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Effect of Maturation and Aging on the Rate of Fast Axoplasmic Transport in Mammalian Nerve
Effect of Maturation and Aging on the Rate of Fast Axoplasmic Transport in Mammalian Nerve SIDNEY OCHS Department of Ph.vsiology, Indiana University Medical School, Indianapolis, Inn. (U.S.A.)
INTRODUCTION
Both the fast and slow transport systems present within nerve fibers carry materials synthesized in the cell body down inside the axons in order to maintain their viability, to supply materials to the nerve terminals required for transmission, and trophic materials to the cells with which the nerve terminals synapse or to cells with which they are in ~ o n t i g u i t y l - ~ . Fast axoplasmic transport is a fundamental process which, if interrupted, either completely or partially, can have profound effects on neuronal function. It was of interest, therefore, to determine if there are changes in the rate of transport associated with maturation and aging. Several recent reports have indicated that changes in transport do occur during early life. An increase in the rate of fast transport of the optic system of the rabbit which takes place in the first few weeks of life was reported by Hendrickson and Cowan4. Injections of [3H]leucine were made into the eye for uptake by ganglion cells of the retina and following incorporation the rate of outflow in the optic nerves was estimated by the earliest time of accumulation of labeled proteins in the superior colliculus. A fast transport rate of 120 mm/day was obtained on the 6th day, rising to 150 mm/day at the end of the 3rd week. A more rapid rise occurred at the end of the 4th week, when a rate of 200 mm/day was attained which then remains fairly level at between 200 and 240 mm/day, the adult rate. Slow transport, on the other hand, declined from 5 mm/day at the end of the 1st week of life to 2 mm/day in animals over 4 weeks of age with a more rapid change at the 4th week. An increase in the rate of a fast component of transport was also found in the chick optic system by Marchisio and Sjostrand5. Rate changes for slow transport were found in the weanling kitten after injecting the dorsal root ganglion with [3H]leucine6. The slope of the activity in the sciatic nerve at 14 hours after injection was 2-3 times greater in the kitten than in the adult cat. Droz’ reported slow transport to be faster in the immature rat with a rate of 2-2.5 mm/day as compared to a rate of 0.6-0.9 mm/day found for the adult. Because fast axoplasmic transport is closely related to a variety of neuronal functions, the present studies to be described were directed to the possibility of a rate change of fast axoplasmic transport with age. In our laboratory fast axoplasmic References a. 362
350
S. OCHS
transport is measured by the position of the crest of activity found in the sciatic nerve after injection of the [3H]leucine either into the L7 dorsal root or the L7 spinal cord for uptake by motoneuron somas3* ’. This technique clearly differentiates between possible changes relating to the level of synthesis of materials in the cell bodies as caused by maturation or aging’, l o , from changes in the mechanism responsible for the transport of materials in the fibers per se. Hopefully, studies on the rate of fast axoplasmic transport in the fibers as affected by maturation and aging might give us additional information on the mechanism underlying fast axoplasmic transport. An hypothesis of a “transport filament” was recently proposed as the mechanism underlying fast axoplasmic transport3, 8 , and this mechanism and the relation of maturation and aging to it will be discussed.
