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A radiolabelled pulse for the simultaneous study of anterograde and retrograde axonal transport
Journal of Neuroscience Methods, 17 (1986) 109-119
109
Elsevier NSM 00595
A radiolabelled pulse for the simultaneous study of anterograde and retrograde axonal transport R.E.
Snyder
Department of Applied Sciences in Medicine, University of Alberta, Edmonton, Alta. (Canada)
(Received 29 April 1985) (Revised 24 April 1986) (Accepted 26 April 1986)
Key words: Axonal transport - Position-sensitive detector - Retrograde transport
A technique is described for producing a pulse of [35S]methionine-labelled material which is axonally transported in amphibian sciatic nerve maintained in vitro. Using a position-sensitive detector of ionizing radiation, it is possible to continuously observe the movement of the pulse in the anterograde direction and, following turnaround at a ligature, the movement of a fraction of the pulse in the retrograde direction. Two sources of contaminant activity, which would otherwise interfere with observation of the pulse, are discussed and shown to be avoidable.
Introduction C o n t i n u o u s monitoring of the axonal transport of a radiolabel in a living preparation m a y be accomplished by placing a nerve in opposition to a positionsensitive detector of ionizing radiation (Snyder et al., 1976a,b; Widen et al., 1976; T a k e n a k a et al., 1978). In an earlier report (O'Brien and Snyder, 1982), it was demonstrated possible to continuously m o n i t o r the anterograde m o v e m e n t of a pulse of radiolabelled material which had been created using a cold-blocking technique ( E d s t r o m and Hanson, 1973; Brimijoin, 1975). However, it was f o u n d that the retrograde transport of labelled material, turned a r o u n d at a distal ligature, was observed in only 15% of the preparations. Relatively little is k n o w n of the dynamics of t u r n a r o u n d and retrograde transport since most studies are performed using the double-ligature technique (Bisby, 1982; Schwab and Thoenen, 1983). Frequently, it is impossible to dissociate the two processes. This has led us to develop a new m e t h o d of forming a pulse, one which has consistently resulted in the retrograde m o v e m e n t of radiolabel turned a r o u n d at a distal ligature. The m e t h o d does not require the concentrating of material, as does cold blocking, thus avoiding
Correspondence: R.E. Snyder, Department of Applied Sciences in Medicine, University of Alberta,
Edmonton, Alta., Canada T6G 2G3, 0165-0270/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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the question of concentration-dependent effects (Goldberg et al.. 1978: Brimijoin, 1979; Bisby, 1983; Litchy and Brimijoin, 1983). Pulse formation consists of incubating a dorsal root ganglion-sciatic nerve preparation in a two-compartment tray, exposing the ganglion cells to a radiolabel. and later severing the nerve at two different times proximal to the moving activity. Special attention must be given to the suppression of contaminant activity by which we mean any activity ultimately monitored by the detector which is not directly associated with transport. We have found there to be two primary sources of such activity which, if not controlled, will result in interference with detection of the transported activity. The first is radiolabel which enters the nerve via a 'diffusion-like' process during the pulse-creation stage. The second is associated with transported label which is expelled from the axons (Hines and Garwood, 1977; Tedeschi et al., 1981).
Methods
Nerve preparation A sciatic nerve in continuity with its ninth dorsal root ganglion and spinal roots was removed from adult female specimens of the amphibian Xenopus laevis. A ligature was placed 65-70 mm distal to the gan~on. The perineural Sheath was not removed at this time. During the dissection and pulse,creation stage the preparation was bathed in an oxygenated saline solution of composition (mM): NaCI 112, KC1 3.0, MgSO4 1.6, CaC12 3.0, glucose 5.0, HEPES (N-2'hydroxyethylpiperaz~ne-N'-2ethanesulphonic acid) 3.0. The pH was adjusted to 7.38-7.42 with NaOH, Following pulse creation (see below) the nerve was normally bathed in a solution of the above composition which contained in addition non-radioactive metl',ionine (1.0 mmol/1). Additional details pertaining to the dissection may be found in the literature (O'Brien and Snyder, 1982). Pulse procedure Immediately following its dissection the preparation was placed in a two-compartment plastic tray which contained saline solution, the ganglion and its root in one compartment (0.025 ml) and the nerve in the second (20 ml). The solution was removed from the compartment containing the ganglion and replaced with 0.025 ml of saline solution to which had been added 0.15-0.25 mCi of L-[aSS]methionine at a specific activity of 1000-1400 Ci/mmol (Amersham or New Fag,land Nuclear). One hour was allowed for the [35S]methionine to become incorporated into protein following which the ganglion compartment was rinsed twice and refilled! with radiolabel-free saline solution. Following an additional 3.25 h duriag which labelled material was exported from the cell bodies, the nerve was severed immediately distal to the septum separating the compartments, approximately 4-5 mm distal to the ganglion, thus creating a pulse of radiolabeHed material at the proximal end of a 60-65 mm length of nerve. In a few experiments the time between refill and severing was extended to 3.50-3.75 h.
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Immediately following its being severed the nerve was rinsed by being placed in 1 ml of saline solution and gently agitated for 5 min. Beginning with this rinse and onwards the bathing saline solution normally contained non-radioactive methionine as noted above. The nerve was next transferred to a dish containing fresh saline solution and, with the perineural sheath still in place, tensioned to a standard length using the technique described by Chan et al. (1980), the length being noted. The ligature near the distal end of the nerve was removed, the perineurium removed, and the ligature replaced. The nerve was then placed in 200 ml of saline solution and gently agitated until 130 min following its being severed, at which time the most proximal 10-mm section of nerve was removed and discarded. Approximately 10 min following removal of the 10-mm segment a thread was attached to the remaining nerve < 1 mm from its proximal end. The nerve was placed in a one-compartment tray with a 3.6-/tm mylar floor and containing saline solution. Using the proximal thread and a second thread used to form the distal ligature the nerve was stretched to its measured length and positioned in contact with the floor of the tray (Snyder et al., 1980). A piece of gauze stretched over an acrylic frame served to lightly press the nerve against the floor. The tray was placed over the entrance window of a multiple proportional counter (Snyder, 1984) and the nerve bathed in a circulating volume of approximately 175 ml of saline solution. Experiments were performed at a temperature of 22.7-23.3 ° C, maintained to within + 0 . 2 ° C for any given experiment.
