendrites (dendrite bases). Zone 2 consisted of the stratum radiatum (except for its initial part), showing predominantly initial dendrite parts. Zone 3 contained the stratum lacunosum and the initial part of the stratum moleculare. The midportions of the dendrites predominate in this region. Zone 4 included the molecular layer containing thin dendrites which presumably correspond to terminal dendrite branches. Each zone was identified on the basis of the thickness of the dendrite profiles most frequently encountered in the region. The characteristic sizes were as follows:
240 zone 1, 7-10/xm thick (category 1); zone 2, 4-6.9 Mnl thick (category 11): zone 3: 2-3.9 # m thick (category Ill); zone 4, 0.5 1.9 Mm thick (category IV) Qualitative evaluation of the autoradiographs comprised the examination of the labelling of the dendrite portions predominant in the different zones and c,f the location of the silver grains over the different cell organelles. Quantitative evaluation of the autoradiographs was concerned with (l) determination of the concentration of radioactivity of the total dendritic tree; (2) estimation of the radioactive concentrations over dendrite portions with different diameters in the various zones; (3) counting the number of grains in the distal and the terminal dendrite branches (categories 111 and IV) over areas containing rilzc,somes (ACR) al~,d in dendrite areas free of ribosomes (AFR). ]-he aim was to obtain an approximate evaluation of the behaviour of the 'sedentary' and 'migratory' proteins in the dendritesa, s. In addition the number of grains was separately determined in the ribosome free areas of the distal and the terminal dendrite portions, over regions containing smooth endoplasmic reticulum (SER) cisterns and over areas free of these elements. Evaluation of the probable location of the grains was performed according to Caro and Nadler (cited by Whur et al.2S). A circle of 225 nm radius was made around each grain. A grain in a circle without any SER cistern was considered as one grain in the area free of SER. Another grain which was in a circle containing SER cisterns and also an area free of SER was partly aligned to the SER and partly to the SER free area. This was made on the basis of the estimated percentage of the two areas in the circle. If for example 40 i'.~,of the circle was occupied by SER and 60"; by the area free of SER, 0.4 grain was aligned to the SER while 0.6 grain was aligned to the area free of SER. Labelling of these two areas was expressed as a percentage of the total grain count over both regions. All examinations were carried out for a period between 5 rain and the 4th day after the injection of the labelled leucine.
RESULTS Qualitative observations Zone 1. Injection of the labelled leucine resulted in 5 rain in a considerable accumulation of silver grains in the area containing the Nissl bodies and the free ribosomes. A more restricted labelling could be seen above the Golgi apparatus (Fig. 2). Thirty minutes after [3H]leucine administration, numerous labelled Golgi areas were evident (Fig. 3). The number of silver grains decreased remarkably in all areas mentioned above 6 h after the injection. No considerable change could be noted at later time intervals. Zone 2. In these dendrite parts, a considerable number of silver grains could be observed 5 and 30 min after the injection of labelled leucine. The majority of the grains occurred in the cytoplasmic areas containing ribosomes. Areas with few or no ribosomes showed only slight labelling. This difference was particularly marked at the bifurcation of dendrites, where areas free of ribosomes could easily be distinguished
Figs. 2 and 3. These and figures up to Fig. 9 are electron microscope autoradiographs of hippocampal pyramidal cells and pyramidal dendrite regions at the indicated time intervals after [3H]leucine administration. Abbreviations: pKy, perikaryon; N, nucleus; Ns, Nissl body; pD, proximal dendrite part; Go, Golgi apparatus; r, free ribosomes; SER, smooth endoplasmic reticulum; ACR, area containing ribosomes; AFR, area free of ribosomes; mt, microtubules. Fig. 2. Pyramidal cell 5 min after the injection of [3H]leucine. Silver grains are abundant over the dendrite. Most of the silver grains overly the Nissl bodies in the perikaryon and in the proximal dendrite part. Fig. 3. At 30 min, radioactivity can be seen in the proximal dendrite parts over the Nissl body and over the Golgi regions.
Figs. 4 and 5. Initial dendrite parts in zone 2 (for details see text). Fig. 4. Five minutes after [~H]leucine administration. The angle of a dendritic bifurcation shows n u m e r o u s stacks of rough endoplasmic reticulum over which silver grains are accumulated. Tile areas free of ribosomes are very distinct. Few silver grains occur over these areas; some are located in the vicinity of the cistern of the s m o o t h endoplasmic reticulunl. Fig. 5. Thirty minutes after [:~H]leucine injection, n u m e r o u s silver grains are distributed over the areas containing ribosomes. Few grains are also seen over areas free of ribosomes.
Fig. 6. As Figs. 4 and 5. At 6 h the label is mostly over the ribosome free areas. Only few grains are located over the ribosomes. Fig. 7. Distal dendrite part belonging to zone 3. Five minutes after the injection, silver grains are found over the areas free of ribosomes, near the smooth endoplasmic reticulum cisterns and over cytoplasm rich in microtubules.