REGULARITY OF RATE OF FAST AXOPLASMIC TRANSPORT
IN THE MATURE ANIMAL
To study fast axoplasmic transport of materials in mammalian nerves, the L7 dorsal root ganglia were injected with [3H]leucine, and after allowing time for downflow of labeled proteins in the nerves, the nerves were removed and cut into 5-mm sections to determine the pattern of activity in them. The rate is determined by the distance to which the crest of labeled material has moved down the nerve fibers during the time allowed, and it was found to have a regular rate close to 410 mm/day (Fig. 1)”. This same rate of fast axoplasmic transport was found in the sciatic nerve of a number of different species differing markedly in size (Table 1) and as shown in Fig. 2 for a large dog. As noted in Table 1, the rate is the same for both the sensory and in the motor fibers of the sciatic nerve. The latter was shown by injecting the L7 segment of the ventral horns of the spinal cord with [3H]leucine in the vicinity of the motoneuron cell bodies and after a period of downflow, finding a similar crest of activity which had moved down to the distance expected of fast axoplasmic transport. The fast downflow thus found for the motor fibers of the rat has the expected fast rate (Fig. 3). The same rate of fast axoplasmic transport was found present in myelinated nerve fibers ranging in diameter from 3 to 23 p. This was shown by taking small pieces of sciatic nerve at the crest of downflowing activity and preparing them for radioautography by freeze-substitution”. In such preparations the osmium tetroxide of the substitution medium stains the myelin sheaths dark and grains representing labeled proteins were found present within fibers over the whole range of diameters from 3 to 23 1.1 (ref. 11). If the large diametered fibers had a faster rate, no grains would be seen in the smaller diameter fibers and vice versa. Thus the rate is the same in all these myelinated fibers. Recently the same idea was pursued using electron microscopy and radioautography, and grains found in non-myelinated fibers indicating these also have the same fast rate close to 410 mm/day. The rate of fast axoplasmic transport is markedly affected by temperature with a Q, of 2-2.3 (ref. 13). At a Q1 of 2 this means that a change in temperature as small
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Fig. 1. Fast axoplasmic transport in cat nerves. Distribution of radioactivity in the dorsal root ganglia and sciatic nerves of five cats taken between 2 to 10 hours after injecting [3H]leucineinto the L7 ganglia (G). The activity present in 5-mm segments of roots, ganglia, and nerves (abscissa) is given on the ordinate in logarithmic divisions. The ordinate scale for the nerve 2 hours after injection is given at the bottom left with divisions in cpm. At the top left a scale is given for the nerve taken 10 hours after injection. Only partial scales are shown at the right for the nerves taken 4, 6 and 8 hours after injection. TABLE 1 RATES OF FAST AXOPLASMIC TRANSPORT
Rate values given as means and standard deviations. Sensory nerves after L7 dorsal root ganglia injection with [3H]leucine, motor nerves after [3H]leucineuptake by L7 motoneuron. _ _ Number of nerves
Species
Motor (sciatic)
18 5
Rat Monkey
411 -+ 50 417 38 (400 f 35)
Sensory (sciatic)
14 26
4
Monkey Cat Rabbit Goat Dog
416 409 394 389 423
3 4
Monkey Cat
397 57 391 f 59
~~
Nerve type
f f
Dorsal column spinal cord
References p . 362
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(360, 415) (382, 396) f 15
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60
45
30
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15
30
I
45
I
I mm
15
60
I
90
I
I
105
120
I
135
I
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165
150
Fig. 2. Fast axoplasmic transport in dog nerves. The same procedure was used as for the cat in Fig. 1 and the usual crest and rate of transport was found. I '
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Fig. 3. Fast axoplasmic transport in rat motor nerves. Thc L7 region of the spinal cord was injected with [3H]leucineand downflow in the nerves of two rats at 5- and 6-hour intervals is seen. The crest positions (arrows 1 and 2, respectively) are at the front of the crest at a position expected of the usual rate of fast transport. The slope of activity near the cord represents leakage.
353
AGING AND FAST TRANSPORT
as 0.5"C will change the calculated rate of transport by 15 mm/day. It is likely, therefore, that the rate of fast axoplasmic transport of close to 410 mm/day so far found may be even more constant than indicated by the experimental variability and that it is a general feature of mammalian nerve. A contribution to this concept of generality for the rate is the finding that the same fast rate is present in the dorsal roots as in the sciatic nerve. The dorsal root ga'iglia contains T-shaped neurons with one branch descending in the sciatic nerve to terminate as a receptor in the periphery and the other branch ascending via the dorsal root to enter the central nervous system (CNS) and subserve synaptic transmission. Thus, different kinds of substances are likely to be transported into the two branches of the ganglion neurons, i.e. into the sensory terminal for transduction and into the presynaptic terminals for synaptic transmission. It was possible, in the longer lengths of dorsal root present in the larger rhesus monkeys, to determine the rate from the crest of the usual form which was seen to advance to the same distance in the dorsal root as in the corresponding part of the sciatic nerve after injection of the L7 ganglia with
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Fig. 4. Fast axoplasmic transport in monkey nerve and in spinal cord. The L7 dorsal ganglia were injected with [3H]leucine and 6 hours later the typical outflow is seen in the two nerves (A). In addition, the long dorsal root was taken and part of the dorsal columns and arranged so that it corresponds with the nerve (B). A crest (arrow 2) is shown at the same distance from the ganglia as in the nerves (arrow 1). Refer e/ices P . 362
354
S. OCHS
[3H]leucine". These experiments indicate that the rate does not depend on the function of the nerve fiber or, incidentally, on the direction of nerve impulse conduction. The dorsal root fibers entering the spinal cord ascend in the dorsal columns of the spinal cord. After L7 ganglion injection with [3H]leucine the usual crest of activity was also found to ascend in this CNS tract and to have the same fast rate (Fig. 4). The figure is arranged so that the crest of activity in the dorsal columns can be directly compared with the crest in the sciatic nerves and the similarity in their rates shown''. The finding that the rate in nerves taken from large animals such as the goat and monkey is the same as the rate in an animal as small as the rat indicates that size per se is not a factor controlling the rate of fast axoplasmic transport. This is of prime importance in a study of the effect of immaturity on changes in rate insofar as the nerves in the weanling kitten are very much smaller than those of the adult or aged cat.