Position-sensitive detector analysis The one-compartment tray containing the nerve was placed in contact with the entrance window of a multiple proportional counter (MPC). The MPC is composed of a series of radiation counters spaced at 3.18-mm intervals and serves to detect those B-particles (typically 0.5-1.5% of the total) which have their origin in the nerve and enter the counter. Counts vs position spectra were recorded for 20-22 h in 1000-s blocks. Prior to placing the nerve in the tray, 2 - 4 background spectra were recorded with the tray in position on the detector. The average of these spectra was used to correct all counts versus position spectra from an experiment for room background. At the conclusion of an experiment the nerve was removed from the tray and 2-3 additional spectra recorded for comparison to the final spectrum obtained with the nerve in position. Finally, the series of counts vs position spectra were arranged so that the counting rate for each 3.18-mm region of nerve could be plotted as a function of time.
Diffusion studies In a variation of the pulse procedure, the inhibitors of protein synthesis cycloheximide ( 1 2 5 / t g / m l ) and puromycin (500/~g/ml) (Snyder et al., 1984) were both added to the solution bathing the ganglion 0.5 h prior to introduction of the [3~S]methionine, remaining throughout the course of the incubation in order to retard the axonal transport of any radiolabel. During some experiments the radiolabel was not removed 1 h following its introduction.
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Efflux studies Four, 10-18 mm sections of unbranched sciatic nerve were removed from a frog and the perineural sheath removed from two sections. Each section was placed in 2 ml of saline solution which contained either non-radioactive methionine (100 /zg/ml) or puromycin (500/~g/ml), and 1 h later 2/~Ci of L-[methyl-3H]methionine (New England Nuclear) was added to the solution. Following 16-17 h, during which time radiolabel entered the nerve, the sections were immersed sequentially in a series of 1-ml volumes of saline solution at room temperature (22-24°C) during a 5 6 h period, followed by overnight immersion in a final volume. At the conclusion of the experiment the samples were treated as will be described below. For additional details, see Snyder and O'Brien (1983).
Liquid scintillation analysis All nerves were assayed by liquid scintillation counting at the completion of an experiment. Nerves monitored with the MPC were cut into segments which corresponded to the detector regions; sections used in the efflux study were counted whole; nerves used in the remaining diffusion studies were cut into 2 or 5 mm segments with a precision of +0.1 mm. All segments were treated with 5% trichloroacetic acid (TCA) solution for 1-2 h at 80°C and dissolved in Protosol (this and other scintillation reagents were obtained from New England Nuclear). Econofluor was added to the dissolved samples and Aquasol to the soluble fractions. Samples of various bathing solutions were counted by addition of Aquasol. All specimens were assayed by liquid scintillation analysis.
Results
Transport studies Sixteen preparations were studied using the normal pulse-creation procedure. The experiments differed only in the use of non,radioactive r n e ~ o m n e in the bathing solutions and in the time elapsed between the introduction of [3~S]methionine and severing of the nerve. For studies in which the elapsed time was 4,25 h, liquid scintillation counting revealed the nerve to contain a total activity of 0,022 5: 0,013% (5: S.D.) of that introduced into the g ~ o n compartment. The anterograde movement of a pulse of activity culminated in a build'up of activity at the distal ligature, followed by the retrograde movement of a pulse of activity (Figl 1). This second, or retrograde pulse, was present in all preparations studied. At the conclusion of each MPC experiment, spectra were obtained with the nerve removed from the one-compartment tray in order to evaluate the extent of contarnination recorded by the MPC due to activity not within the nerve. The total counting rate observed following removal of ~ nerve was d i ~ e d : by that obtained from the last spectrum acquh,~xl with the nerve in ptace and fountl to be 7,2± 3,7% when non-radioactive methionine was absent from the bathing s ~ f i o n , but only 1.2 5: 0.5% with methionine present. Nerves incubated with ii~ibitors of protein
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Fig. 1. Counts vs time plots. Each plot corresponds to a 3.18-mm segment of nerve (e) whose mean distance from the proximal end of the nerve is indicated on each plot in mm. Sixteen plots were obtained; only 8 are shown. A pulse can be seen to move in the anterograde direction, resulting in accumulation of radiolabel at the distal ligature between 6-8 h, following which a pulse of smaller amplitude moves in the retrograde direction (arrows). The retrograde pulse is generally discernible in 8-12 plots. Open circles (O) are either background data obtained prior to placing the nerve in the tray or contamination data obtained following removal of the nerve from the tray.
synthesis present in the ganglion compartment showed no observable contamination following removal from the tray. In order to better understand the nature of this contaminant activity, an experiment was performed in which one nerve was monitored by the MPC while the contralateral nerve, which had been treated with inhibitors of protein synthesis during the pulse-creation procedure, was maintained in a separate tray. Samples of the two bathing solutions were drawn at regular intervals; the results are shown in Fig. 2. As is evident, relatively little activity entered the bathing solution from the
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Fig. 2. Total activity in the bathing solution expressed as parts-per-million (ppm) of the quantity of [35S]methionine used during the pulse-creation procedure: closed circles (O), nerve containing normal radiolabelled pulse; open circles (©), similar nerve in which transport of radiolabdted material was suppressed by the presence of inhibitors of protein synthesis in the ganglion compartment during the pulse-creation procedure. The nerves were placed in their bathing solutions at time zero.
treated nerve compared to that from the nerve containing the pulse of activity. This finding was substantiated by assaying the activity in the MPC bathing solution at the beginning and at the end of other experiments. The m o u n t of activity present in the bathing solution at the conclusion of a pulse experiment was found to be 4.0 + 1.0% of that in the nerve when non-radioactive methionine was absent from the bathing solution, but 29 + 12% when methionine was present. In an attempt to determine the physical location within the tray of the contaminant activity, spectra were obtained following removal of the nerve in which strips of 25-#m thick copper foil, sufficient to reduce the 35S beta-spectrum by < 98%, were imposed between the MPC window and the tray floor. Such studies i n d i c t e d that < 20% of the recorded contaminant activity is due to activity in the bathing solution, with the remainder due to activity which adheres to the mylar floor of the tray adjacent to where the nerve was situated. These studies lead us to conclude that the most likely source of this contaminant activity recorded by the MPC is axonal transport. The major of the recorded activity does not r e s e t from activity within the bathing solution, but rather from that adhering to the mylar floor. Finally, an experiment was performed to determine the extent to which activity might gain access to a nerve after having entered the bathing solution. Such contamination would not appear in recordings obtained with the neree removed. A desheathed nerve, which h a d not been e x p o s ~ to ra~aotai~l, was position¢~l in the one-compartment tray in tl~ normal maaner. A second nerve, whicheontained a radiolabelled pulse, was placed in the bathing solution a b o v e the first such that
115 activity f r o m the second could not enter the MPC. N o increase in counting rate could be observed outside of the statistical limits due to the r o o m background.