Figs. 8 and 9. Terminal dendrite branches from zone 4. Fig. 8. Five minutes after leucine administ[ation, silver grains are present near the cisterns oF the s m o o t h e n d o p l a s m i c reticulum a n d over the areas containing microtubules. Fig. 9. Thirty minutes after the injection of tritiated leucine, relatively intense a c c u m u l a t i o n s of silver grains are visible. T h e grains are frequently located in the vicinity of the cisterns of the s m o o t h e n d o p l a s m i c reticulum.
245 from the areas containing ribosomes located mainly at the angle of the bifurcations (Figs. 4 and 5). At later time intervals, i.e. 6 h and 4 days after [3H]leucine injection, silver grains were frequently seen over ribosome-free areas (Fig. 6). Zone 3. Five and 30 min after the injection of labelled leucine, the majority of labelling was located, as in the more proximal dendrite portions, predominantly over the cytoplasmic areas containing ribosomes. Silver grains occurred in small number, even in ribosome-free areas, in the vicinity of the cisterns of the smooth endoplasmic reticulum and over areas rich in microtubules (Fig. 7). Four days and 6-24 h after the injection, the majority of the labelling appeared in the ribosome-free areas, near the smooth endoplasmic reticulum cisterns, but silver grains were present also in the areas rich in microtubules. Zone 4. Administration of labelled amino acid resulted after 5 and 30 min in the appearance of silver grains both in the areas containing a few ribosomes as well as in the cytoplasmic areas free of ribosomes but containing predominantly smooth endoplasmic reticulum cisterns and microtubules (Figs. 8 and 9). At 6 h and later, the number of silver grains increased and labelling could be observed mainly in the vicinity of the smooth endoplasmic reticulum cisterns. Quantitative observations
Table I shows in terms of grain counts the concentration of radioactivity in the entire pyramidal dendritic tree. The number of silver grains did not change significantly from 5 min to 24 h following the injection of labelled leucine; between 24 h and day 4 there was a slight decrease in their quantity. In contrast, the amount of radioactivity measured in the entire cytoplasmic area of the dendrite parts located in different zones (i.e. categories) changed with time (Table I, Fig. 10). In the area of the most proximal dendrite parts, i.e. category I, the concentration of radioactivity reached a peak at 5 min, then decreased till 24 h and did not change significantly up to the 4th day. In category II the concentration of radioactivity was maximal 30 min after the injection and did not change significantly at later time intervals. TABLEI Radioactivity of the whole cytoplasmic area from the entire dendritic tree and the different dendrite portions (categories I-IV) at the indicated time intervals after injection of [aH]leucine
Results are expressed as number of silver grains per 100 sq./~m (mean ± S.E.M.). Number of rats in brackets. Dendrite category
5 rain (4)
30 rain (3)
6 h (5)
24 h (3)
4 days (3)
Entire dendritic tree Cat. I Cat. II Cat. III Cat. IV
35.9 ± 4.5 52.0 -4- 6.8 31.7 4- 4.2 28.7 -4- 3.5 23.1 ± 2.8
35.6 ± 5.2 49.8 ± 7.2 33.0 ± 5.4 30.2 ± 3.1 28.8 :k 4.2
35.1 ± 3.8 30.0 ± 4.6 33.3 ± 2.9 33.4 -4-4.1 40.5 -q- 5.2
34.2 ± 5.4 23.6 ± 3.9 33.8 -+-4.2 31.7 ± 4.7 39.2 ± 4.1
28.6 ± 5.4 24.7 ± 4.4 32.8 -q-4.0 24.9 ± 2.8 35.5 ± 3.9
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Fig. 10. Time course of the concentration of radioactivity in the whole cytoplasmic matrix for different categories of dendrite diameters and for ribosome-containing (ACR) and ribosome-free (AFR) areas of categories III and IV. In categories III and IV, the radioactivity maximum was found 6 h after leucine administration. It did not change appreciably up to 24 h, but decreased thereafter. In the areas containing ribosomes in dendrite categories III and IV (ACR categories III and IV), the changes of radioactive concentrations were similar (Table II. Fig. 10). The highest value of radioactivity was found 5 min after [SH]leucine injection and this value did not change significantly between 5 and 30 min, while 6 h later the activity decreased considerably. Between 6 h and 4 days the further decrease was only very slight. In contrast to the changes in the ribosome containing areas, a marked change of the radioactivity could be observed in the areas free of ribosomes (AFR categories I l l and IV) (Table II, Fig. 10). Five minutes after tritiated leucine administration, there was a low but unequivocal activity in both categories. The concentration of radioactivity was somewhat higher in category III than in the terminal branches (category IV). Between 30 min and 6 h, the concentration of radioactivity in-
247 TABLE II Radioactivity contents in areas containing ribosomes (ACR) and free of ribosomes (AFR) from different dendrite portions (categories Ill and IV) at the indicated time intervals after the injection of [ aH j leucine
Results are expressed as number of silver grains per 100 sq./~m. Dendrite category
5 min
30 min
6h
24 h
4 days
ACR AFR ACR AFR
19.1 10.6 18.2 6.8
17.4 12.5 18.0 11.6
6.1 26.3 4.5 36.9
6.8 25.0 3.6 30.8
5.2 19.8 3.7 31.8
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creased in both parts, particularly in the terminal branches. Subsequently, no marked changes could be detected except for a slight decrease between 24 h and the 4th day. In the areas free of ribosomes in dendrite categories I I I and IV, the percentage of grains associated with the cisterns of the smooth endoplasmic reticulum increased slightly until 30 min and decreased afterwards. The rate of grain counts over regions containing microtubules but free of smooth endoplasmic reticulum cisterns did not change between 5 and 30 min, and then went up continuously up to 24 h (Fig. 11).
