Effects of temperature on axonal transport and turnover of protein in goldfish optic system

EXPERIMENTAL

34, 1.58-170

NEUROLOGY

Effects

of

Temperature

Turnover

of

BERNICE Departrmxt New

I*ork

on

Protein

GRAFSTEIN,

(1972)

Axonal

in Goldfish

Optic

DAVID S. FORMAN, MCEWEN r

of Physiology, Cornell 10021 and Rockefeller Received

University University, October

Transport

Medical New

and

System

AND BRUCE S. College, New York, York, New York.

4,197l

Tritiated leucine was injected into one eye of goldfish maintained at 20.5 C. After allowing 2 hr for incorporation of the labeled amino acid, one group of fish was transferred to 9 C. The rate of the fast component of protein transport in the optic axons was reduced from about 60 mm/day at 20.5 C to about 20 mm/ . day at 9 C, grvmg a Q,, of at least 2.6. The rate of appearance of the initial portion of the slow component remained unchanged, which emphasizes the difference in the mechanisms underlying the two components. At the lower temperature, the fast component showed a sharp early peak not seen at 20.5 C. The turnover of retinal protein showed two phases, a decrease of about 30% over 5-10 days, then an exponential decline with a half-life of about 3 weeks at 20.5 C and a Q,, of 4.5. The locally synthesized proteins in the tectum showed, in addition to the initial synthesis period, an increase in radioactivity between 3 and 10 days after the injection. Eventually the tectal radioactivity declined exponentially with a half-life of about 7 weeks at 20.5 C and a Q,, of 2.2. The total transported radioactivity, on the other hand, showed an over-all decline which was close to linear rather than exponential, with a half-time of about 6 weeks. This lifetime was so much prolonged at the lower temperature that the Q,, could not be determined. From autoradiograms of the tectum it was found that the increase in radioactivity signaling the arrival of the slow component could not be detected in the synaptic endings of the optic fibers where the fast component accumulated. The radioactivity in the endings declined to half in about 90 days at 15 C. Introduction

Axonal transport of protein in the optic nerve of the goldfish has been shown to have two components, differing in rate and composition. The fast component advances along the axon at a rate of up to 70-100 mm/day and consists ahnost entirely of particulate protein (6, 31). It includes gly1 This work was supported by U. S. Public Health Service grants NS-09015, NS-07080 and MH-13189. Dr. Forman was supported by a Graduate Fellowship from the National Science Foundation. 158 0

1972

by

Academic

Press,

Inc.

AXONAL

TRANSPORT

159

coproteins ( 10) and sulfated mucopolysaccharide proteins (8). The slow component moves at a rate of about 0.4 mm/day (13) and contains nearly 50% soluble protein in addition to particulate material (1, 21, 31, 33). At least part of the slow component is microtubule protein ( 14,19,22). In the present study we investigated the effects of lowered temperature on both fast and slow components of protein transport in goldfish optic axons. In the course of this study we have also made some interesting observations on the turnover of protein in retina and brain. Methods Goldfish 55-65 mm in body length were used. An injection of 4 &i of 4,5JH-leucine (specific activity 5Ci/mmole, New England Nuclear) was made into the posterior chamber of the right eye of each fish with a microliter syringe. All fish were kept at 20.5 C for 2 hr after the injection, then the fish in the low-temperature series were transferred to water in a refrigerator at 9 C. For each time point after the injection five or six fish were killed. The fish were decapitated, the brains exposed, and the heads fixed for 48 hr in Bouin’s solution, then transferred to 70% alcohol. This procedure preserves the proteins but washes out the soluble amino acids (5). The retinas and tecta were dissected out, air-dried and weighed, then the radioactivity was measured with Gupta’s incineration technique ( 17) at about 30% efficiency for tritium. All measurements have been expressed in counts per minute per milligram dry weight of tissue. Values obtained from different experiments were normalized to compensate for different levels of incorporation. Some animals were prepared for light microscopic autoradiography by standard techniques (24). For each time point, two to four tecta were examined, and for each tectum grain counts were made over an area of 14,600~” at each of four to eight points. Results