Diffusion studies During the period when the ganglion is exposed to the radiolabel, activity can enter the nerve via b o t h transport and diffusion (Chihara, 1979; Haley et al., 1979). A series of experiments was thus undertaken to estimate the magnitude of diffusion-related contamination. Experiments were performed using nerves incubated as in normal pulse studies, but with inhibitors of protein synthesis present in the c o m p a r t m e n t containing the ganglion to inhibit formation of a radiolabelled pulse. The nerves were severed at 4.25 h following the introduction of [35S]methionine and either (1) rinsed for 0.5-1.0 min, frozen, and divided into segments, or (2) following the n o r m a l 5-min rinse, desheath, 130-min rinse pattern, divided into segments for liquid scintillation counting. In all cases the TCA-insoluble activity in a nerve was < 3% of its total activity at the time of division. For nerves divided immediately following removal
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Fig. 3. TCA-soluble activity in nerves in which inhibitors of protein synthesis were present in the ganglion compartment during the pulse-creation procedure: triangles (&), [35S]methionine remained in the ganglion compartment until severing, nerve assayed immediately following severing; closed circles (e), [35S]methionine remained in the ganglion compartment until severing, nerve assayed following 130-min rinse procedure; open circles (O), [35S]methionine removed 1 h following its introduction, nerve assayed following 130-min rinse procedure. Cross-hatched areas depict pulse of axonally-transported radiolabelled-material immediately following severing (0-6 ram) and following 130-min rinse procedure (10-22.5 mm). Shapes and amplitudes of the pulses were estimated by extrapolation of the data recorded from 16 normal pulse experiments, such as that shown in Fig. 1.
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from the two-compartment tray the activity in the 0.5-1.0 min rinse plus that lost during the process of division was < 15% of that remaining in the nerve. Representative studies are shown in Fig. 3. Plots for similarly treated nerves were identical in shape, but displaced by + 1 mm along the abscissa. We attribute these displacements to an uncertainty in the position of severing of the nerves. By assaying the various rinse solutions it was determined that 30-50% of the activity present in a nerve immediately following severing remained in the nerve at the conclusion of the 130-min rinse. Based upon MPC recordings (Fig. 1), it is possible to estimate by extrapolation the size and shape of the anterograde pulse at times prior to those at which data were recorded. Two such 'extrapolated' pulses are shown in Fig. 3, one at the time of the 4.25-h severing and the second at the time of removal of the 10-mm segment. As is evident, at the time of the 4.25-h severing, diffusion-related activity within the nerve is approximately one order of magnitude greater than activity in the moving pulse. By allowing the pulse to propagate for 130 min, and removing and discarding the, proximal 10-mm section, it is possible to obtain a length of nerve in which the quantity of activity associated with transport is at least an order of magnitude greater than that related to diffusion. In order to determine the quantity of diffusion-related activity recorded by the MPC during a typical experiment, two preparations incubated with inhibitors of protein synthesis, and otherwise prepared as in the normal pulse procedure, were studied using the MPC. Both yielded initial MPC counting rates of 100-150 counts per hour in the most proximal 3 - 4 segments, decreasing to zero in approximately 10 h. Comparison to MPC studies of axonally transported pulses of activity (Fig. 1) showed that activity which enters the nerve due to means other than transport accounts for < 2-5% of the counting rate observed in the presence of transport, and only in the most proximal 3-4 segments of nerve during the initial 10 h.
Efflux studies In 4 efflux studies it was found in each case that < 5% of the total activity initially present was in the TCA-insoluble form, suggesting that the experiments served to measure the diffusion of [3H]methionine with minimal interference due to incorporation. N o significant differences were observed between the use of non-radioactive methionine or puromycin to retard incorporation. Data from each section of nerve was plotted as the rate of loss of activity versus time (data not shown) and fitted to a mathematical expression of the form 3
E A i e -Bit, i=l
where A i and B i are coedfu~ats and t is the time. For details, the reader is referred to the literature (Snyder and O'Brien, 1983). To a first approximation, each term in the above expression corresponds to a single efflux compartment with a corresponding half, life of In 2/Bi. Analysis of the two de,sheathed sections of nerve yielded half-lifes of 3.5 + 0.8 ( + S.D,), 36 4- 12, and 248 + 66 rain with correrpondiag fraetions of 0.18 + 0.11, 0.22 4- 0 . t l , and 0.60 + 0.23 r~l~etively. Similar analysis of the two sha~taed sections of nerve
117 yielded half-lifes of 3.8 + 1.7, 53 + 6, and 432 + 12 min with corresponding fractions of 0.09 + 0.04, 0.09 + 0.01, and 0.82 + 0.04 respectively.