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248 DISCUSSION The autoradiographic technique used demonstrates the presence of newly synthesized proteins in the dendrites. The very early appearance of the labelled proteins in some cell organelles points either to the sites of protein synthesis or indicates a very fast movement of the newly formed labelled molecules. The almost unchanged radioactivity of the whole dendrite arborization of the pyramid cells 5 rain to 4 days after the injection of [3H]leucine suggests that the majority of the dendrite proteins are synthesized in these processes, particularly in the area of the most proximal parts of the dendrites, i.e., in the dendrite base. This view is supported by the findings that the decrease in radioactivity in the different dendrite parts 5 min after the injection appears to be in relation to the ribosome content of the dendrite portion and, further, by the time course of the radioactivity changes in the ribosome-containing areas. The radioactivity detected at later time intervals shows the actual localization of the previously synthesized labelled proteins. On the basis of our investigations, it may be concluded that a part of the new proteins synthesized in the most proximal parts of dendrites flows continuously toward distal parts. This assumption is in accordance with our observations that there is a maximum of radioactivity in the dendrite mid-portions and especially in the terminal dendrite branches 6 h following the injection of the labelled leucine. Increase in radioactivity of the areas free of ribosomes also supports the existence of the dendritic protein transport. Furthermore, it may be assumed that the radioactivity detected over the ribosome-free areas 5 and 30 min after the injection of the labelled leucine is due to the fast phase of dendritic protein transport. Considering the 700 #m distance of dendrite arborization from the soma to the terminal branches, the velocity of the fast phase of the dendritic protein transport is estimated to be 100-200 mm/day. The increase of radioactivity between 30 rain and 6 h also shows the presence of proteins moving with a slow rate of approximately 2.8-100 mm/day. On the basis of our examinations, it is probable that the majority of the proteins flowing toward the distal parts of the dendrites is transported by the slow phase. As already mentioned in the introduction there are light microscopic data concerning the dendritic transport of newly synthesized proteins. The decrease in this transport under the influence of colchicine indirectly suggests that, in the dendrites as in the axons, the transport process depends on the integrity of the microtubular system25. This assumption is supported by our electron microscopic autoradiographic examinations; in the ribosome-free dendrite areas, we found numerous silver grains over the cytoplasm rich in microtubules. Outside the areas containing microtubules, silver grains were often seen near the cistern of the smooth endoplasmic reticulum. These findings are consistent with the view that, in addition to microtubules, smooth endoplasmic reticulum may also participate in dendritic protein transport. The increase in the early labelling of the cisterns of the smooth endoplasmic reticulum suggests that these elements may be involved in the fast phase of dendritic protein transport.
249 REFERENCES 1 Barondes, S. H., Axoplasmic transport. In A. Lajtha (Ed.), Handbook ofNeurochemistry, Vol. 2, Plenum Press, New York, 1969, pp. 435--445. 2 Bergeron, M. et Droz, B., Analyse critique des conditions de fixation et de pr6paration des tissus pour la d6tection radioautographique des prot6ines n6oform~s en microscopie 61ectronique, J. Microscopie, 7 (1968) 51-62. 3 Descarries, L. et Schr6der, J. M., Fixation du tissu nerveux par perfusion ~ grand d6bit, J. Microscopie, 7 (1968) 281-286. 4 Droz, B., Synth~se et transfert des prot6ines cellulaires dans les neurones ganglionnaires; 6tude radioautographique quantitative en microscopic 61ectronique, J. Microscopie, 6 (1967) 201-228. 5 Droz, B., Fate of newly synthetized proteins in neurons. In C. P. Leblond and K. B. Warren (Eds.), The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 159-175. 6 Droz, B. and Koenig, H. L., Dynamic condition of protein in axons and axon terminals, Acta neuropath. (Berl.), Suppl. V, (1971) 109-118. 7 Droz, B., Koenig, H. L. and di Giamberardino, L., Axonal migration of protein and glicoprotein to nerve endings. I. Autoradiographic analysis of the removal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 (1973) 93-127. 8 Droz, B., Pisam, M. and Chretien, M., Morphological aspects of protein synthesis. In E. Diczfalusy (Ed.), Protein Synthesis in Reproductive Tissue, Bogtrykkeriet Forum, Copenhagen, 1973, pp. 13-32. 