RATES OF FAST AXOPLASMIC TRANSPORT IN THE NEWBORN KITTEN
A number of profound neuronal changes occur within the first 4 weeks of life in the kitten, including a considerable growth of dendritic and axon processes. Kittens as young as 2 weeks had L7 ganglia large enough to be injected with C3H]leucine by the usual procedure. The shorter length of their sciatic nerves, however, precluded downflow times longer than 3-3.5 hours. Fortunately, the nerves are long enough to observe the whole of the characteristic crest of downflow. An example is shown in 106
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Fig. 5. Fast axoplasmic transport in a 12-day-old kitten. The L7 ganglia are injected and the usual pattern of outflow is seen.
355
AGING AND FAST TRANSPORT
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Fig. 6. Fast axoplasmic transport in sensory and motor fibers of 6-week kittens. The L7 ganglia in one animal (No. 1) were injected and the usual outflow is seen. III addition the outflow in nerves taken from another kitten (No. 2) is presented and it shows the usual crest positions indicated by the arrows.
Fig. 5 where the usual high level of activity is seen remaining in the ganglion with a fall of activity to the plateau level and a rise to a crest before a fall to base-line levels. The rate of fast axoplasmic transport is determined as usual by the distance from the intersection of front of the crest with the base-line level of activity back to the center of high activity in the ganglia and the amount of time allowed for downflow. The rate of transport while close to that of the adult cat is a little lower (Table 2). Another example taken from a 6-week kitten is shown with, in addition, the outflow pattern in the motor fibers of a sciatic nerve after injection of the L7 motoneuron region with [3H]leucine (Fig. 6). The similarity in the positions of the crests indicates that the rate of fast axoplasmic transport in the motor fibers are the same as that of the sensory fibers. The crest positions are determined by taking into account the more anterior position of the L7 motoneuron as compared to the L7 ganglion". 14. The slope of References P. 362
356
S. OCHS
TABLE 2 FAST AXOPLASMIC DOWNFLOW RATES IN NEWBORN KITTENS
Expt . No.
Downflow time (hours)
Distance
f mmi
(mmldayl
56 58 48 55
384 398
15 15
3.5 3.5 3.5 3.5
453
13
3
454 454
13 13
3 3
45 45 45
360 360 360
455 455
14 14
3 3
408 416
456 456
15 15
3 3
51 52 51 52
451 457
3 3 3
49 46 48
392 368
465
15 15 14
466 466 467 461
14 14 14 14
3 3 3 3
48 49
384 392
47 48
316 384
468 468 469 469
14 14 15 15
3 3 3 3
49 47 52 51
392 316 416 408
410
14
3
49
392
471 47 1
15 15
3 3
48 52
408 416
416 416
12 12
3 3
49 50
392 400
( A ) Two weeks 446 446 447 441
15 15
No. nerves 27
(B) Three weeks 452 452 492 492 493 493 494 494 503 503 504 504 505
Rate
330 371
408 416
384
Mean i S.D. 389 1 29 22 22 21 21 21 21
4 4
21 21 21 21
3 3 3 3 3 3 3.5 3.5
62 62 45 45 45 42 45 39 60 60
21 21
3.5 3.5
60 60
360 336 360 312 41 1 41 1 41 1 41 1
21
3.5
60
41 1
372 372 360 360
357
AGING AND FAST TRANSPORT
TABLE 2 (continued) ~~
-
~
-
~
Expt . No.