Discussion
Using the procedure described, it is possible to produce a pulse of radiolabelled material which yields data rates sufficient for quantitative studies without the need for either cold blocking or concentrating material. The retrograde movement of radiolabel turned around at a ligature is a regular occurrence, facilitating the study of both turnaround and retrograde transport. However, attention must be given to the reduction of contaminant activity. Two significant sources of contamination exist, one present during the pulsecreation procedure and the other related to transport itself. The activity profiles of nerves for which axonal transport of labelled material was inhibited (Fig. 3) are qualitatively similar to those associated with diffusion: monotonically decreasing curves which may be approximated by a Gaussian distribution. From the results of the efflux study we estimate the diffusion for methionine in the interstitial space of the nerve to be 1.7 x 10 -6 cm2//s and the permeability of the perineural sheath to be 7.4 x 10 -7 c m / s (see Snyder and O'Brien, 1983 for details). However, using the model of a long cylinder of fluid bounded by a thin permeability barrier (Carslaw and Jaeger, 1959; Crank, 1959), it is possible to show that the above values will not result in sufficient diffusion to account for the observed profiles, although increasing the value of the diffusion coefficient by a factor of 2-10 does produce profiles which agree qualitatively with the observed results. Diffusion coefficients for amino acids in water at 25°C are typically 8-10 × 10 -6 cm2/s (Gosting, 1956). However, the present situation is more complex than this, both in structure and in factors such as viscosity (Haak et al., 1976). Thus, while having the general appearance of diffusion, it is not possible to account for this type of contamination solely based upon a simple model of diffusion. As is evident from Fig. 3, the pulse of transported radiolabel is engulfed at the time of severing by non-transport related activity. Since 30-50% of this activity remains in the nerve 130 min later, either it does not exist as free methionine, although TCA soluble, or more likely, does not reside in the interstitial space from which free methionine is cleared with a half-life of 3 - 4 rain. Thus, even removal of the perineurium prior to introduction of the radiolabel will not circumvent this type of contaminant activity, and we must allow the pulse to propagate beyond the front of non-transported label. We have found a distance of 10 mm to be sufficient with a propagation time of 130 min. This corresponds to a transport rate of 110 m m / d a y , compared to a slowest rate for fast transport of 119 + 10 m m / d a y measured for cold-blocked nerves (O'Brien and Snyder, 1982). The second source of contaminant activity stems from transport itself. As shown in Fig. 2, radiolabel associated with transport enters the bathing solution over a period of 20 h, this activity being approximately an order of magnitude greater than that resulting from non-transported activity. While we do not wish at this time to
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comment upon the nature of this phenomenon, it should be noted that two earlier reports investigated a closely related point, the release of transported proteins from nerve, arriving at opposing conclusions (Hines and Garwood, 1977: Tedeschi et al., 1981). Activity within the bathing solution itself contributes relatively little to the contamination; the majority of the contaminant activity appears to be associated with the mylar floor in proximity to the nerve. This may be reduced to an acceptable level by the addition of non-radioactive methionine to the bathing solution. As demonstrated, activity from the bathing solution does not result in significant contaminant activity by re-entering the nerve.
Acknowledgements This study was supported in part by a grant from the Medical Research Council of Canada.
References Bisby, M.A. (1982) Ligature techniques. In D.G. Weiss (Ed.), Axoplasmic Transport, Springer, Berlin, pp. 193-199. Bisby, M.A. (1983) Velocity of axoplasmic transport of labelled protein is not dependent on local concentration, Exp. Neurol., 79: 168-175. Brimijoin, S. (1975) Stop-flow: a new technique for measurement of axonal transport, and its application to the transport of dopamine fl-hydroxylase. J. Neurobiol., 6: 379-394. Brimijoin, S. (1979) On the kinetics and maximal cal~city of the system for rapid axonal transport in mammalian neurons, J. Physiol. (London), 292: 325-337. Carslaw, H.S. and Jaeger, J.C. (1959) Conduction of heat in solids, 2nd edn., Clarendon Press, Oxford. Chan, S.Y., Ochs, S. and Worth, R.M. (1980) The requirement for calcium ions and the effect of ions on axoplasmic transport in mammalian nerve, J. Physiol. (London), 301: 477-504. Chihara, E. (1979) Axoplasmic and nonaxoplasmic transport along the optic pathway of albino rabbits: a theoretical pattern of distribution, Invest. Ophthalmol. Visual Sci., 18: 339-345. Crank, J. (1959) The Mathematics of Diffusion, Clarendon Press. Oxford. Edstrom, A. and Hanson. M. (1973) Temperature effects on fast axonat transport of proteins in vitro in frog sciatic nerves, Brain Res., 58: 345-354. Goldberg, D.J.. Schwartz, J.H. and Sherbany, A.A. (1978) Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon, J. Physiol. (London), 281: 559-579. Gosting, L.J. (1956) Measurement and interpretation of diffusion coefficients in proteins, Adv. Prot. Chem.. 11: 429-554. Haak, R.A.. Kleinhans, F.W. and Ochs, S. (1976) The viscosity of mammalian nerve axoplasm measured by electron spin resonance, J. Physiol. (London), 263: 115-137. Haley, J.E.. Wisniewski, H.M. and Ledeen, R.W. (1979) Extra-axonal diffusion in the rabbit optic system: a caution in axonal transport studies, Brain Res., 179: 69-76. Hines, J.F. and Garwood, M.M. (1977) Relea~ of protein from axons during rapid axonal transport: an in vitro preparation. Brain Res, 125: 141-148. Litchy, W. and Brimijoin, S. (1983) Concentration dependence of rapid axonal transport: a study of transport kinetics of [35S]methionine-tabdled protein in postganglionic sympathetic fibres of the bullfrog, J. Neurosci., 3: 2075-2082. O'Brien, D.W. and Snyder, R.E. (1982) Position-sensitive detector studies of the axonal transport of a pulse of radioisotope, J. Neurobiol., 13: 435-445.
119 Schwab, M.E. and Thoenen, H. (1983) Retrograde axonal transport, A. Laitha (Ed.), In Handbook of Neurochemistry, Vol. 5, Plenum Press, New York, pp. 381-404. Snyder, R.E. (1984) The design and construction of a multiple proportional counter used to study axonal transport, J. Neurosci. Meth. 11: 79-88. Snyder, R.E. and O'Brien, D.W. (1983) Calcium efflux from amphibian sciatic nerve, Can. J. Physiol. Pharmacol., 61: 1085-1089. Snyder, R.E., Reynolds, R.A., Smith, R.S. and Kendal, W.S. (1976a) Application of a multiwire proportional chamber to the detection of axoplasmic transport, Can. J. Physiol. Pharmacol., 54: 238-244. Snyder, R.E., Reynolds, R.A., Smith, R.S. and Kendal, W.S. (1976b) Erratum: application of a multiwire proportional chamber to the detection of axoplasmic transport, Can. J. Physiol. Pharmacol., 54: 945. Snyder, R.E., Nichols, T.R. and Smith, R.S. (1980) Rapid orthograde transport of 32P-labelled material in amphibian sensory axons: a multiwire proportional chamber study, Can. J. Physiol. Pharmacol., 59: 513-524. Snyder, R.E., O'Brien, D.W. and Nihei, T. (1984) A temporal variation in non-neuronal protein synthesis in dorsal root ganglia and nerve and its significance to studies of axonal transport, Exp. Neurol., 83: 518-533. Takenaka, T., Horrie, H. and Sugita, T. (1978) New technique for measuring axonal transport and its application to temperature effects, J. Neurobiol. 9: 317-324. Tedeschi, B., Wilson, D.L., Zimmerman, A. and Perry, G.W. (1981) Are axonally transported proteins released from sciatic nerves?, Brain Res., 211: 175-178. Widen, L., Greitz, T., Michelayananakis, J. and Asard, P.E. (1976) External detection of axoplasmic flow using radionuclides, Neuroradiology, 10: 197-203.