9 Globus, A., Lux, H. D. and Schubert, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1968) 440--445. 10 Granboulan, P., Comparison of emulsion and techniques in electron microscope radioautography. In C. P. Leblond and K. B. Warren (Eds.), The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 43-63. 11 Hendrickson, A. E., Electron microscopic distribution of axoplasmic transport, J. comp. neurol., 144 (1972) 381-397. 12 Karlsson, J. O., Hansson, H. A. and Sj6strand, J., Effect of colchicine on axonal transport and morphology of retinal ganglion cells, Z. Zellforsch., 115 (1971) 265-283. 13 Karlsson, J. O. and Sj6sstrand, J., The effect of colchicine on the axonal transport of protein in the optic nerve and tract of the rabbit, Brain Research, 13 (1969) 617-619. 14 Kiss, J., Nagyfelold~tsfiautoradiogr~tfia elm~leti 6s gyakorlati probl6mfii, MTA Biol. Oszt. Ki~zl., 12 (1969) 186-208. 15 Kiss, J., Lfing, E. and H~lmori, J., The effect of electric stimulation on the axoplasmic transport of newly synthesized neural proteins, J. Neural Transm., Suppl. XI (1974) 125-133. 16 Kreutzberg, G. W., Neuronal dynamics and axonal flow. IV. Blockage of intra-axonal enzyme transport by colchicine, Proc. nat..4cad. Sci. (Wash.), 62 (1969) 722-728. 17 Kopriwa, B. M. and Leblond, C. P., Improvements in the coating technique of radioautography, J. Histochem. Cytochem., 10 (1961) 269-284. 18 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 19 Lux, H. D., Schubert, P., Kreutzberg, G. W. and Globus, A., Excitation and axonal flow: Autoradiographic study on motoneurons intracellularly injected with a 3H-amino acid, Exp. Brain Res., 10 (1970) 197-204. 20 Marchisio, P. C., Aglietta, M. and Rigamonti, D., Short term effect of colcemid on the rapid axonal transport of proteins in the optic pathway of chick embryos, Experientia (Basel), 29 (1973) 1126. 21 Ramon y Cajal, S., Histologie du Systeme Nerveux de l'homme et des Vert~brds, Maloine, Paris, 1911. 22 SchiSnbach, H. and Cu6nod, M., Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retino-tectal pathway, Exp. Brain Res., 12 (1971) 275-282. 23 Sch6nbach, J., SchiSnbach, C. and Cu6nod, M., Rapid phase of axoplasmic flow and synaptic proteins: an electron microscopical autoradiographic study, J. comp. Neurol., 141 (1971) 485-498. 24 Schubert, P., Lux, H. D. and Kreutzberg, G. W., Single cell isotope injection technique, a tool for studying axonal and dendritic transport, Acta neuropath. (Berl.), 5 (1971) 179-186. 25 Schubert, P., Kreutzberg, G. W. and Lux, H. D., Neuroplasmic transport in dendrites: Effect
250 of colchicine on morphology and physiology of motoneurons in the cat, Brain Research, 47 (1972) 331-343. 26 Weiss, P. and Hiscoe, H. B., Experiments on the mechanism of nerve growth, J. exp. Zool., 107 (1948) 315-395. 27 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. 28 Whur, P., Herscovics, A. and Leblond, C. P., Radioautographic visualization of the incorporation of galactose-aH and mannose-SH by rat thyroid in vitro in relation to the stages of thyroglobulin synthesis, J. Cell Biol., 43 (1969) 289-311.
237
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
SYNTHESIS AND TRANSPORT OF NEWLY FORMED PROTEINS IN DENDRITES OF RAT HIPPOCAMPAL PYRAMID CELLS. AN ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY
J.
KISS
Second Department of Anatomy, Histology and Embryology, Semmelweis University Medical School Budapest (Hungary)
(Accepted July 20th, 1976)
SUMMARY The synthesis and transport of newly synthesized proteins in dendrites of rat hippocampal pyramid cells were investigated. Labelled leucine was injected into the left lateral ventricle and the hippocampal region was processed for light and electron microscopic autoradiography. To differentiate between the silver grains originating from 'sedentary' or 'migratory' proteins, the radioactivity in dendritic areas free of ribosomes and rich in ribosomes was determined separately. Several conclusions were reached. (1) Protein synthesis in dendrites takes place mainly in the proximal parts although a slight synthetic activity can be observed along the whole dendritic tree as well. (2) Newly synthesized proteins are transported toward the distal dendritic region; the data obtained suggest that cisterns of the smooth endoplasmic reticulum and the microtubular system may be involved in this transport. (3) Two phases of dendritic transport may be distinguished; a fast phase with a rate of 100-200 mm/day and a slow phase with a rate of 2.8-10 mm/day. It also seems probable that the majority of the proteins newly synthesized in dendrites are transported by the slow phase.