Axonal Transport of Protein. The rate of transport of labeled materials along the fish optic axons may be measured by determining the rate at which the transported materials accumulate in the optic tectum where most of the optic fibers end. After injection of labeled amino acid into the eye, the labeled protein appearing in the tectum consists both of material transported along the optic nerve and of material locally synthesized in the tecturn from labeled amino acid which had escaped into the circulation from the eye. The technique used, therefore, was to inject the label into the right eye only and to measure radioactivity in both tecta. While both contain protein labeled via the circulation, only the left tectum contains the labeled protein transported along the optic nerve, because the nerves are

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MCEWEN

completely crossed at the chiasma. Thus the difference in radioactivity between the two tecta is a measure of the transported protein (3 1). In determining the effects of temperature on transport it is important to keep the incorporation of labeled amino acid constant. This was done by changing the temperature only after incorporation was essentially complete (31), at 2 hr after injection. Under these circumstances, the initial time course of incorporation of radioactivity into protein in the right tectum, which is probably determined largely by the rate of release of radioactivity into the general circulation from the eye, was the same both in animals kept at room temperature and in those transferred to 9 C (Fig. 1B). Only after 2-3 days did the values in the two series of animals diverge. In animals at room temperature, as has previously been found (6, 31)) labeled proteins transported from the eye had already begun to appear in the tectum by 3 hr after intraocular injection (Fig. 1A). The transported radioactivity rose for about 12 hr, then declined slightly over a period of about 10 days. In the animals in the cold series, the transported radioactivity at 3 hr after the injection was only slightly less than at room temperature, preA. TRAiSPORTED 800-

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appearance of (A) transportedradioactiveprotein (differencebetweenright and left tecta) and (B) locally synthesizedradioactive protein (right tectum), after injection of 3H-leucineinto right eye, at 20.5 C (‘O-0) and 9 C (O--O). FIG.

1. Time

course

of

AXONAL

161

TRANSPORT

sumably because the animals had been at room temperature during the first 2 hr. However, the peak of transported radioactivity in the tectum occurred considerably later than in the room temperature series, i.e., at 24 hr after the injection, and was followed by a sharp decline to about 60% of the peak. The level then remained constant for about 10 days (Fig. 1A j. If we compare the time courses by measuring the time to half peak, we get a value of 5 hr for the room temperature series, corresponding to an average rate of transport of about 60 mm/day, and 15 hr for the room temperature series, i.e., an average rate of about 20 mm/day. These numbers give a Qlo for the rate of rapid transport of 2.6, This value is certainly an underestimate of the Qlo, since the times measured above would include the period of incorporation of the isotope before the temperature was changed, as well as a period in which transport was occurring but equilibration to the low temperature was not yet complete. Both these factors would decrease the difference between the rates of transport in the two series. On a different time scale, the rate of slow transport may be seen (Fig. 2). The rise of radioactivity signaling the arrival of the slow component TRANSPORTED ,

iz

20

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(L-R

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1 40 AFTER

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,

80

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1

100

(days)

FIG. 2. Time course of appearance of transported radioactive protein (A) at 20.5 C (OA) and (B) at 9 C (On). The different symbols represent different experiments, normalized on the basis of retinal incorporation. The early part of the time course has been drawn to correspond with Fig. 1.

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FORMAN

AND

MCEWEN

began at the same time at both temperatures, namely between 10 and 15 days after the injection. Even assuming the maximum difference between the two curves during this S-day interval, the Qlo for the first arrival of the slow component would not be greater than 1.4. The peak radioactivity occurred at about 3 weeks in the room temperature series but the temperature sensitivity of its appearance could not be determined, since no clear peak was seen at 9 C. This complicates the interpretation of the data, but it appears that at least a portion of the slow transport was not appreciably sensitive to temperature. Protein Turnover in Retina. At most sites in the nervous system, the picture of turnover of materials is modified by the existence of axonal transport systems which rapidly or slowly remove materials from the site of synthesis independently of their rate of metabolic degradation. In the retina, however, only a small proportion of the neurons, possibly about lo%,, are ganglion cells with axons which leave the retina, so that axonal transport accounts for the disappearance of only a small proportion of locally synthesized material. Thus we expect the disappearanceof radioactive protein from the retina to represent fairly well the time course for metabolic turnover of the retinal proteins. In line with this, we found the time course of disappearance of radioactive protein from the retina showed a relatively simple picture at both temperatures (Fig. 3) : after a loss of about 30% of the initial radioactivity over the first S-10 days, the remain-

2 3

RETINA

L-s 50* 0

5 a

,0

I 40

20 TIME

FIG. 3. Time

course of disappearance (@A) and 9 C (On). The various logarithmic scale of ordinate.