109
Elsevier NSM 00595
A radiolabelled pulse for the simultaneous study of anterograde and retrograde axonal transport R.E.
Snyder
Department of Applied Sciences in Medicine, University of Alberta, Edmonton, Alta. (Canada)
(Received 29 April 1985) (Revised 24 April 1986) (Accepted 26 April 1986)
Key words: Axonal transport - Position-sensitive detector - Retrograde transport
A technique is described for producing a pulse of [35S]methionine-labelled material which is axonally transported in amphibian sciatic nerve maintained in vitro. Using a position-sensitive detector of ionizing radiation, it is possible to continuously observe the movement of the pulse in the anterograde direction and, following turnaround at a ligature, the movement of a fraction of the pulse in the retrograde direction. Two sources of contaminant activity, which would otherwise interfere with observation of the pulse, are discussed and shown to be avoidable.
Introduction C o n t i n u o u s monitoring of the axonal transport of a radiolabel in a living preparation m a y be accomplished by placing a nerve in opposition to a positionsensitive detector of ionizing radiation (Snyder et al., 1976a,b; Widen et al., 1976; T a k e n a k a et al., 1978). In an earlier report (O'Brien and Snyder, 1982), it was demonstrated possible to continuously m o n i t o r the anterograde m o v e m e n t of a pulse of radiolabelled material which had been created using a cold-blocking technique ( E d s t r o m and Hanson, 1973; Brimijoin, 1975). However, it was f o u n d that the retrograde transport of labelled material, turned a r o u n d at a distal ligature, was observed in only 15% of the preparations. Relatively little is k n o w n of the dynamics of t u r n a r o u n d and retrograde transport since most studies are performed using the double-ligature technique (Bisby, 1982; Schwab and Thoenen, 1983). Frequently, it is impossible to dissociate the two processes. This has led us to develop a new m e t h o d of forming a pulse, one which has consistently resulted in the retrograde m o v e m e n t of radiolabel turned a r o u n d at a distal ligature. The m e t h o d does not require the concentrating of material, as does cold blocking, thus avoiding
Correspondence: R.E. Snyder, Department of Applied Sciences in Medicine, University of Alberta,
Edmonton, Alta., Canada T6G 2G3, 0165-0270/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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the question of concentration-dependent effects (Goldberg et al.. 1978: Brimijoin, 1979; Bisby, 1983; Litchy and Brimijoin, 1983). Pulse formation consists of incubating a dorsal root ganglion-sciatic nerve preparation in a two-compartment tray, exposing the ganglion cells to a radiolabel. and later severing the nerve at two different times proximal to the moving activity. Special attention must be given to the suppression of contaminant activity by which we mean any activity ultimately monitored by the detector which is not directly associated with transport. We have found there to be two primary sources of such activity which, if not controlled, will result in interference with detection of the transported activity. The first is radiolabel which enters the nerve via a 'diffusion-like' process during the pulse-creation stage. The second is associated with transported label which is expelled from the axons (Hines and Garwood, 1977; Tedeschi et al., 1981).
Methods
Nerve preparation A sciatic nerve in continuity with its ninth dorsal root ganglion and spinal roots was removed from adult female specimens of the amphibian Xenopus laevis. A ligature was placed 65-70 mm distal to the gan~on. The perineural Sheath was not removed at this time. During the dissection and pulse,creation stage the preparation was bathed in an oxygenated saline solution of composition (mM): NaCI 112, KC1 3.0, MgSO4 1.6, CaC12 3.0, glucose 5.0, HEPES (N-2'hydroxyethylpiperaz~ne-N'-2ethanesulphonic acid) 3.0. The pH was adjusted to 7.38-7.42 with NaOH, Following pulse creation (see below) the nerve was normally bathed in a solution of the above composition which contained in addition non-radioactive metl',ionine (1.0 mmol/1). Additional details pertaining to the dissection may be found in the literature (O'Brien and Snyder, 1982). Pulse procedure Immediately following its dissection the preparation was placed in a two-compartment plastic tray which contained saline solution, the ganglion and its root in one compartment (0.025 ml) and the nerve in the second (20 ml). The solution was removed from the compartment containing the ganglion and replaced with 0.025 ml of saline solution to which had been added 0.15-0.25 mCi of L-[aSS]methionine at a specific activity of 1000-1400 Ci/mmol (Amersham or New Fag,land Nuclear). One hour was allowed for the [35S]methionine to become incorporated into protein following which the ganglion compartment was rinsed twice and refilled! with radiolabel-free saline solution. Following an additional 3.25 h duriag which labelled material was exported from the cell bodies, the nerve was severed immediately distal to the septum separating the compartments, approximately 4-5 mm distal to the ganglion, thus creating a pulse of radiolabeHed material at the proximal end of a 60-65 mm length of nerve. In a few experiments the time between refill and severing was extended to 3.50-3.75 h.
1ll
Immediately following its being severed the nerve was rinsed by being placed in 1 ml of saline solution and gently agitated for 5 min. Beginning with this rinse and onwards the bathing saline solution normally contained non-radioactive methionine as noted above. The nerve was next transferred to a dish containing fresh saline solution and, with the perineural sheath still in place, tensioned to a standard length using the technique described by Chan et al. (1980), the length being noted. The ligature near the distal end of the nerve was removed, the perineurium removed, and the ligature replaced. The nerve was then placed in 200 ml of saline solution and gently agitated until 130 min following its being severed, at which time the most proximal 10-mm section of nerve was removed and discarded. Approximately 10 min following removal of the 10-mm segment a thread was attached to the remaining nerve < 1 mm from its proximal end. The nerve was placed in a one-compartment tray with a 3.6-/tm mylar floor and containing saline solution. Using the proximal thread and a second thread used to form the distal ligature the nerve was stretched to its measured length and positioned in contact with the floor of the tray (Snyder et al., 1980). A piece of gauze stretched over an acrylic frame served to lightly press the nerve against the floor. The tray was placed over the entrance window of a multiple proportional counter (Snyder, 1984) and the nerve bathed in a circulating volume of approximately 175 ml of saline solution. Experiments were performed at a temperature of 22.7-23.3 ° C, maintained to within + 0 . 2 ° C for any given experiment.