INTRODUCTION There is a continuous and very intensive protein synthesis in the perikaryon of neurons. The majority of the synthesized proteins flow continuously toward the most distal parts of the cell processes, primarily in axons. Since the first evidence for axonal protein flow has been reported by Weiss and Hiscoe~6, much effort has been directed towards the clarification of the mechanism underlying this process1,4-s,11,15, 18,27. It is very likely that the microtubules and the smooth endoplasmic reticulum present in the axon play an important role in the mechanism of axonal protein
238 transport. This is suggested by the finding that colchicine inhibits axonal protein transportle,la,16, 2a. The numerous, predominantly electron microscopic autoradiographic investigations demonstrating that in the fast and the slow phase of the axonal flow the labelling occurs primarily around the microtubules, neurofilaments and smooth endoplasmic reticulum cisterns4, 5,s also support this view. Soon after the publication of the axon-flux theory of Weiss the assumption arose that there may also be a protein flow in the dendrites. Autoradiographic examinations have revealed that intracellularly injected [aH]glycine is incorporated into proteins and distributed throughout not only the perikaryon but the whole dendritic tree as well 9. On the basis of light 24,25 and electron microscopic autoradiographic 14 investigations, the appearance of labelled proteins in dendrites was attributed to protein transport from the perikaryon. It was proposed that dendritic transport is either due to simple diffusion or to a mechanism similar to that responsible for axonal flow. In this respect it has been shown that colchicine inhibits not only axonal protein transport but also reduces dendritic transport 25, which suggests that similar or identical mechanisms are operative in both cases. In the present study, dendritic protein transport was examined by electron microscopic autoradiography. The main purpose of the investigation was to provide further characterization of this process, especially by seeking information on the structures involved and on the speed of the transport. MATERIAL AND METHODS Male Wistar rats, weighing 220 zk 5 g, were used. Under Nembutal anaesthesia 1.0 mCi of labelled leucine (OL-4,5-ZH, spec. act. 1.4 Ci/mmole; Radioisotope Institute of the Hungarian Academy of Sciences, Budapest), diluted in 70 #1 of saline was injected under constant pressure into the left lateral ventricle, over a period of 2 min. The animals were perfused 3 with a 4 % paraformaldehyde solution containing phosphate buffer (pH 7.1) and 1.4 mmole of cold leucine, 5 and 30 min, 6 and 24 h and 4 days following [aH]leucine injection. After perfusion the brain was removed and cut into 2 mm thick frontal sections. Four sections containing the hippocampal regions were immersed for 2 h in the same paraformaldehyde solution, postfixed for 2 h in osmium tetroxide, dehydrated, embedded in Epon and polymerized at 60 °C for 16 h. After this polymerization, 1/~m thick sections were cut for light microscopic autoradiography 17. Light microscopic autoradiography was used to select hippocampal areas which contained the Ammon pyramid cell layer, which shaved a longitudinal orientation of the pyramid cell dendrite arborizations and which exhibited a number of silver grains sufficient for electron microscopic autoradiography. The blocks were retrimmed accordingly, and polymerized for a further 20 h at 60 °C. Then, ribbons corresponding to pale gold interference colour were cut and processed for electron microscopic autoradiography 10. After 3 months of exposure the autoradiographs were processed in Kodak Microsol X and subsequently studied under a JEM 6C and JEM 100 B elec-
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Fig. 1. Diagrammatic representation of the hippocampal area examined. The squales show the excised specimens used for light microscopic autoradiography, from which minute areas were retrimmed for electron microscopic autoradiography. The examined areas include the stratum pyramidale (SP); stratum radiatum (SR); stratum lacunosum (SL); stratum moleculare (SM); stratum oriens (SO) and the alveus (a). CA1 and CA~, hippocampal regions (in terminology of Lorente de N6); GD, gyrus dendatus; VL, lateral ventricle; Vm, third ventricle; I, II, III, IV, dendrite categories.
tron microscope. Direct magnifications of 4000 × , 10,000 ×, and final magnifications of 10,000 x and 25,000 × were used in all experiments. The area chosen for examination had the advantage of regular dendrite orientation and relative accessibility to the labelled precursor injected in the lateral ventricle. It contained the alveus hippocampi, the stratum oriens, the stratum pyramidale, the stratum radiatum, the stratum lacunosum and the stratum moleculare. In some cases the layer of the gyrus dentatus was also examined (Fig. 1). The stratum radiatum includes the thick trunks and the branches of the dendrites of the pyramidal cells; the ramification of the dendrites into thin terminal branches occurs in the molecular layer 21. The area extending from the pyramidal layer to the molecular layer, approximately 700-1000 # m wide, was divided into 4 zones and was investigated in both the quantitative and qualitative studies. Zone I contained the pyramidal layer and the initial part of the stratum radiatum, which is mainly composed of the most proximal parts of dendrites (dendrite bases). Zone 2 consisted of the stratum radiatum (except for its initial part), showing predominantly initial dendrite parts. Zone 3 contained the stratum lacunosum and the initial part of the stratum moleculare. The midportions of the dendrites predominate in this region. Zone 4 included the molecular layer containing thin dendrites which presumably correspond to terminal dendrite branches. Each zone was identified on the basis of the thickness of the dendrite profiles most frequently encountered in the region. The characteristic sizes were as follows:
