JOURNAL O F NEUROBIOLOGY, VOL. 7, NO. 4, PP. 339-354

Temperature-Dependenceof Rapid Axonal Transport in Sympathetic Nerves of the Rabbit BARRY COSENS, DAVID THACKER, a n d STEPHEN BRIMIJOIN Department of Pharmacology, Mayo Medical School, Rochester, Minnesota 55901

SUMMARY

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-P-hydroxylase (DBH) activity in rabbit sciatic nerves in uitro. These nerves were cooled locally to 2°C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration a t various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13OC and 42OC. Over this range of temperatures, the results were well described by the equation: V = O.E146(1.09)~, where V is velocity in m m h r , and T is temperature in degrees centigrade. The Qlo between 13' and 42OC was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47OC, although only about half of the DBH activity disappeared during incubation a t this temperature. Another transition occurred at 13OC; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13OC. INTRODUCTION

Rapid axonal transport delivers organelles, proteins, and other key materials to distant parts of nerve cells a t rates up to several hundred millimeters per day (for recent reviews, see Ochs, 1974; Heslop, 1975). One reason for exploring the effect of temperature on this process might be to determine if transport could reflect a purely physical phenomenon like diffusion, which is relatively insensitive to temperature (Christensen, 1974). However, unlike diffusion, rapid axonal transport of proteins requires metabolic energy (Ochs, 1972; Garcia, Kirpekar, Prat, and Wakade, 1974). And diffusion can not generate the sharp fronts and distinct crests typically seen in waves of labeled protein moving down nerve axons whose cell bodies have been exposed to labeled amino acids (Copeland and Reiner, 1974). Thus, studies of temperature are hardly needed to rule out diffusion as a mechanism for rapid axonal transport. On the other hand, such 339 0 1976 by John Wiley & Sons, Inc.

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COSENS, THACKER, AND BRIMIJOIN

studies might yield further clues to the mechanism of transport. For example, Ochs and Smith (1975) have found similarities in the effects of temperature on transport and muscular contraction; these similarities could mean that axonal transport involves contractile proteins like actomyosin. The dependence of rapid axonal transport on temperature has been studied a few times (Grafstein, Forman, and McEwen, 1972; Heslop and Howes, 1972; Edstrom and Hanson, 1973; Gross, 1973; Ochs and Smith, 1975). However, only Ochs and Smith (1975) have examined this dependence in a mammal. No one has observed the effects of temperature on transport of an identified protein. It is uncertain if temperature affects the proportion of material that is transported in addition to the speed of transport. Reports conflict as to whether transport velocity is a linear or an exponential function of temperature and whether transport stops below some threshold temperature. Finally, the behavior of transport at high temperatures is unknown. Because of these limitations in our understanding of such a fundamental phenomenon, we decided to investigate thoroughly how temperature affected the transport of a specific protein in a mammalian nerve. Stop-flow techniques were recently developed in this laboratory to analyze rapid axonal transport of specific endogenous substances (Brimijoin, 1975). These techniques use local cooling to block transport reversibly: during blockade, transported substances accumulate at the edges of a cooled region; but when nerves are rewarmed, waves of accumulated substances resume migration up or down. Transport velocities can thus be measured directly, without artifacts arising from ligation and without introduction of radioactive labels. With stop-flow techniques, we have characterized rapid orthograde and retrograde transport of dopamine-P-hydroxylase (DBH) in rabbit sciatic nerves incubated in uitro at 37°C (Brimijoin, 1975;Brimijoin and Helland, 1976). DBH is a useful marker for transport since, in the peripheral nervous system, this enzyme is confined to postganglionic sympathetic neurons (Hartman, 1973), in which it is bound to storage vesicles for norepinephrine (Hortnagl, Hortnagl, and Winkler, 1969). Therefore, one can use DBH to study transport of a particular organelle in a largely homogeneous population of nerve axons. In the present experiments, we have tried to determine how temperature affects the velocity and proportion of DBH transported by rabbit sciatic nerves in uitro. Stop-flow techniques were used to measure the velocity of migrating waves of enzyme activity. Average transport velocity was computed from separate measurements of the rate at which DBH activity accumulated at the edge of a region cold enough to block transport. By comparing average velocity with that of the migrating waves at a given temperature, we estimated what proportion of the enzyme was moving. METHODS Preparation of nerues Adult New Zealand white rabbits (3 to 5 kg) were killed by sodium pentobarbital (300 mg) injected into an ear vein. Previous results indicated that this method of killing did not affect transport of