AFTER

1

60

INJECTION

1

00

1 loo

(days)

of radioactive protein from retina at 20.5 c symbols represent different experiments. Note

AXONAL

163

TRANSPORT

der showed an exponential decline with a half-life of about 3 weeks at room temperature and about 17 weeks at 9 C, giving a net Qlo for the degradative process of about 4.5. Protein Turnover in Right Tectunz. In the right tectum the level of locally synthesized radioactive protein increased over the first 12 hr, then declined over a period of 23 days (Fig. 1U). Unlike the retina, however, the radioactivity then rose again over the next 2 weeks (Fig. 4). This rise was somewhat slower at the lower temperature, but otherwise there was little difference between the two series of fish during the first 4 weeks after the injection. Thereafter the values in each series followed an apparently simply exponential decline but with different half-lives, namely 7 weeks at 20.5 C and 17 weeks at 9 C, to give a net Qlo of 2.2. Transported Material. After the arrival of the slowly transported protein, the transported radioactivity measured in the tectum represents a combination of the fast and slow components of the transport. The disappearance of this combined material at room temperature, as indicated by the decline of radioactivity beginning 3 weeks after the injection, followed a nearly linear time course with a half-time of about 6 weeks (Fig. 2A). At 9 C, the total tectal radioactivity showed no appreciable decline even at 86 days after the injection (Fig. ZB), making it impossible to estimate a Q10 for the turnover of the slowly transported material. To separate the individual contributions from the fast and slow components, the time course of appearance of radioactivity was compared in the BACKGROUND

(R TECTUM)

. 300-

*A ofi

250e 0

20.5*C 9*c

20 TIME

\

40 AFTER

60 INJECTION

80

loo

(days)

FIG. 4. Time course of disappearance of locally synthesized radioactive protein in right tectum, after injection of 3H-leucine into right eye, at 20.5 C (.A,) and 9 C (OA). The symbols as in Figs. 2 and 3. Note logarithmic scale of ordinate.

164

GRAFSTEIN.

FORMAN

AND

MCEWEN

layer of the tectum in which the synaptic endings of the optic fibers are concentrated ( 13, 31) and in nearby regions containing closely packed optic axons, particularly the main dorsomedial and ventrolateral optic fiber bundles. In autoradiograms of the tectum obtained from fish at 15 C, the arrival of the slowly transported material in the fiber layers was seen as an increase in radioactivity between 25 and 50 days after the injection (Fig. 5). No corresponding increase was seen, however, in the synaptic layer, which suggests that the slowly transported material did not penetrate into the terminals of the optic fibers. The final decline of radioactivity showed the same time course in both regions and was grossly different from the exponential decline seen in either the retina or right tectum, but could be reconciled with the decline seen in the total radioactivity in the tectum (Fig. 2A). In the synaptic layer, the radioactivity had fallen to half by about 90 days after its initial appearance, although there was relatively little decline during the first half of this period. Discussion

Fast and Slow Protein Transport. The temperature coefficient of a particular process may give us some clue to the nature of the limiting step in that process. Low temperature coefficients are generally associated with

.*\.\ pq 6oop-P---__ i \ -

0 Synoplic l

0

TIME

Fiber

\

loyer

I

layer

I

I

I

I

I

20

40

60

80

100

AFTER

INJECTION

(days)

FIG. 5. Radioactive protein in fiber layer (0) and synaptic layer (0) of tectum after injection of sH-leucine into contralateral eye, determined from grain counts in autoradiograms. Values represent mean 2 standard error of mean count over an area of 14,600 fi2. The counts were made on sections taken about mid-way along the length of the tectum.