240 zone 1, 7-10/xm thick (category 1); zone 2, 4-6.9 Mnl thick (category 11): zone 3: 2-3.9 # m thick (category Ill); zone 4, 0.5 1.9 Mm thick (category IV) Qualitative evaluation of the autoradiographs comprised the examination of the labelling of the dendrite portions predominant in the different zones and c,f the location of the silver grains over the different cell organelles. Quantitative evaluation of the autoradiographs was concerned with (l) determination of the concentration of radioactivity of the total dendritic tree; (2) estimation of the radioactive concentrations over dendrite portions with different diameters in the various zones; (3) counting the number of grains in the distal and the terminal dendrite branches (categories 111 and IV) over areas containing rilzc,somes (ACR) al~,d in dendrite areas free of ribosomes (AFR). ]-he aim was to obtain an approximate evaluation of the behaviour of the 'sedentary' and 'migratory' proteins in the dendritesa, s. In addition the number of grains was separately determined in the ribosome free areas of the distal and the terminal dendrite portions, over regions containing smooth endoplasmic reticulum (SER) cisterns and over areas free of these elements. Evaluation of the probable location of the grains was performed according to Caro and Nadler (cited by Whur et al.2S). A circle of 225 nm radius was made around each grain. A grain in a circle without any SER cistern was considered as one grain in the area free of SER. Another grain which was in a circle containing SER cisterns and also an area free of SER was partly aligned to the SER and partly to the SER free area. This was made on the basis of the estimated percentage of the two areas in the circle. If for example 40 i'.~,of the circle was occupied by SER and 60"; by the area free of SER, 0.4 grain was aligned to the SER while 0.6 grain was aligned to the area free of SER. Labelling of these two areas was expressed as a percentage of the total grain count over both regions. All examinations were carried out for a period between 5 rain and the 4th day after the injection of the labelled leucine.
RESULTS Qualitative observations Zone 1. Injection of the labelled leucine resulted in 5 rain in a considerable accumulation of silver grains in the area containing the Nissl bodies and the free ribosomes. A more restricted labelling could be seen above the Golgi apparatus (Fig. 2). Thirty minutes after [3H]leucine administration, numerous labelled Golgi areas were evident (Fig. 3). The number of silver grains decreased remarkably in all areas mentioned above 6 h after the injection. No considerable change could be noted at later time intervals. Zone 2. In these dendrite parts, a considerable number of silver grains could be observed 5 and 30 min after the injection of labelled leucine. The majority of the grains occurred in the cytoplasmic areas containing ribosomes. Areas with few or no ribosomes showed only slight labelling. This difference was particularly marked at the bifurcation of dendrites, where areas free of ribosomes could easily be distinguished
Figs. 2 and 3. These and figures up to Fig. 9 are electron microscope autoradiographs of hippocampal pyramidal cells and pyramidal dendrite regions at the indicated time intervals after [3H]leucine administration. Abbreviations: pKy, perikaryon; N, nucleus; Ns, Nissl body; pD, proximal dendrite part; Go, Golgi apparatus; r, free ribosomes; SER, smooth endoplasmic reticulum; ACR, area containing ribosomes; AFR, area free of ribosomes; mt, microtubules. Fig. 2. Pyramidal cell 5 min after the injection of [3H]leucine. Silver grains are abundant over the dendrite. Most of the silver grains overly the Nissl bodies in the perikaryon and in the proximal dendrite part. Fig. 3. At 30 min, radioactivity can be seen in the proximal dendrite parts over the Nissl body and over the Golgi regions.
Figs. 4 and 5. Initial dendrite parts in zone 2 (for details see text). Fig. 4. Five minutes after [~H]leucine administration. The angle of a dendritic bifurcation shows n u m e r o u s stacks of rough endoplasmic reticulum over which silver grains are accumulated. Tile areas free of ribosomes are very distinct. Few silver grains occur over these areas; some are located in the vicinity of the cistern of the s m o o t h endoplasmic reticulunl. Fig. 5. Thirty minutes after [:~H]leucine injection, n u m e r o u s silver grains are distributed over the areas containing ribosomes. Few grains are also seen over areas free of ribosomes.
Fig. 6. As Figs. 4 and 5. At 6 h the label is mostly over the ribosome free areas. Only few grains are located over the ribosomes. Fig. 7. Distal dendrite part belonging to zone 3. Five minutes after the injection, silver grains are found over the areas free of ribosomes, near the smooth endoplasmic reticulum cisterns and over cytoplasm rich in microtubules.
Figs. 8 and 9. Terminal dendrite branches from zone 4. Fig. 8. Five minutes after leucine administ[ation, silver grains are present near the cisterns oF the s m o o t h e n d o p l a s m i c reticulum a n d over the areas containing microtubules. Fig. 9. Thirty minutes after the injection of tritiated leucine, relatively intense a c c u m u l a t i o n s of silver grains are visible. T h e grains are frequently located in the vicinity of the cisterns of the s m o o t h e n d o p l a s m i c reticulum.