Age (days)
Downflow time
Distance
(hours)
(trim)
Rate (mmldayi
506 506
21 21
3.5 3.5
60 66
41 1 452
501 507
22 22
3.5 3.5
60 57
41 1 390
508 508
22 22
3.5 3.5
66 66
452 452
509 509
20 20
3.5 3.5
60 56
41 1 3 84
510 510
21 21
3.5 3.5
60 57
41 I 391
51 1 511
21 21
3.5 3.5
62 63
425 432
512 512
21 21
3.5 3.5
57 51
390 390
519 519 520 520
21 21 21 21
3.5 3.5
58 58 60 60
398 398 41 1 41 1
~-
3.5 3.5
._
~.
.
Mean & S.D. 397 t 32
No. nerves 31 ( C ) Four weeks 442 442
~~
27 21
4 4
73 13
432 432
443 443
21 27
4 4
444 444
21 21
4 4
12 72 65 65
432 432 390 390
488 488 489 489
28 28 28 28
3.5 3.5 3.5 3.5
51 51
350 350 391 370
57 54
Mean
No. nerves 10 ( D ) Six weeks 430 430
S.D. 397
42 42
4 4
70 10
420 420
433 433
42 42
4 4
68 68
408 408
413 413
42 42
4 4
74 14
444 440
474 474
42 42
4 4
72 74
432 444
415 415
42 42
4 4
10 11
420 426
No. nerves 10 References p . 362
Mean
-+ 40
S.D. 426 f 26
~~-
358
S . OCHS
activity near the cord is due to leakage as a result of the relatively high level of precursor injected (cf. Fig. 11, ref. 14). The rates of fast axoplasmic transport in kittens 2 and 3 weeks of age are just a little less than the adult rate of 410 mm/day and do not statistically differ from adult animals. TABLE 3 FAST AXOPLASMIC DOWNFLOW RATES I N AGED ANIMALS
Weight (kg)
Downflow time (hours)
8 8
4.55 4.55
5.25 5.25
361 367
9-1 0 9-10
3.7 3.7
409 409
16 16
791 791
20 20
Expt. No.
Age (years)
-
( A ) Aged cats 366 366
Distance (mm)
96 95
439 434
1 1
135 138
463 473
4.3 4.3
6 6
102 102
408 408
3.6 3.6
8 8
132 140
396 420
No. nerves 8
Mean f S.D. 430 i 21
( B ) Control dogs 478 478
6 6
1 7
122 122
490
21.5
6
120
491 491
11.5 17.5
6 6
125 125
No. nerves 5
( C ) Aged dogs 450 450
418 41 8 480 499 499
-
60 60
=
439 439
S.D. 439 k 34
Mean 13.5 13.5
20 20
7 7
152 153
521 525
464 464
12 12
21.5 21.5
6 6
108 123
432 492
479 419
12 12
11 11
7 7
149 150
51 1 514
485
12
18.5
1
130
452
486 486
8-9 8-9
5 5
6 6
100
105
400 420
495 495
> 12
23 23
1 1
140 140
496 496
> 12
31 31
6 6
117 117
18.5
6
105
487 487 No. nerves 15
12
12
Prematurely Aged 1
490 490
420 420
-
45 45
+ 90 + 90
=
445 445 468 468
= =
510 510
Mean & S.D. 469
+ 47
AGING AND FAST TRANSPORT
359
The rates found for the 4-6-week kittens were, as can be seen, close to the rate of transport seen for the adult, i.e. statistically they were not significantly different.