Position-sensitive detector analysis The one-compartment tray containing the nerve was placed in contact with the entrance window of a multiple proportional counter (MPC). The MPC is composed of a series of radiation counters spaced at 3.18-mm intervals and serves to detect those B-particles (typically 0.5-1.5% of the total) which have their origin in the nerve and enter the counter. Counts vs position spectra were recorded for 20-22 h in 1000-s blocks. Prior to placing the nerve in the tray, 2 - 4 background spectra were recorded with the tray in position on the detector. The average of these spectra was used to correct all counts versus position spectra from an experiment for room background. At the conclusion of an experiment the nerve was removed from the tray and 2-3 additional spectra recorded for comparison to the final spectrum obtained with the nerve in position. Finally, the series of counts vs position spectra were arranged so that the counting rate for each 3.18-mm region of nerve could be plotted as a function of time.
Diffusion studies In a variation of the pulse procedure, the inhibitors of protein synthesis cycloheximide ( 1 2 5 / t g / m l ) and puromycin (500/~g/ml) (Snyder et al., 1984) were both added to the solution bathing the ganglion 0.5 h prior to introduction of the [3~S]methionine, remaining throughout the course of the incubation in order to retard the axonal transport of any radiolabel. During some experiments the radiolabel was not removed 1 h following its introduction.
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Efflux studies Four, 10-18 mm sections of unbranched sciatic nerve were removed from a frog and the perineural sheath removed from two sections. Each section was placed in 2 ml of saline solution which contained either non-radioactive methionine (100 /zg/ml) or puromycin (500/~g/ml), and 1 h later 2/~Ci of L-[methyl-3H]methionine (New England Nuclear) was added to the solution. Following 16-17 h, during which time radiolabel entered the nerve, the sections were immersed sequentially in a series of 1-ml volumes of saline solution at room temperature (22-24°C) during a 5 6 h period, followed by overnight immersion in a final volume. At the conclusion of the experiment the samples were treated as will be described below. For additional details, see Snyder and O'Brien (1983).
Liquid scintillation analysis All nerves were assayed by liquid scintillation counting at the completion of an experiment. Nerves monitored with the MPC were cut into segments which corresponded to the detector regions; sections used in the efflux study were counted whole; nerves used in the remaining diffusion studies were cut into 2 or 5 mm segments with a precision of +0.1 mm. All segments were treated with 5% trichloroacetic acid (TCA) solution for 1-2 h at 80°C and dissolved in Protosol (this and other scintillation reagents were obtained from New England Nuclear). Econofluor was added to the dissolved samples and Aquasol to the soluble fractions. Samples of various bathing solutions were counted by addition of Aquasol. All specimens were assayed by liquid scintillation analysis.
Results
Transport studies Sixteen preparations were studied using the normal pulse-creation procedure. The experiments differed only in the use of non,radioactive r n e ~ o m n e in the bathing solutions and in the time elapsed between the introduction of [3~S]methionine and severing of the nerve. For studies in which the elapsed time was 4,25 h, liquid scintillation counting revealed the nerve to contain a total activity of 0,022 5: 0,013% (5: S.D.) of that introduced into the g ~ o n compartment. The anterograde movement of a pulse of activity culminated in a build'up of activity at the distal ligature, followed by the retrograde movement of a pulse of activity (Figl 1). This second, or retrograde pulse, was present in all preparations studied. At the conclusion of each MPC experiment, spectra were obtained with the nerve removed from the one-compartment tray in order to evaluate the extent of contarnination recorded by the MPC due to activity not within the nerve. The total counting rate observed following removal of ~ nerve was d i ~ e d : by that obtained from the last spectrum acquh,~xl with the nerve in ptace and fountl to be 7,2± 3,7% when non-radioactive methionine was absent from the bathing s ~ f i o n , but only 1.2 5: 0.5% with methionine present. Nerves incubated with ii~ibitors of protein
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Fig. 1. Counts vs time plots. Each plot corresponds to a 3.18-mm segment of nerve (e) whose mean distance from the proximal end of the nerve is indicated on each plot in mm. Sixteen plots were obtained; only 8 are shown. A pulse can be seen to move in the anterograde direction, resulting in accumulation of radiolabel at the distal ligature between 6-8 h, following which a pulse of smaller amplitude moves in the retrograde direction (arrows). The retrograde pulse is generally discernible in 8-12 plots. Open circles (O) are either background data obtained prior to placing the nerve in the tray or contamination data obtained following removal of the nerve from the tray.
synthesis present in the ganglion compartment showed no observable contamination following removal from the tray. In order to better understand the nature of this contaminant activity, an experiment was performed in which one nerve was monitored by the MPC while the contralateral nerve, which had been treated with inhibitors of protein synthesis during the pulse-creation procedure, was maintained in a separate tray. Samples of the two bathing solutions were drawn at regular intervals; the results are shown in Fig. 2. As is evident, relatively little activity entered the bathing solution from the
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Fig. 2. Total activity in the bathing solution expressed as parts-per-million (ppm) of the quantity of [35S]methionine used during the pulse-creation procedure: closed circles (O), nerve containing normal radiolabelled pulse; open circles (©), similar nerve in which transport of radiolabdted material was suppressed by the presence of inhibitors of protein synthesis in the ganglion compartment during the pulse-creation procedure. The nerves were placed in their bathing solutions at time zero.
treated nerve compared to that from the nerve containing the pulse of activity. This finding was substantiated by assaying the activity in the MPC bathing solution at the beginning and at the end of other experiments. The m o u n t of activity present in the bathing solution at the conclusion of a pulse experiment was found to be 4.0 + 1.0% of that in the nerve when non-radioactive methionine was absent from the bathing solution, but 29 + 12% when methionine was present. In an attempt to determine the physical location within the tray of the contaminant activity, spectra were obtained following removal of the nerve in which strips of 25-#m thick copper foil, sufficient to reduce the 35S beta-spectrum by < 98%, were imposed between the MPC window and the tray floor. Such studies i n d i c t e d that < 20% of the recorded contaminant activity is due to activity in the bathing solution, with the remainder due to activity which adheres to the mylar floor of the tray adjacent to where the nerve was situated. These studies lead us to conclude that the most likely source of this contaminant activity recorded by the MPC is axonal transport. The major of the recorded activity does not r e s e t from activity within the bathing solution, but rather from that adhering to the mylar floor. Finally, an experiment was performed to determine the extent to which activity might gain access to a nerve after having entered the bathing solution. Such contamination would not appear in recordings obtained with the neree removed. A desheathed nerve, which h a d not been e x p o s ~ to ra~aotai~l, was position¢~l in the one-compartment tray in tl~ normal maaner. A second nerve, whicheontained a radiolabelled pulse, was placed in the bathing solution a b o v e the first such that
115 activity f r o m the second could not enter the MPC. N o increase in counting rate could be observed outside of the statistical limits due to the r o o m background.