245 from the areas containing ribosomes located mainly at the angle of the bifurcations (Figs. 4 and 5). At later time intervals, i.e. 6 h and 4 days after [3H]leucine injection, silver grains were frequently seen over ribosome-free areas (Fig. 6). Zone 3. Five and 30 min after the injection of labelled leucine, the majority of labelling was located, as in the more proximal dendrite portions, predominantly over the cytoplasmic areas containing ribosomes. Silver grains occurred in small number, even in ribosome-free areas, in the vicinity of the cisterns of the smooth endoplasmic reticulum and over areas rich in microtubules (Fig. 7). Four days and 6-24 h after the injection, the majority of the labelling appeared in the ribosome-free areas, near the smooth endoplasmic reticulum cisterns, but silver grains were present also in the areas rich in microtubules. Zone 4. Administration of labelled amino acid resulted after 5 and 30 min in the appearance of silver grains both in the areas containing a few ribosomes as well as in the cytoplasmic areas free of ribosomes but containing predominantly smooth endoplasmic reticulum cisterns and microtubules (Figs. 8 and 9). At 6 h and later, the number of silver grains increased and labelling could be observed mainly in the vicinity of the smooth endoplasmic reticulum cisterns. Quantitative observations
Table I shows in terms of grain counts the concentration of radioactivity in the entire pyramidal dendritic tree. The number of silver grains did not change significantly from 5 min to 24 h following the injection of labelled leucine; between 24 h and day 4 there was a slight decrease in their quantity. In contrast, the amount of radioactivity measured in the entire cytoplasmic area of the dendrite parts located in different zones (i.e. categories) changed with time (Table I, Fig. 10). In the area of the most proximal dendrite parts, i.e. category I, the concentration of radioactivity reached a peak at 5 min, then decreased till 24 h and did not change significantly up to the 4th day. In category II the concentration of radioactivity was maximal 30 min after the injection and did not change significantly at later time intervals. TABLEI Radioactivity of the whole cytoplasmic area from the entire dendritic tree and the different dendrite portions (categories I-IV) at the indicated time intervals after injection of [aH]leucine
Results are expressed as number of silver grains per 100 sq./~m (mean ± S.E.M.). Number of rats in brackets. Dendrite category
5 rain (4)
30 rain (3)
6 h (5)
24 h (3)
4 days (3)
Entire dendritic tree Cat. I Cat. II Cat. III Cat. IV
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28.6 ± 5.4 24.7 ± 4.4 32.8 -q-4.0 24.9 ± 2.8 35.5 ± 3.9
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247 TABLE II Radioactivity contents in areas containing ribosomes (ACR) and free of ribosomes (AFR) from different dendrite portions (categories Ill and IV) at the indicated time intervals after the injection of [ aH j leucine
Results are expressed as number of silver grains per 100 sq./~m. Dendrite category
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248 DISCUSSION The autoradiographic technique used demonstrates the presence of newly synthesized proteins in the dendrites. The very early appearance of the labelled proteins in some cell organelles points either to the sites of protein synthesis or indicates a very fast movement of the newly formed labelled molecules. The almost unchanged radioactivity of the whole dendrite arborization of the pyramid cells 5 rain to 4 days after the injection of [3H]leucine suggests that the majority of the dendrite proteins are synthesized in these processes, particularly in the area of the most proximal parts of the dendrites, i.e., in the dendrite base. This view is supported by the findings that the decrease in radioactivity in the different dendrite parts 5 min after the injection appears to be in relation to the ribosome content of the dendrite portion and, further, by the time course of the radioactivity changes in the ribosome-containing areas. The radioactivity detected at later time intervals shows the actual localization of the previously synthesized labelled proteins. On the basis of our investigations, it may be concluded that a part of the new proteins synthesized in the most proximal parts of dendrites flows continuously toward distal parts. This assumption is in accordance with our observations that there is a maximum of radioactivity in the dendrite mid-portions and especially in the terminal dendrite branches 6 h following the injection of the labelled leucine. Increase in radioactivity of the areas free of ribosomes also supports the existence of the dendritic protein transport. Furthermore, it may be assumed that the radioactivity detected over the ribosome-free areas 5 and 30 min after the injection of the labelled leucine is due to the fast phase of dendritic protein transport. Considering the 700 #m distance of dendrite arborization from the soma to the terminal branches, the velocity of the fast phase of the dendritic protein transport is estimated to be 100-200 mm/day. The increase of radioactivity between 30 rain and 6 h also shows the presence of proteins moving with a slow rate of approximately 2.8-100 mm/day. On the basis of our examinations, it is probable that the majority of the proteins flowing toward the distal parts of the dendrites is transported by the slow phase. As already mentioned in the introduction there are light microscopic data concerning the dendritic transport of newly synthesized proteins. The decrease in this transport under the influence of colchicine indirectly suggests that, in the dendrites as in the axons, the transport process depends on the integrity of the microtubular system25. This assumption is supported by our electron microscopic autoradiographic examinations; in the ribosome-free dendrite areas, we found numerous silver grains over the cytoplasm rich in microtubules. Outside the areas containing microtubules, silver grains were often seen near the cistern of the smooth endoplasmic reticulum. These findings are consistent with the view that, in addition to microtubules, smooth endoplasmic reticulum may also participate in dendritic protein transport. The increase in the early labelling of the cisterns of the smooth endoplasmic reticulum suggests that these elements may be involved in the fast phase of dendritic protein transport.