AXONAL

TRANSPORT

165

phenomena involving purely physical interactions among molecules ; simple chemical interactions have higher coefficients ; and still higher coefficients are characteristic of enzymatic processes in which a cascading of the temperature effect may occur in a series of successive reactions (12). Our data indicate that the fast transport of protein in goldfish optic nerve is a temperature-sensitive process, while the slow transport is relatively temperature-insensitive. The fact that the fish were kept in the dark while they were in the cold does not affect our results. We have found that in short-term experiments the absence or presence of light had no effect on either the fast or slow protein transport in goldfish optic nerve (B. S. McEwen and B. Grafstein, unpublished results). Long-term exposure to cold causes degeneration of the visual receptor cells (4) but this is irrelevant, since the slow transport rate was evidently utlchanged by the change in temperature. For the fast transport the Q,,, value of over 2.6 that we have found is close to that which can he obtained from the data of Elam and Agranoff. Although they did not keep the temperature of incorporation constant, it is possible to calculate a Q10 for the transport rate by comparing their data for fish at 23 C or 16 C (6) and for fish that they injected at 1s C then transferred to 11 C (7) ; the value thus obtained is 2.5. These values are high enough to encompass an oxidative process, since the Qlo for osygen consumption in goldfish brain (calculated by us from data provided by Freeman, 11) is 2.7. Thus the QIo that we have obtained is consistent with the evidence that the fast transport is dependent on oxidative metabolism (32, 34). With the observed degree of temperature sensitivity, we would expect that the fast transport which has an average rate of about 60 mm/day at 20.5 C would be equivalent to about 290 mm/day at mammalian body temperature. This is somewhat in excess of the rates that have been found in the optic fibers in either mouse (16) or adult rabbit (41 ; but this value has been challenged as being too low, 18). However, the discrepancy is probably not significant, since the Ql,, obtained by Ochs and Smith (36) in a mammalian preparation was only 2-2.3, indicating that the temperature coefficient probably decreases at increasing temperatures, a not uncommon phenomenon. Moreover, the maximum values that have been found for goldfish optic nerve, i.e., 70-100 mm/day (6 ; D. Forman, unpublished results) would be equivalent to rates of 300-500 mm/day at 37 C, equal to those observed in a number of mammalian nerves (27, 35). The temperature-independence of the slow component, on the other hand, emphasizes the fact, already apparent from other evidence (1, 6-8, 10, 13, 14, 19, 30, 31), that the two components are distinct entities. The low QIo that we have found suggests that either (a) at least some of the

166

GRAFSTEIN,

FORMAN

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MCEWEN

slow component is transported by a physical-chemical mechanism, e.g., diffusion; or (b) the slow transport is subject to temperature acclimation (37) which returns the net transport rate to a nearly constant level ; or (c) the transport mechanism is inherently temperature-compensated. Of these three, diffusion is the least likely, for the following reasons: about half of the slowly transported protein is particulate in nature (31, 33, 41) and would not participate in diffusion ; in regenerating goldfish optic nerve the slow transport rate increases to nearly three times normal, which would not be expected with a diffusional process (15) ; the peak of radioactivity in the slow transport does not spread out in time (9, 27) as would be predicted from diffusional thermodynamics (3). The process of temperature acclimation has been demonstrated for a number of functions in goldfish (2, 11, 20, 37-39), but many processes, including fast transport (7), do not acclimate, so it cannot be assumed that the slow transport would do so. As for the possibility of an inherently temperature-compensated process, on the other hand, there is only some indirect evidence: R. Biondi, P. Weiss, and M. Levy (personal communication) have constructed a hydrodynamic model for the peristaltic mechanism that Weiss has postulated as the basis of slow transport (42), and have found its operation to be independent of temperature. Finally, since we were able to ascertain the Q,, only of the beginning of the arrival of the slow component, we must consider the possibility that slow transport might be a hybrid process in which the initial phase has the low Qlo that we have observed, but other fractions of which are more temperature-sensitive. This would reconcile our present results with those of Fernandez, Huneeus, and Davison (9), who found that the rate of translocation of the peak of slowly transported radioactivity is linearly related to temperature. An intriguing aspect of our present findings is the early peak associated with the fast transport at lowered temperature. Although a rise to a maximum followed by a slow decline has usually been seen at room temperature ( 16, 31), the change was relatively small, and hardly detectable at all in our present room temperature data (Fig. 1A). A sharp spike similar to that appearing here at 9 C has been reported in goldfish at room temperature under two other circumstances: in tecta with regenerated optic nerves in which the rate of fast transport was about twice normal (15), and in normal tecta when proline or arginine was used as precursor instead of leucine (6, 7). The difference between the results obtained with proline and with leucine has also been seen in mice (16). In some other species a short-lasting early peak in the fast transport is a characteristic occurrence even with leucine (21, 40). Our present tentative view is that the early peak represents a fraction of the fast transport that either has a very short half-life, due to metabolic destruction, or is diluted as it comes down the