TEMPERATURE AND RAPID TRANSPORT

341

DBH (Brimijoin, 1975). We took 5 to 6 cm lengths of sciatic nerve from the thighs, trimmed away stray fascicles and excess connective tissue, tied the isolated nerves a t both ends, put a thin reference mark of powdered graphite about midway between the ends, and then transferred the nerves to incubation chambers. Apparatus The incubation chambers for these experiments have previously been described in detail (Brimijoin, 1975; Brimijoin and Helland, 1976). Nerves were mounted in plexiglass tubes (6 mm i d . ) with ports for inflow and slits for outflow of physiologic salt solution. Steep temperature gradients, arising from the pattern of flow of separate currents of solution, were confined to the vicinity of the outflows. By means of these gradients we selectively cooled or warmed specific regions of nerve. This technique of temperature compartmentation avoided mechanical compression and anoxia, along with many associated artifacts. One of our chambers was divided into two sections by a single outflow slit; this chamber was used to incubate proximal and distal regions of nerve a t distinctly different temperatures. The other chamber was similar but was divided into three sections; the center section, defined by two outflow slits only 3 mm apart, served to cool a short region while the outer sections kept the rest of the nerve a t 37°C. Thermistor probes implanted near the outflow slits of each chamber allowed continuous monitoring of temperature. Incubation Nerves were incubated in bicarbonate-buffered physiologic salt solution of the following composition: Na+, 122 meq/liter; k+,5 meqhiter; Ca2+,3 meqhiter; Mg2+,2 meqhiter; C1-, 102 meqhiter; 2 meqhter; and glucose, 5.6 mM. This solution HP0d2-, 4 meqhiter; HCOf, 24 meq/liter; was continuously aerated in a reservoir with 95% 0 2 and 5% C02. Flowing by gravity from the reservoir, the solution was divided by Y-junctions in latex tubing. Solution then passed through glass-coil heat exchangers immersed in water baths of desired temperature and entered the incubation chambers a t appropriate points. A peristaltic pump returned the outflow to the reservoir. Assay for DBH activity After incubation, nerves were straightened along the base of a cutting apparatus consisting of a hinged array of razor blades. This apparatus chopped the nerves into consecutive 3 mm segments, which were homogenized in all-glass homogenizers containing 1.5 ml of ice-cold buffer (0.005 M Tris HC1, pH 7.4; 0.1% (v/v) Triton X-100; 0.2% (w/v) bovine serum albumin). Homogenates were centrifuged a t 10,000 X g for 10 min a t 4°C. Duplicate 200 fi1portions of the supernatant fractions were assayed for DBH activity by the method of Molinoff, Weinshilhoum, and Axelrod (1971), with tyM ) to overcome ramine as the substrate. We added Cu2+SOa2- (final concentration, 1.3 X endogenous inhibitors of DBH. Essentially complete activity was recovered from partially purified bovine adrenal DBH added to duplicate tissue samples, and no local variations in inhibition were detected along the length of the nerves. Analysis DBH activity was calculated as pmoles of octopamine formed per hour of incubation, per mm of nerve assayed. For each nerve we determined the overall average activity first and then expressed the activity in each 3 mm segment as a percentage of this average. The resulting “relative DBH activities” were used to compare data from different nerves. Previous experiments with these nerves (Brimijoin, 1975) have shown that the initial distribution of DBH activity is flat; i.e., when nerves are cut up just after removal from the rabbit, each 3 mm segment has a relative activity of 100. It also appears that the total DBH activity of rabbit nerves does not change during several hours of incubation in uitro (Brimijoin, 1975). Therefore, changes in relative activity probably reflect redistribution of DBH: values above 100 in a given segment reflect net accumulation in that segment; values below 100, net loss.