TRANSPORT RATES IN AGED ANIMALS
Cats 8 years and older of verified age were difficult to obtain. In the small sample so far studied the unexpected finding was that some of these aged animals had a greater rate of transport seen (Table 3). Aged dogs which were easier to obtain were used to further augment the group of aged animals studied. A higher rate was also found for several of the aged dogs (Table 3). It is possible that some of the rate increase in the larger dogs was due to their higher body temperature, which, in some cases, rose to a temperature of 40°C. Considering the Q1 of 2-2.3 for fast axoplasmic transport, this could account for part of the rate difference. However, higher body temperatures may not account for all the rate increases seen. Possibly the nerves may have been more easily elongated in these aged animals. In any case, as indicated in the preceding section, size per se does not account for the higher rates found. The finding of a higher rate in the nerves of some animals and not in others indicates that the rate increase is not due to aging itself but that it may likely be caused by a process associated with aging. This interpretation suggested the use of the prematurely aged dog (the juvenile amaurotic familiar idiocy-like syndrome) which had been genetically isolated by Koppang. ' '. This genetic defect causes changes which mimic the group of ceroid-lipofuscinosis diseases, and it could be a model of agingI6. One such animal 12 months of age made available for study of fast transport showed an increased rate when compensation was made for the low body temperature at which downflow occurred, and another showed a normal rate (Table 3) (Ochs and Koppang, unpublished observations). The neurological signs of CNS or peripheral nerve involvement are not fully developed until these genetically defective animals are approximately 18 months of age, and the variability of rate change again suggest some concomitant changes and not one regularly associated with an aging process.
MECHANISM OF FAST AXOPLASMIC TRANSPORT
Leaving to one side the possible small reduction in rate seen in the 2-3-week kittens and some evidence of increased rates seen in some of the aged animals, the results show a remarkable uniformity in the rate of fast axoplasmic transport in the nerves from kittens of 2 weeks of age through the adult cats of approximately 8 years of age. The age of adult cats obtained without a known history is difficult to determine, and probably these animals are not much beyond 3-5 years of age. The regularity of the rate of fast axoplasmic transport seen within this large age range conforms to the similar rate seen in the nerves of various other species and in differing nerve types. Such a close similarity in the rate implies that there is one underlying mechanism of fast axoplasmic transport present in these nerves. A model for the mechanism of fast References P. 362
360
S. OCHS
\
-\-NF a
b
C
Fig. 7. Model for fast axoplasmic transport. Transport filament hypothesis of fast axoplasmic transport. Glucose (G) enters the fiber and after glycolysis and oxidation in the mitochondrion (Mit), the resulting ATP supplies the sodium pump (dashed arrow) controlling Na+ and K+ in the fiber. The ATP is also shown supplying energy to the “transport filament” which is indicated by a black bar to which particulates (a), soluble protein (b), and other undesignated substances (c) are bound, and in this way the various species are carried down the axon at the same fast rate. The crossbridges shown between the transporting filament and the microtubules (M) (or neurofilaments (NF)), effect this downward movement and require ATP as a source of energy in analogy to cross-bridge action in muscle.
transport, a “transport filament” hypothesis based closely on the sliding filament theory of muscle was recently proposed3, *. The transport filament is considered to be synthesized in the soma and to move out into the axons along the microtubules and/or neurofilaments by means of cross-bridges (Fig. 7). A source of energy is required at the cross-bridges for chemomechanical transduction. The close dependence of fast axoplasmic transport on oxidative metabolism and an ATP involvement was shown in in vitro experiments18 - 2 1 . The heterogenous range of small particulates, proteins and polypeptides synthesized in the soma along with some free amino acids is considered to be bound to a transport filament and, thus, all these components are carried down the nerve fibers all at the same fast rate22*2 3 . In further analogy to the sliding filament in muscle, a Mg2+-Ca2+-activated ATPase with the characteristics of an actomyosin was found present in cat sciatic nerve24. It has many of the properties reported for the actomyosin-like material which had been found present in the brain2532 6 . This ATPase can use ATP for the movement of the transport filament down the nerve fiber. Cross-bridges between the transport filament and the microtubules and/or neurotubules are postulated. Most likely the microtubules are the stationary element. The composition of the protein monomers and dimers which combine to form the long length of the microtubules