249 REFERENCES 1 Barondes, S. H., Axoplasmic transport. In A. Lajtha (Ed.), Handbook ofNeurochemistry, Vol. 2, Plenum Press, New York, 1969, pp. 435--445. 2 Bergeron, M. et Droz, B., Analyse critique des conditions de fixation et de pr6paration des tissus pour la d6tection radioautographique des prot6ines n6oform~s en microscopie 61ectronique, J. Microscopie, 7 (1968) 51-62. 3 Descarries, L. et Schr6der, J. M., Fixation du tissu nerveux par perfusion ~ grand d6bit, J. Microscopie, 7 (1968) 281-286. 4 Droz, B., Synth~se et transfert des prot6ines cellulaires dans les neurones ganglionnaires; 6tude radioautographique quantitative en microscopic 61ectronique, J. Microscopie, 6 (1967) 201-228. 5 Droz, B., Fate of newly synthetized proteins in neurons. In C. P. Leblond and K. B. Warren (Eds.), The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 159-175. 6 Droz, B. and Koenig, H. L., Dynamic condition of protein in axons and axon terminals, Acta neuropath. (Berl.), Suppl. V, (1971) 109-118. 7 Droz, B., Koenig, H. L. and di Giamberardino, L., Axonal migration of protein and glicoprotein to nerve endings. I. Autoradiographic analysis of the removal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 (1973) 93-127. 8 Droz, B., Pisam, M. and Chretien, M., Morphological aspects of protein synthesis. In E. Diczfalusy (Ed.), Protein Synthesis in Reproductive Tissue, Bogtrykkeriet Forum, Copenhagen, 1973, pp. 13-32. 9 Globus, A., Lux, H. D. and Schubert, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1968) 440--445. 10 Granboulan, P., Comparison of emulsion and techniques in electron microscope radioautography. In C. P. Leblond and K. B. Warren (Eds.), The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 43-63. 11 Hendrickson, A. E., Electron microscopic distribution of axoplasmic transport, J. comp. neurol., 144 (1972) 381-397. 12 Karlsson, J. O., Hansson, H. A. and Sj6strand, J., Effect of colchicine on axonal transport and morphology of retinal ganglion cells, Z. Zellforsch., 115 (1971) 265-283. 13 Karlsson, J. O. and Sj6sstrand, J., The effect of colchicine on the axonal transport of protein in the optic nerve and tract of the rabbit, Brain Research, 13 (1969) 617-619. 14 Kiss, J., Nagyfelold~tsfiautoradiogr~tfia elm~leti 6s gyakorlati probl6mfii, MTA Biol. Oszt. Ki~zl., 12 (1969) 186-208. 15 Kiss, J., Lfing, E. and H~lmori, J., The effect of electric stimulation on the axoplasmic transport of newly synthesized neural proteins, J. Neural Transm., Suppl. XI (1974) 125-133. 16 Kreutzberg, G. W., Neuronal dynamics and axonal flow. IV. Blockage of intra-axonal enzyme transport by colchicine, Proc. nat..4cad. Sci. (Wash.), 62 (1969) 722-728. 17 Kopriwa, B. M. and Leblond, C. P., Improvements in the coating technique of radioautography, J. Histochem. Cytochem., 10 (1961) 269-284. 18 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 19 Lux, H. D., Schubert, P., Kreutzberg, G. W. and Globus, A., Excitation and axonal flow: Autoradiographic study on motoneurons intracellularly injected with a 3H-amino acid, Exp. Brain Res., 10 (1970) 197-204. 20 Marchisio, P. C., Aglietta, M. and Rigamonti, D., Short term effect of colcemid on the rapid axonal transport of proteins in the optic pathway of chick embryos, Experientia (Basel), 29 (1973) 1126. 21 Ramon y Cajal, S., Histologie du Systeme Nerveux de l'homme et des Vert~brds, Maloine, Paris, 1911. 22 SchiSnbach, H. and Cu6nod, M., Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retino-tectal pathway, Exp. Brain Res., 12 (1971) 275-282. 23 Sch6nbach, J., SchiSnbach, C. and Cu6nod, M., Rapid phase of axoplasmic flow and synaptic proteins: an electron microscopical autoradiographic study, J. comp. Neurol., 141 (1971) 485-498. 24 Schubert, P., Lux, H. D. and Kreutzberg, G. W., Single cell isotope injection technique, a tool for studying axonal and dendritic transport, Acta neuropath. (Berl.), 5 (1971) 179-186. 25 Schubert, P., Kreutzberg, G. W. and Lux, H. D., Neuroplasmic transport in dendrites: Effect
250 of colchicine on morphology and physiology of motoneurons in the cat, Brain Research, 47 (1972) 331-343. 26 Weiss, P. and Hiscoe, H. B., Experiments on the mechanism of nerve growth, J. exp. Zool., 107 (1948) 315-395. 27 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. 28 Whur, P., Herscovics, A. and Leblond, C. P., Radioautographic visualization of the incorporation of galactose-aH and mannose-SH by rat thyroid in vitro in relation to the stages of thyroglobulin synthesis, J. Cell Biol., 43 (1969) 289-311.