342

COSENS, THACKER, AND BRIMIJOIN

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RESULTS

Stop-flow Experiments Each experiment had two stages. In stage one, nerves were incubated in the three-section chambers for 1.5 hr. Temperature was 37°C everywhere except in a 3 mm region just distal to the graphite reference mark; this region was cooled to 2"C, making it a local "cold-block." Stage one was the "stop" phase of the experiment, during which DBH activity accumulated a t the cold-block. At the end of stage one, we transferred nerves t o beakers containing an aerated saline solution identical to that used in the incubation chambers. A second stage incubation was then carried out a t some constant temperature. In this "flow" phase of the experiment, the effects of temperature on migration of DBH were tested. The distribution of DBH activity along nerves at the end of stage one revealed substantial accumulation in the marked segment, just proximal to the cooled region (Fig. 1). On each side of this sharply localized peak, up to 50% of the DBH activity disappeared from zones of clearance. In the segment just above the distal

TEMPERATURE AND RAPID TRANSPORT

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mm FROM MARK Fig. 2. Transport of DBH activity a t selected temperatures. Second stage incubations lasted 1 hr a t the indicated temperatures. Distances from the graphite mark, in mm, are shown on the horizontal axis (proximal to the left, distal to the right). Symbols (- 0 -) indicate mean positions of peak activities 1SD.

*

ligature (not shown) DBH activity always increased greatly, to levels averaging 213 f 24% above those initially present ( n = 6, p < 0.001). Redistribution of DBH activity during second stage incubations for 1 and 2 hr at 37°C clearly indicated a wave of activity moving steadily down the nerve (Fig. 1). The position of peak activity was measured in each rewarmed nerve in order to calculate a transport velocity for DBH in that nerve. The mean positions of these peaks after l and 2 hr of rewarming are shown in Figure 1, as are the standard errors of these means. Actual peak activity in each of the rewarmed nerves was more than two standard deviations greater than the mean activity in corresponding segments from control nerves that were not rewarmed. Spatial distribution of DBH activity after l - h r second stage incubations depended on the incubation temperature (Fig. 2). During 1hr of incubation at 19OC, the peak of DBH activity shifted 3 mm distally from its position a t the end of stage one. This compared with a shift of about 12 mm a t 37OC. As a t the higher temperature, peak activities in individual nerves rewarmed to 19°C were all more than two standard deviations above the mean in corresponding segments from control nerves that were not rewarmed. A t 42OC, DBH appeared to move much faster than a t lower temperatures. One hour after rewarming locally cooled

344

COSENS, THACKER, AND BRIMIJOIN

nerves to 42OC, the peak of DBH activity was about 2 1 mm distal to its position a t the end of stage one. Again, individual peak activities were more than two standard deviations above the corresponding mean from control nerves. In striking contrast, 1hr of heating to 47OC scarcely altered the distribution of DBH activity from that a t the end of stage one. A peak appeared to move 3 mm distally in two of the four nerves; however, throughout the length of each nerve heated to 47OC, DBH activity remained within one standard deviation of mean activity in corresponding segments from control nerves. Transport at this high temperature was not statistically significant. In total, 60 stop-flow experiments were performed on the temperature-dependence of transport (summarized in Table 1). Second stage incubations lasted longer, in general, as temperature was lowered; thus we increased precision to measure progressively slower transport. Well-defined peaks migrated a t temperatures from 42' to 13°C. By means of second stage incubations up to 12 hr long, we detected some transport even at 10°C. At this temperature, the position of peak DBH activity moved in only 2 of 8 nerves; however, DBH activity did increase in some distal segments as compared to mean levels a t the end of stage one. By far the largest increase occurred 6 mm distal to the position of peak activity at the end of stage one (A = 51 f 15, p < 0.01). This corresponds to transport at about 0.5 mmkr. Data from individual nerves were compared with the same baseline to determine the mean position of the largest increase. The results indicated distal migration of 4.92 f 1.2 mm during incubation a t 10°C ( p < 0.005). The best estimate of transport velocity at this temperature is 0.41 mm/hr (Table 1). Two different durations were used for second stage incubations at 37OC and three for incubations at 19OC (Table 1); these represent attempts to detect delays in the resumption of transport after blockade by local cooling. A t a given temperature, however, we calculated similar velocities, which certainly did not tend to increase with the duration of incubation. TABLE 1 Summary of Stop-Flow Experiments Temperature ("C) 47 42