AXONAL

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nerve, due to exchange with the unlabeled constituents of the axon. At normal temperatures it would therefore be largely attenuated even before it reached the nerve terminals. At lower temperatures either the destruction or exchange might be slowed to a greater degree than the rate of transit along the nerve. In regeneration, the increased transport rate might be sufficient to compensate for the rapid disappearance. Substitution of proline for leucine might produce a label of longer lifetime, as it does in the case of the slow component of transport (16). Still another explanation that we may entertain is that the sharp early peak may represent material that is rapidly transported to the nerve endings, then removed by retrograde transport and recirculated throughout the neuron. However, the present picture of the retrograde process (23, 25, 29) is still so incomplete that any analysis in these terms would be premature. Profcis Tumoz~r. The relatively simple two-phase picture of disappearance of radioactivity that we have described for the retina has also been seen in rabbit retina (22), and the values that we have obtained for the second phase, a half-life of about 3 weeks at 20.5 C with a Qlo of 4.5, are consistent with the estimate for the half-life of about a week at rabbit body temperature. It is likely, however, that even the relatively simple picture seen in the retina is a composite of many processes involving proteins of different turnover times (26,28). A s an indication, for example, of the degree to which the visual receptor cell proteins dominate the picture of total retinal protein turnover, it has been found that in mice lacking visual receptor cells, the half-life for retinal protein in the second phase is only 2 days, compared to a week in normal mice (B. Grafstein, unpublished resuits)

.

In most parts of the brain the picture would be expected to be even more complicated than in the retina because of the arrival of transported material from other regions of brain. This may explain the increase in the radioactivity in the right tectum that begins about 3 days after the injection. However, an increase of this magnitude could be produced only by a large, relatively uniform group of fibers whose cells of origin had been nearly as intensely labeled as the retinal ganglion cells. A more likely explanation, therefore, it that the increase may be built up from the discharge into the circulation of label from a pool outside the brain. Turnover times of label in viscera and muscles are shorter than in the brain (26, 25)) and the radioactivity liberated from these and other tissues might become available for incorporation into the brain. Presumably a corresponding increase in retinal labeling would also occur, but would not be detectable in our experiments since the retina is already so intensely labeled. Another possible complication, the local reutilization of label, has been ruled out, in the case of leucine at least (28). In summary, then, the interpretation of the values

168

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FORMAN

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MCEWEN

that we have obtained for the half-lives and temperature coefficients of protein turnover of various tissues is complicated by the heterogeneity of the turnover processes, by the arrival of transported material, and by the continued synthesis of protein from labeled material released by other tissues. In the case of the transported protein, interpretation of the results is further complicated by the presence of at least two components of transport. Thus the nearly linear decline in total transported radioactivity that was seen after the arrival of the slow component may be determined by the rate of disappearance of either the fast or slow component, or both. The radioautographic data do not resolve this matter: the level of radioactivity in the synaptic layer, initially comprising only the fast component, showed little decline for about 7 weeks, but this level may have been maintained by the continual entry of small amounts of the slow component into the endings. Thus although we might conclude from these data, as we have previously done (30)) that the fast component in the synaptic endings has “a half-life of around 100 days,” it may in fact be considerably shorter than this. Experiments in which the fast component alone was examined by labeling only glycoproteins have produced an estimate of only about a month for its half-life (lo), but this need not be characteristic of the whole of the rapidly transported material. Our finding that the arrival of the slow wave is undetectable in the synaptic layer of the tectum is consistent with other observations of the decrease in the height of the slow wave relative to the rapid component as the nerve terminals of the optic fibers are approached (21, 40). This apparent “dissipation” of the slow component may be due to three factors: (a) compared to the axon trunks, the endings contain relatively less of the materials that comprise the slow component; (b) the slow component is continually metabolized as it descends the nerve; (c) there is a continual exchange between the labeled materials in transit and the unlabeled materials of the axons, so that while the level of radioactivity decreases as the slow component approaches the’ nerve endings, the amount of material moving in the transport stream does not. With this last mechanism, it would be necessary to suppose that the large amount of material conveyed to the synaptic endings by the slow transport was continually being liberated at the endings. References 1. BRAY, J. J., and L. AUSTIN. 1969. Axoplasmic transport of 1°C proteins at two rates in chicken sciatic nerve. Brain Res. 12 : 23&233. 2. DAS, A. B., and C. L. PROSSER. 1967. Biochemical changes in tissues of goldfish acclimated to high and low temperatures. I. Protein synthesis. Ca~fp. Bio,chem.