Diffusion studies During the period when the ganglion is exposed to the radiolabel, activity can enter the nerve via b o t h transport and diffusion (Chihara, 1979; Haley et al., 1979). A series of experiments was thus undertaken to estimate the magnitude of diffusion-related contamination. Experiments were performed using nerves incubated as in normal pulse studies, but with inhibitors of protein synthesis present in the c o m p a r t m e n t containing the ganglion to inhibit formation of a radiolabelled pulse. The nerves were severed at 4.25 h following the introduction of [35S]methionine and either (1) rinsed for 0.5-1.0 min, frozen, and divided into segments, or (2) following the n o r m a l 5-min rinse, desheath, 130-min rinse pattern, divided into segments for liquid scintillation counting. In all cases the TCA-insoluble activity in a nerve was < 3% of its total activity at the time of division. For nerves divided immediately following removal
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Fig. 3. TCA-soluble activity in nerves in which inhibitors of protein synthesis were present in the ganglion compartment during the pulse-creation procedure: triangles (&), [35S]methionine remained in the ganglion compartment until severing, nerve assayed immediately following severing; closed circles (e), [35S]methionine remained in the ganglion compartment until severing, nerve assayed following 130-min rinse procedure; open circles (O), [35S]methionine removed 1 h following its introduction, nerve assayed following 130-min rinse procedure. Cross-hatched areas depict pulse of axonally-transported radiolabelled-material immediately following severing (0-6 ram) and following 130-min rinse procedure (10-22.5 mm). Shapes and amplitudes of the pulses were estimated by extrapolation of the data recorded from 16 normal pulse experiments, such as that shown in Fig. 1.
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from the two-compartment tray the activity in the 0.5-1.0 min rinse plus that lost during the process of division was < 15% of that remaining in the nerve. Representative studies are shown in Fig. 3. Plots for similarly treated nerves were identical in shape, but displaced by + 1 mm along the abscissa. We attribute these displacements to an uncertainty in the position of severing of the nerves. By assaying the various rinse solutions it was determined that 30-50% of the activity present in a nerve immediately following severing remained in the nerve at the conclusion of the 130-min rinse. Based upon MPC recordings (Fig. 1), it is possible to estimate by extrapolation the size and shape of the anterograde pulse at times prior to those at which data were recorded. Two such 'extrapolated' pulses are shown in Fig. 3, one at the time of the 4.25-h severing and the second at the time of removal of the 10-mm segment. As is evident, at the time of the 4.25-h severing, diffusion-related activity within the nerve is approximately one order of magnitude greater than activity in the moving pulse. By allowing the pulse to propagate for 130 min, and removing and discarding the, proximal 10-mm section, it is possible to obtain a length of nerve in which the quantity of activity associated with transport is at least an order of magnitude greater than that related to diffusion. In order to determine the quantity of diffusion-related activity recorded by the MPC during a typical experiment, two preparations incubated with inhibitors of protein synthesis, and otherwise prepared as in the normal pulse procedure, were studied using the MPC. Both yielded initial MPC counting rates of 100-150 counts per hour in the most proximal 3 - 4 segments, decreasing to zero in approximately 10 h. Comparison to MPC studies of axonally transported pulses of activity (Fig. 1) showed that activity which enters the nerve due to means other than transport accounts for < 2-5% of the counting rate observed in the presence of transport, and only in the most proximal 3-4 segments of nerve during the initial 10 h.
Efflux studies In 4 efflux studies it was found in each case that < 5% of the total activity initially present was in the TCA-insoluble form, suggesting that the experiments served to measure the diffusion of [3H]methionine with minimal interference due to incorporation. N o significant differences were observed between the use of non-radioactive methionine or puromycin to retard incorporation. Data from each section of nerve was plotted as the rate of loss of activity versus time (data not shown) and fitted to a mathematical expression of the form 3
E A i e -Bit, i=l
where A i and B i are coedfu~ats and t is the time. For details, the reader is referred to the literature (Snyder and O'Brien, 1983). To a first approximation, each term in the above expression corresponds to a single efflux compartment with a corresponding half, life of In 2/Bi. Analysis of the two de,sheathed sections of nerve yielded half-lifes of 3.5 + 0.8 ( + S.D,), 36 4- 12, and 248 + 66 rain with correrpondiag fraetions of 0.18 + 0.11, 0.22 4- 0 . t l , and 0.60 + 0.23 r~l~etively. Similar analysis of the two sha~taed sections of nerve
117 yielded half-lifes of 3.8 + 1.7, 53 + 6, and 432 + 12 min with corresponding fractions of 0.09 + 0.04, 0.09 + 0.01, and 0.82 + 0.04 respectively.