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Time,a (hr)

Number in Group

DBH Activityb

1 1 1 2 2 1 2 5 10 11-12 3

4 4 10 9 4 5 4 3 4 8 5

228 411 4 48 606 330 362 485 602 703 575 493

Velocity i S.E.M. mm/hr 1.5 -t 21.8 + 12.3 i 12.8 ? 6.0 f 3.6 -f 2.1 i3.2 k 1.7 i 0.4 * 0.3 f

0.9 N.S.C 3.0 0.7 0.6 0.9 0.6 0.4 0.6 0.2 0.1 0.2 N.S.

Duration of the second stage incubation. Given in units of picomoles of octopamine produced per hr of incubation per mm of nerve. c Not statistically significant. a

b

TEMPERATURE AND RAPID TRANSPORT

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TEMPERATURE ('C)

Fig. 3. Semilogarithmic plot of transport velocity versus incubation temperature. Each point represents the mean velocity obtained by averaging results of all experiments a t a given temperature (Table 1). Vertical bars indicate the standard errors of these means. The solid regression line was fitted by the method of least squares to log transport velocity between 13" and 42"C, inclusive.

A semilogarithmic plot of our results shows that transport velocity was an exponential function of temperature between 13' and 42°C (Fig. 3). Over this range, a regression line fitted to log velocity by the method of least squares has a slope of 0.03675 f 0.00183 log units per degree. The equation that defines the relation between velocity and temperatures is V = O.E146(1.09)~, where V has units of mm/hr and T is in degrees centigrade. The Qlo indicated by this result is 2.33, with 95% confidence limits of 2.14 to 2.54. Dramatic breakdown of transport between temperatures of 42' and 47'C is shown by Figure 3. Although a smaller effect, it also appears from this figure that transport velocity fell sharply between 13' and 10°C. The regression line recalculated to include data obtained a t l0'C has a slope of 0.04717 log units per degree. However, an analysis of variance (Table 2) shows that the mean square deviation about this new regression line is significantly greater than the mean square deviation within groups ( p < 0.005). This indicates that a straight line does not adequately fit these data (Dixon and Massey, 1969) and lends statistical support to the conclusion that velocity falls especially rapidly a t the lower end of the temperature range. By contrast, a similar analysis supports a linear relation between log velocity and temperature over the range from 13" to 42°C (Table 2). At least one second stage incubation at each temperature was long enough for accumulated DBH to move about 15 mm, a t the velocity predicted by the equation above. Figure 4 shows how much DBH activity remained in the marked

346

COSENS, THACKER, AND BRIMIJOIN

segment after such incubations (lower panel); for comparison, measured transport velocity is shown as a percentage of that predicted by the equation (upper panel). During second stage incubations at temperatures from 19" to 42"C, DBH activity in the marked segment fell by more than 50% from the mean level a t the end of stage one. At all of these temperatures, measured and predicted transport velocities matched closely. During incubations at 47" or at 1O"C, on the other hand, transport velocity was much lower than predicted and activity in the marked segment hardly changed. Incubation at 13°C led to intermediate results: transport was as fast as predicted, but DBH activity fell only modestly in the marked segment. Average transport ue 1ocit y

Lower than expected transport velocity in a stop-flow experiment could equally well reflect primary failure of transport or difficulty with resuming transport after blockade by local cooling. Therefore, we measured velocity of transport by a procedure that did not require recovery from low temperatures. Nerves were removed from the rabbit, placed in beakers of isotonic NaCl a t room temperature, trimmed within 10 min, and given a preliminary incubation for 1/2 hr in physiological saline solution at the test temperature. They were then put into two-section incubation chambers so that the proximal 3 cm were maintained a t the test temperature while the distal parts were cooled to about 1°C. Incubations lasted 4.5 hr at temperatures of 19°C and below. When the test temperature was 19OC, a sizeable peak of DBH activity accuTABLE 2 Test for Linearity of Regression A. Transport velocities from 10" to 42°C. Slope of regression line: 0.04717 log units per degree. ~