Physiol. 21: 449467. 3. DAVISON,

P. F.

1970.

Axoplasmic

transport:

physical

and

chemical

aspects,

pp.

AXONAL

4. 5.

6. 7. 8. 9.

10.

11.

12. 13. 14. 15. 16. 17.

18.

851-857. In “The Neurosciences: Second Study Program.” F. 0. Schmitt [Ed.]. Rockefeller Univ. Press, New York. DAWSON. W. W., G. M. HOPE, and J. J. BERNSTEIN. 1971. Goldfish retina structure and function in extended cold. Erp. Ncwol. 31: 368-382. DROZ, B.. and H. WARSHA~SKY. 1963. Reliability of the radioautographic technique for the detection of newly synthesized protein. J. Nistocherlr. Cyforhor~. 11: 426-435. ELAM, J. S., and B. W. AGRANOFF. 197la. Rapid transport of protein in the optic system of the goldfish. J. ~VmrohwI. 18 : 375-387. ELAM, J. S., and B. \V:. AGRANOFF. 1971b. Transport of proteins and sulfated mucopolysaccharides in the goldfish visual system. J. Nrrwobiol. 2: in press. ELAM, J. S., J. M. GOLDBERG, N. S. RADIN, and B. W. AGRANOFF. 1970. Rapid axonal transport of sulfated mucopolysaccharide proteins. Scieltcc 1711: 458-460. FERNANDEZ, H. L., F. C. HUNEECS, and P. F. DAVISON. 1970. Studies on the mechanism of axoplasmic transport in the crayfish cord. J. Nrrrrobiol. 1 : 395409. FORMAN, D. S., B. S. MCEWEN, and B. GRAFSTEIN. 1971. Rapid transport of radioactivity in goldfish optic nerve following injections of labeled glucosamine. Brain Res. 28 : 119-130. FREEMAN, J. A. 1950. Oxygen consumption, brain metabolism, and respiratory movements of goldfish during temperature acclimation, with special reference to lowered temperature. Biol. Bull. 99 : 416-424. GIESE, A. C. 1962. “Cell Physiology.” Saunders, Philadelphia. GRAFSTEIN. B. 1967. Transport of protein by goldfish optic nerve fibers. Sriellce 157 : 196-198. GRAFSTEIN, B., B. S. MCEWN, and M. SHELANSKI. 1970. Axonal transport of neurotubule protein. Nature 227 : 28’+290. GRAFSTEIN, B., and M. MURRAY. 1969. Transport of protein in goldfish optic nerve during regeneration. Exp. Newel. 25 : 494-508. GRAFSTEIN, B., M. MURRAY, and N. INGOGLIA. 1972. Protein synthesis and axonal transport in optic system of mice lacking visual receptors. Brain Res. In press. GUPTA, G. N. 1966. A simple in-vial combustion method for assay of hydrogen-3, carbon-14 and sulfur-35 in biological, biochemical and organic materials. -gtu~l. ChVl. 38 : 1356-1359. HENDRICKSON, A., and W. M. COWAS. 1971. Changes in the rate of axoplasmic transport during postnatal development of the rabbit’s optic nerve and tract.

E.rp. Nrurol. 19. 20.

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30 : 403-422.

K., and L. AUSTIN. 1970. The binding i)t a&o of colchicine to axoplasmic proteins from chicken sciatic nerve. Biochcrrl. /. 117 : 773-777. KANUGO, M. S., and C. L. PROSSER. 1959. Physiological and biochemical adaptation of goldfish to cold and warm temperatures. I. Standard and active oxygen consumptions of cold- and warm-acclimated goldfish at various temperatures. J. Cdl. Conzp. Physiol. 54: 259-263. II. Oxygen consumption of liver homogenate ; oxygen consumption and oxidative phosphorylation of liver mitochondria, JAMES,

A.