Discussion
Using the procedure described, it is possible to produce a pulse of radiolabelled material which yields data rates sufficient for quantitative studies without the need for either cold blocking or concentrating material. The retrograde movement of radiolabel turned around at a ligature is a regular occurrence, facilitating the study of both turnaround and retrograde transport. However, attention must be given to the reduction of contaminant activity. Two significant sources of contamination exist, one present during the pulsecreation procedure and the other related to transport itself. The activity profiles of nerves for which axonal transport of labelled material was inhibited (Fig. 3) are qualitatively similar to those associated with diffusion: monotonically decreasing curves which may be approximated by a Gaussian distribution. From the results of the efflux study we estimate the diffusion for methionine in the interstitial space of the nerve to be 1.7 x 10 -6 cm2//s and the permeability of the perineural sheath to be 7.4 x 10 -7 c m / s (see Snyder and O'Brien, 1983 for details). However, using the model of a long cylinder of fluid bounded by a thin permeability barrier (Carslaw and Jaeger, 1959; Crank, 1959), it is possible to show that the above values will not result in sufficient diffusion to account for the observed profiles, although increasing the value of the diffusion coefficient by a factor of 2-10 does produce profiles which agree qualitatively with the observed results. Diffusion coefficients for amino acids in water at 25°C are typically 8-10 × 10 -6 cm2/s (Gosting, 1956). However, the present situation is more complex than this, both in structure and in factors such as viscosity (Haak et al., 1976). Thus, while having the general appearance of diffusion, it is not possible to account for this type of contamination solely based upon a simple model of diffusion. As is evident from Fig. 3, the pulse of transported radiolabel is engulfed at the time of severing by non-transport related activity. Since 30-50% of this activity remains in the nerve 130 min later, either it does not exist as free methionine, although TCA soluble, or more likely, does not reside in the interstitial space from which free methionine is cleared with a half-life of 3 - 4 rain. Thus, even removal of the perineurium prior to introduction of the radiolabel will not circumvent this type of contaminant activity, and we must allow the pulse to propagate beyond the front of non-transported label. We have found a distance of 10 mm to be sufficient with a propagation time of 130 min. This corresponds to a transport rate of 110 m m / d a y , compared to a slowest rate for fast transport of 119 + 10 m m / d a y measured for cold-blocked nerves (O'Brien and Snyder, 1982). The second source of contaminant activity stems from transport itself. As shown in Fig. 2, radiolabel associated with transport enters the bathing solution over a period of 20 h, this activity being approximately an order of magnitude greater than that resulting from non-transported activity. While we do not wish at this time to
118
comment upon the nature of this phenomenon, it should be noted that two earlier reports investigated a closely related point, the release of transported proteins from nerve, arriving at opposing conclusions (Hines and Garwood, 1977: Tedeschi et al., 1981). Activity within the bathing solution itself contributes relatively little to the contamination; the majority of the contaminant activity appears to be associated with the mylar floor in proximity to the nerve. This may be reduced to an acceptable level by the addition of non-radioactive methionine to the bathing solution. As demonstrated, activity from the bathing solution does not result in significant contaminant activity by re-entering the nerve.
Acknowledgements This study was supported in part by a grant from the Medical Research Council of Canada.
References Bisby, M.A. (1982) Ligature techniques. In D.G. Weiss (Ed.), Axoplasmic Transport, Springer, Berlin, pp. 193-199. Bisby, M.A. (1983) Velocity of axoplasmic transport of labelled protein is not dependent on local concentration, Exp. Neurol., 79: 168-175. Brimijoin, S. (1975) Stop-flow: a new technique for measurement of axonal transport, and its application to the transport of dopamine fl-hydroxylase. J. Neurobiol., 6: 379-394. Brimijoin, S. (1979) On the kinetics and maximal cal~city of the system for rapid axonal transport in mammalian neurons, J. Physiol. (London), 292: 325-337. Carslaw, H.S. and Jaeger, J.C. (1959) Conduction of heat in solids, 2nd edn., Clarendon Press, Oxford. Chan, S.Y., Ochs, S. and Worth, R.M. (1980) The requirement for calcium ions and the effect of ions on axoplasmic transport in mammalian nerve, J. Physiol. (London), 301: 477-504. Chihara, E. (1979) Axoplasmic and nonaxoplasmic transport along the optic pathway of albino rabbits: a theoretical pattern of distribution, Invest. Ophthalmol. Visual Sci., 18: 339-345. Crank, J. (1959) The Mathematics of Diffusion, Clarendon Press. Oxford. Edstrom, A. and Hanson. M. (1973) Temperature effects on fast axonat transport of proteins in vitro in frog sciatic nerves, Brain Res., 58: 345-354. Goldberg, D.J.. Schwartz, J.H. and Sherbany, A.A. (1978) Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon, J. Physiol. (London), 281: 559-579. Gosting, L.J. (1956) Measurement and interpretation of diffusion coefficients in proteins, Adv. Prot. Chem.. 11: 429-554. Haak, R.A.. Kleinhans, F.W. and Ochs, S. (1976) The viscosity of mammalian nerve axoplasm measured by electron spin resonance, J. Physiol. (London), 263: 115-137. Haley, J.E.. Wisniewski, H.M. and Ledeen, R.W. (1979) Extra-axonal diffusion in the rabbit optic system: a caution in axonal transport studies, Brain Res., 179: 69-76. Hines, J.F. and Garwood, M.M. (1977) Relea~ of protein from axons during rapid axonal transport: an in vitro preparation. Brain Res, 125: 141-148. Litchy, W. and Brimijoin, S. (1983) Concentration dependence of rapid axonal transport: a study of transport kinetics of [35S]methionine-tabdled protein in postganglionic sympathetic fibres of the bullfrog, J. Neurosci., 3: 2075-2082. O'Brien, D.W. and Snyder, R.E. (1982) Position-sensitive detector studies of the axonal transport of a pulse of radioisotope, J. Neurobiol., 13: 435-445.
119 Schwab, M.E. and Thoenen, H. (1983) Retrograde axonal transport, A. Laitha (Ed.), In Handbook of Neurochemistry, Vol. 5, Plenum Press, New York, pp. 381-404. Snyder, R.E. (1984) The design and construction of a multiple proportional counter used to study axonal transport, J. Neurosci. Meth. 11: 79-88. Snyder, R.E. and O'Brien, D.W. (1983) Calcium efflux from amphibian sciatic nerve, Can. J. Physiol. Pharmacol., 61: 1085-1089. Snyder, R.E., Reynolds, R.A., Smith, R.S. and Kendal, W.S. (1976a) Application of a multiwire proportional chamber to the detection of axoplasmic transport, Can. J. Physiol. Pharmacol., 54: 238-244. Snyder, R.E., Reynolds, R.A., Smith, R.S. and Kendal, W.S. (1976b) Erratum: application of a multiwire proportional chamber to the detection of axoplasmic transport, Can. J. Physiol. Pharmacol., 54: 945. Snyder, R.E., Nichols, T.R. and Smith, R.S. (1980) Rapid orthograde transport of 32P-labelled material in amphibian sensory axons: a multiwire proportional chamber study, Can. J. Physiol. Pharmacol., 59: 513-524. Snyder, R.E., O'Brien, D.W. and Nihei, T. (1984) A temporal variation in non-neuronal protein synthesis in dorsal root ganglia and nerve and its significance to studies of axonal transport, Exp. Neurol., 83: 518-533. Takenaka, T., Horrie, H. and Sugita, T. (1978) New technique for measuring axonal transport and its application to temperature effects, J. Neurobiol. 9: 317-324. Tedeschi, B., Wilson, D.L., Zimmerman, A. and Perry, G.W. (1981) Are axonally transported proteins released from sciatic nerves?, Brain Res., 211: 175-178. Widen, L., Greitz, T., Michelayananakis, J. and Asard, P.E. (1976) External detection of axoplasmic flow using radionuclides, Neuroradiology, 10: 197-203.