Analysis of Variance Degrees of Sum of Squares Freedom

Source Within groups Regression About regression Total

1.15 14.88 1.2 17.23 F = 0.300/0.026 = 11.54,

Mean Square

45 1 4 50 p < 0.005

0.026 0.300

B. Transport velocities only from 13°C to 42°C. Slope of regression line: 0.03675 log units per degree.

Source Within groups Regression About regression Total

Analysis of Variance Degrees of Sum of Squares Freedom 0.53 5.55 0.02 6.10 F = 0.007/0.014 = 0.5,

38 1 3 42 p > 0.25

Mean Square 0.014 0.007

347

TEMPERATURE AND RAPID TRANSPORT 1 T

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Fig. 4. Comparison of transport velocity with the amount of stationary DBH activity. Horizontal axis in each panel is temperature in degrees centigrade. Upper panel: measured velocity of transport in stop-flow experiments as a percentage of the velocity predicted from the regression line (Fig. 3). Lower panel: DBH activity remaining in the marked segment (just proximal to the cooled region) after second stage incubations a t the indicated temperatures. Durations of incubation are indicated by numbers next to each data point. Accompanying these numbers are the distances which the peak of DBH activity would have moved if it had traveled a t the expected velocity.

mulated in the segment that crossed the boundary between sections (Fig. 5). Previous measurements have shown that about 80% of the total gradation in temperature occurs across this segment (Brimijoin and Helland, 1976). As the test temperature was reduced, so was the size of the peak at the boundary. Even after incubation at 13"C, however, this peak was highly significant when compared with activity in corresponding segments from nerves incubated for 4.5 hr a t a uniform temperature of 1°C (A = 64 f 8.2, p < 0.001). At 11°C and below, DBH activity did not accumulate a t the boundary to a significant degree. One can calculate a velocity of transport from these results as follows: V = (A.L)/(100 T ) ,where V is in m m k r , A is accumulation of relative DBH activity, L is length of segments assayed (in mm), and T is incubation time in hours. Because some DBH may have moved slightly past the boundary, we defined

348

COSENS, THACKER, AND BRIMIJOIN DBH ACTIVITY

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Fig. 5. Accumulation of DBH activity as a function of temperature. The proximal parts of nerves were incubated a t test temperatures indicated on the left-hand margin. Distal parts were incubated a t 1 O C . The horizontal axis indicates the distance from the midpoint of each nerve segment to the midpoint of the boundary between regions of different temperature. Relative DBH activity is drawn to a scale indicated by the vertical bar a t the top. Arrows indicate the overall mean activities for each group of nerves. Each group contained four nerves, except for the one tested a t l l ° C , which contained eight nerves. The data points are means of activity in each group and the vertical bars represent standard errors of these means.

accumulation as the excess activity (above a presumed initial level of 100) in both the segment a t the boundary and the next segment distal to it. Note that this calculation of velocity makes no allowance for the likelihood that DBH is divided into rapidly moving and slow moving or stationary compartments in sympathetic nerve axons (Brimijoin, 1974). Such compartmentation means that V, in the equation above, is an average velocity that is probably a mean of high and low rates. Average velocities calculated from the 4.5 hr incubations between 9" and 19"C, and from a series of experiments a t 35°C (four nerves each, incubated for 0.5, 1, 1.5, and 2 hr), are plotted on a semilogarithmic scale against temperature in Figure 6. For comparison, the profile obtained from the stop-flow experiments

TEMPERATURE AND RAPID TRANSPORT

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Fig. 6. Semilogarithmic plot of average velocity versus temperature. Average velocity was computed from measurements of basal activity and from the rate of accumulation of activity against a cold-block (see text). Means 5 standard errors of the means (vertical bars) of average velocity are shown. The solid regression line was fitted to the points between 13' and 35OC, inclusive, by the method of least squares. The dotted line indicates velocity of moving peaks (redrawn from Fig. 3).