Ibid. 265-274. 21. 22.

KARLSSON, J.-O., and J. SJGSTRAND. 1971. Synthesis, migration and protein in retinal ganglion cells. J. Ncurochcm. 18 : 749-767. KARLSSON, J.-O., and J. SJ~STRAND. 1971. Transport of microtubular axons of retinal ganglion cells. J. Nezwoclzctrr 18: 975-982

turnover protein

of in

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G. A., A. SHAPIRA, and R. J. WALKER. 1967. The transport of labeled material from CNS + muscle along a nerve trunk. Cow@. Biochem. Physiol. 23 : 729-748. 24. KOPRIWA, B., and C. P. LEBLOND. 1962. Improvements in the coating technique of radioautography. J. Histochern. Cytoc/rem. 10 : 269-284. 25 KRISTENSSON, K, and Y. OLSSON. 1971. Retrograde axonal transport of protein. KERKUT,

Brain

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Res. 29 : 36s365.

A. 1964. Protein metabolism of the nervous system. Znf. Rev. Newol-98. LASEK, R. 1970. Protein transport in neurons. Int. Rev. Newobiol. 13: 289-321. LIM, R., and B. W AGRANOFF. 1969. Protein metabolism in goldfish brain. J. Neurochem. 16 : 431445. LUBI~SKA, L. 1964. Axoplasmic streaming in regenerating and in normal nerve fibres, pp. l-66. In “Mechanisms of Neural Regeneration.” M. Singer and J. P. Schade [Eds.]. (Progr. Brain Res., 13). Elsevier, Amsterdam. MCEWEN, B. S., D. FORMAN, and B. GRAFSTEIN. 1971. Components of fast and slow axonal transport in the goldfish optic nerve. J. Nezlrobiol. 2. In press. MCEWEN, B. S., and B. GRAFSTEIN. 1968. Fast and slow components in axonal transport of protein. J. Cell Biol. 39 : 494-508. OCHS, S., and D. HOLLINGSWORTH. 1971. Dependence of fast axoplasmic transport in nerve on oxidative metabolism. J. Neurochenr. 16: 107-114 OCHS, S., J. JOHNSON, and M.-H. Nc. 1967. Protein incorporation and axoplasmic flow in motoneuron fibres following intra-cord injection of labeled leucine. J. Neurchem. 14 : 317-331. OCHS, S., and N. RANISH. 1970. Metabolic dependence of fast axoplasmic transport in nerve. Science 167 : 878879. OCHS, S., M. I. SABRI, and J. JOHNSON. 1969. Fast transport system of materials in mammalian nerve fibers .Science 163 : 686687. OCHS, S., and C. SMITH. 1971. Effect of temperature and rate of stimulation on fast axoplasmic transport in mammalian nerve fibers. Fed. Proc. 39: 665. PROSSER, C. L. 1961. Temperature, pp. 238-284. III “Comparative Animal Physiology,” 2nd ed. C. L. Prosser and F. A. Brown [Eds.]. Saunders, Philadelphia. RENTS, B. I. 1968. Phospholipids of goldfish (Carassius arrratus L.) brain: the influence of environmental temperature. Contp. Biochem. Physiol. 25 : 457-466. ROOTS, B. I., and C. L. PROSSER. 1962. Temperature acclimation and the nervous system in fish. J. E.@. Biol. 39 : 617-629. SCHONBACH, J., and M. C&NOD. 1971. Axoplasmic migration of protein. A light microscopic study in the avian retino-tectal pathway. E.rP. Brain Res. 12: 275282. SJ~STRAND, J., and J.-O. KARLSSON. 1969. Axoplasmic transport in the optic nerve and tract of the rabbit: a biochemical and radioautographic study. J. Neurochem. 16 : 833844. WEISS, P. 1967. Neuronal dynamics. Neurosci. Res. Prog. Bull. 5: 371-400. LAJTHA,

biol. 6 :

27. 28. 29.

30. 31. 32. 33.

34. 35. 36. 37 38. 39. 40. 41. 42.