is redrawn on the same figure. The solid line was fitted by the method of least squares to log average velocity over the range from 13" to 35°C. The slope of this regression line, 0.03652 f 0.00402 log units per degree, is essentially identical to the slope obtained from stop-flow experiments over the same range of temperatures. Below 13"C, average velocity fell steeply to levels not significantly different from zero; this transition closely matched the behavior of migrating peaks of DBH activity a t these temperatures. On the other hand, average velocity never equalled the velocity of:migrating peaks; over the entire tested range, in fact, average velocity was a steady 35% of peak velocity. This probably means that only about 35% of the axonal DBH was undergoing rapid transport down these axons. DISCUSSION

It has been suggested that axonal transport involves the binding of migrating materials to contractile proteins, either directly (Schmitt, 1968) or via intermediate transport filaments (Ochs, 1971). We found that the velocity of transport of DBH was highly sensitive to temperature, but that the fraction of DBH transported did not vary perceptibly from 35% over the range of temperatures tested. Therefore, if DBH must bind to contractile proteins in order to

350

COSENS, THACKER, AND BRIMIJOIN

be transported, its binding must be largely insensitive to 30" changes in temperature. Several possibilities are consistent with this conclusion: ( I ) Transport of DBH involves no binding; (2) Binding is proportional to absolute temperature, and the 10% change on this scale produces an effect too small to detect with our technique; ( 3 )Binding is nearly saturated at physiological temperatures so that moderate changes in affinity hardly change total binding. Further studies on the kinetics of transport are needed to test these possibilities. We found that transport velocity of DBH activity in sympathetic nerves was a simple exponential function of temperature over a wide range. This behavior qualitatively resembles that of labeled proteins that are rapidly transported along sensory nerves of the cat (Ochs and Smith, 1975) and frog (Edstrom and Hanson, 1973). Labeled proteins in the garfish olfactory tract, on the other hand, apparently move at velocities that are linearly related to temperature down to 5°C (Gross, 1973). We cannot account for this divergent finding, which may depend on some unusual feature of neurons in the garfish or, perhaps, of olfactory neurons in general. Arrhenius plots of the natural logarithm of velocity versus the reciprocal of absolute temperature can yield apparent activation energies for axonal transport. Such a plot (Fig. 7) reveals that the activation energy derived from our data is remarkably similar to those derived from the data of Ochs and Smith (1975) and of Edstrom and Hanson (1973). Based on measurements from 13°C and up, these energies ranged only from 13.1 to 14.8 kcal, while corresponding Qlo values were 2.28 to 2.33. If the data taken at 13°C by Ochs and Smith are excluded, one calculates a Q l o of 2.0 and an apparent activation energy of 12.7 kcal for transport in the cat sciatic nerve, still not greatly different from our results. Although the regression lines in Figure 7 have nearly the same slope, their intercepts differ somewhat. Temperature thus appears to affect transport velocity similarly in all three species, but the actual velocity at any given temperature depends on the species. Ochs has repeatedly found rates close to 410 mmlday for axonal transport in cat sensory nerves at 38°C (e.g., Ochs and Smith, 1975). Using the Arrhenius plot to extrapolate Edstrom and Hanson's data to 38°C gives a velocity of 606 mmlday for frog sensory nerves; treating our results in the same way gives a velocity of 330 mmlday for rabbit sympathetic nerves. These differences in calculated transport velocity could reflect real biological variation or could represent methodological artifacts. One should especially consider the possibility of systematic errors in stop-flow experiments. Artifacts from altered transport in previously cooled regions of nerve did not influence our study since we measured velocity in regions well distal to the cold block. These regions were never cooled below the test temperature. But when the cold block was released, local concentrations of moving material were probably higher than normal, which might conceivably have retarded transport. However, Edstrom and Hanson (1973) raised the local concentrations of moving, labeled proteins by cooling frog nerves for 7 hr while the ganglia incorporated .?H-leucine. Yet they found transport velocities slightly higher than did Ochs and Smith (1975) at comparable temperatures. Although all these discrepancies are modest, further tests should be made to see whether or not transport velocity depends on the amount of material in motion.

TEMPERATURE AND RAPID TRANSPORT

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Fig. 7. Arrhenius plot of transport velocity. The vertical axis is the natural logarithm of transport velocity; the horizontal axis is the reciprocal of absolute temperature. Present results (rabbit) are compared with those of previous experiments on migrating labeled proteins in sciatic nerves of cat (data taken from a table in Ochs and Smith, 1975) and frog (data derived from a figure in Edstrbm and Hanson, 1973). Regression lines were fitted to the data by the method of least squares; the lowest temperature point in each series was arbitrarily excluded, and mean values were weighted according to the number of observations a t the temperature in question. Apparent energies of activation were calculated from the slopes of these regression lines according to the equation: In V = In A - (Ea/RT); where Ea is the activation energy, R is the gas constant, and A is a constant (Johnson, Eyring, and Stover, 1974).

In general, our results indicated that DBH would resume migration at any temperature that could normally sustain transport (compare Figs. 4 and 6). Recovery of transport, when it occurred, must have been fast since calculated transport velocity did not depend on time allowed for flow, at least a t 19” and 37°C. At 13OC, recovery was incomplete. Some accumulated DBH activity clearly resumed migration at the expected velocity during second stage incubations a t this temperature, but after 10 hr much of the activity was still where it had been a t the end of stage one (Fig. 4). One might explain this by supposing that some of the blockade from local cooling to 2°C persisted during subsequent incubation at 13°C. However, a few “channels” must have quickly reopened to allow a little DBH to escape into regions where previous cooling had not impaired transport. Ochs and Smith (1975) found that the migrating front of labeled proteins became less well defined (more sloping) at 18OC and below, and they found steeply sloping fronts in nerves incubated a t 13°C. These observations may relate to the partial failure we observed in recovery of transport at this temperature. In our experiments on DBH, 13°C was a transitional temperature for transport,

COSENS, THACKER, AND BRIMIJOIN not only because recovery from a cold-block was incomplete, but also because transport velocity fell abruptly when temperature was further reduced. Since average velocity of transport also fell sharply below 13°C (Fig. 6), the transition was not simply an artifact of the stop-flow system (e.g., a persistent local coldblock). Ochs and Smith's (1975) observations on cat sensory nerves agree with ours on rabbit sympathetic nerves in revealing a sharp break a t 13°C in the relation between transport velocity and temperature (see Fig. 7). Even if the Arrhenius plot of their data is redrawn to fit points at 18°C and above separately from points a t 13°C and below, the regression lines intersect near 13°C. Thus, both studies of mammalian nerves point to 13°C or thereabouts as a transitional temperature for transport. I t remains unclear, however, whether this temperature represents a true threshold; although Ochs and Smith found no transport at all below l l ° C , we cannot exclude a slow residual transport at IOOC. This discrepancy might mean simply that sensory and sympathetic nerve axons respond differently to low temperatures. Frog nerves may likewise have a transitional temperature for axonal transport, but the break in the Arrhenius plot of transport velocity in these nerves occurs at 65°C (Fig. 7, data from Edstrom and Hanson, 1973). Thus frog nerves seem to resist cold distinctly better than do nerves from cats and rabbits. Perhaps transitional temperatures for transport are characteristically lower in nerves from poikilotherms than in nerves from mammals. What could account for marked reductions of axonal transport velocity at low temperatures? One possibility is phase-changes in lipid membranes. Often, over a narrow range of temperatures, changes in membrane function coincide with changes in the mobility of spin-labeled probes incorporated in the same membrane (Raison, 1973). Apparently, various membrane functions, including the activity of associated enzymes, critically depend on whether the membrane is fluid or crystalline. If the smooth endoplasmic reticulum forms a pathway for rapid axonal transport, as Droz, Rambourg, and Koenig (1975) suggest, crystallization of its lipid components at low temperatures would probably affect velocity in a dramatic way. It may be significant that protoplasmic streaming in cells of chilling-sensitive plants suddenly stops below 10°C (Lewis, 1956),the temperature at which phase-changes occur in membrane lipids of these species (Raison, 1973). We know of no mammalian membrane, however, which changes phase a t temperatures so low. Lipid phase-changes in membranes from mammalian cells typically occur a t or above room temperature (Raison, 1973; Hazel and Prosser, 1974). Sarcoplasmic reticulum from rabbit muscle is a good example: in this membrane, transitions in temperature-dependence of Ca+ accumulation, Ca+ +-stimulated ATPase activity, and mobility of spin-labeled probes all take place at 20°C (Inesi, Millman, and Eletr, 1973). Current evidence, therefore, does not favor phase-changes in membranes as an explanation for sharp reductions in the velocity of axonal transport in mammalian nerves cooled below 13°C. Failure of transport at low temperatures is probably unrelated to decreased neuronal stores of high energy phosphate (WP). Ochs and Smith (1975) have shown that combined levels of ATP and creatine phosphate in cat sciatic nerve remain near normal for up to 64 hr of incubation in uitro at 8"C, and for up to 34 hr a t 0°C. Assuming that an unchanged proportion of -P is available to the transport apparatus, slowed transport at temperatures below 13°C must reflect +-

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decreased ut,ilization rather than decreased production of energy. Microtubules dissociate at low temperatures in many types of cells (Tilney and Porter, 1967; Behnke, 1967), including neurons of the toad sciatic nerve (Rodriguez-Echandia and Piezzi, 1968), although apparently not neurons from the nerve cord of cold-adapted crayfish (Fernandez, Huneeus, and Davison, 1970). In view of the evidence suggesting a role for microtubules in axonal transport (Dahlstrom, 1971; Heslop, 1975) one wonders how the effects of temperature upon microtubules and transport compare. Unfortunately, we can find no quantitative information concerning graded effects of temperature on microtubules in live mammalian axons. It has been reported that microtubules prepared in uitro from extracts of pig brain partly depolymerize at 20°C and do so rapidly at 15°C (Olmsted and Borisy, 1973). These findings do not strongly support depolymerization of microtubules as the cause of decreased transport velocity at temperatures below 13°C. Nevertheless, an in uiuo comparison is clearly warranted. The rate and temperature-dependence of repolymerization should also be studied in intact nerves since transport recovers so quickly when cooled nerves are rewarmed. Breakdown of the axonal transport of DBH at 47°C (Fig. 3) is easy to dismiss as a nonspecific effect. But nerves heated to this temperature for 1hr lost only about half of their DBH activity (as compared to the overall mean activity in Table 1). Thus, not all neural proteins are rapidly denatured a t 47°C. Transport at high temperatures probably fails for different reasons than it does at low temperatures, and microtubules may not be involved a t all. Yet it is interesting that, after heating to 5OoC, microtubules prepared from sea urchin suddenly lose their normal structure when they are mildly shaken, whereas tubules heated to 45°C resist such treatment (Ventilla, Cantor, and Shelanski, 1972). Kinetic studies like the present one provide a base for future investigations of transport. Although temperature is a nonspecific probe that affects all neuronal properties, the abrupt changes that occur in transport at low and high temperatures lend themselves to correlative studies. In particular, comparing temperature-dependent transitions in transport velocity, microtubular state, and membrane fluidity in several animal species might yield new insights into the mechanism of axonal transport. This work was supported in part by NIH grant, NS 11855, and by an NIH Career development Award, NS 00119 (to S.B.). REFERENCES BEHNKE,0. (1967). Incomplete microtubules observed in mammalian blood platelets during microtubule polymerization. J . Cell Biol. 34: 697-701. BRIMIJOIN,S. (1974). Local changes in subcellular distribution of dopamine-0-hydroxylase (EC 1.14.2.1) after blockade of axonal transport. J . Neurochem. 22: 347-353. BRIMIJOIN,S. (1975). Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-6-hydroxylase. J . Neurobiol. 6: 379-394. BRIMIJOIN,S. and HELLAND,L. (1976). Rapid retrograde transport of dopamine-0-hydroxylase as examined by the stop-flow technique. Brain Res. 102: 217-228. CHRISTENSEN,H. N. (1975). Biological Transport, 2nd ed., Benjamin, Reading, Massachusetts. COPELAND,A. R. and REINER,J. M. (1974). Mathematical constructs of axonal transport. Fed. Proc. 33: 319. DAHLSTR~M A., (1971). Axoplasmic transport (with particular respect to adrenergic neurons). Phil. Trans. Roy. Soc. Lond. R. 261: 325-358.

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