likely undergoes a turnover, and it is composed of colchicine-binding protein. There is evidence that the colchicine-binding protein is carried from the cell body by the slow transport system27. This protein is presumed to form the microtubules in the nerve which undergo a turnover in the axon and if there is a disassembly of microtubules in mammalian nerve at low temperatures, a fairly rapid reassembly must occur (Ochs, unpublished). The possibly slight low rate of transport in the newborn kitten may have a counterpart in the lower rate of fast axoplasmic transport found for retrograde transport. Acetylcholinesterase transport has a fast forward rate28 (cf. ref. 29). In our studies, acetylcholinesterase was shown to be carried down the fibers at a rate close to that of the usual fast axoplasmic
AGING AND FAST TRANSPORT
361
transport rate (a mean of 431 mm/day). In addition, as Lubiliska and N i e m i e r k ~ ~ ~ found, there is a retrograde transport of acetylcholinesterase at half this rate, at 230 mm/day. This suggests that transport filaments originating in the nerve terminal and ascending in the fiber to the soma can, in some cases, have different properties, possibly in their cross-bridge spacing3. It would suggest that possible changes in cross-bridge properties could occur as a function of maturation or aging. These remarks are admittedly speculative and reflect the limited structural knowledge we have so far of the linearly organized elements in the axon and their exact relationship to transport. The difference in rates found for kittens 2 weeks old is not significantly large to encourage a search for such possible differences. If there is a real increase in rate associated with aged animals, this would represent more of a challenge to the present concept of the underlying mechanism of transport. As already noted, the significant variable is not likely to be age per se because of the normal rate found in several of the older animals. If age were a variable we might also expect to see a progressive increase in the rate of transport with age. Possibly some condition associated with age is responsible for the few cases of increased rate seen. Possibly the overproduction of neurofibrillatory elements in the neurons such as seen in Alzheimer’s disease, the senile or presenile dementias of the aged3’ or in other degenerative diseases could contribute to increases in rate. Or, an overproduction of neurofibrillatory material elicited by local injections of aluminum salts3” 3 2 might be a model for such studies. Another possibility could relate to the accumulation of lipofuscin in neurons, a pigment found in increased amounts in aged animals33. In electron micrograph preparations the lipofuscin granules are seen as a peculiar lamellated organelle possibly related to l y s o ~ o m e s In ~ ~ the . two prematurely aged dogs so far studied a defect related to ceroid lipofuscin accumulation was not consistent with regard to an effect on the rate of transport. Admittedly, these few examples make it difficult to draw conclusions and a more extensive experimental evaluation is required to make a proper analysis. It is, however, especially noteworthy that the few rate changes seen were of an increase rather than a decrease. Every other variable so far studied in this laboratory (except for elevated temperature) produced a decrease in the rate of transport3. Further study along these lines could, if rate changes are found, lead to a better understanding of the fast axoplasmic transport mechanism. So far, studies indicate that for the most part, the rate from the young kitten through aged animals remains remarkably close to 410 mm/day.
SUMMARY
The effect of maturation and aging on the rate of fast axoplasmic transport was studied in the cat and a group of dogs used to augment the age studies. The most significant finding was of a similarity in the rate found over a wide span of age. This is in conformity to recent studies made in the nerves of a variety of species when the References P. 362
362
S. OCHS
regularity of the rate of fast axoplasmic transport at close to 410 k 50 mm/day was found. A possible slightly lower rate was seen in the newborn kitten and in some aged animals a somewhat higher rate was encountered. These were too few to be significant and the results further emphasize the regularity of the fast axoplasmic transport mechanism.
ACKNOWLEDGMENTS
This work was supported by N.S.F. GB 28664 X, P.H.S. R01-8706 and John A. Hartford Foundation, Inc. REFERENCES
8 9 10 11 12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
B. GRAFSTEIN, Adv. Biochem. Psychopharm., 1 (1969) 1 1 . A. DAHLSTROM, Phil. Trans. R. Soc. London, Ser. B, 261 (1971) 325. S. OCHS,Science, 176 (1972) 252. A. E. HENDRICKSON AND W. M. COWAN, Exp. Neurol., 30 (1971) 403. P. C. MARCHISIO AND J. SJOSTRAND, Brain Res., 26 (1971) 204. R. J. LASEK, Brain Res., 20 (1970) 121. B. DROZ,in C. P. LEBLOND AND K. B. WARREN (eds.), The Use of Radioautography in Investigating Protein Synthesis, Symp. SOC.Cell Biol., Vol. 4, Academic Press, New York, N.Y., 1965, p. 159. S. OCHS,J. Neurobiol., 2 (1971) 331. N. MIANI,A. RIZZOLIAND G. BUCCIANTE, J. Neurochem., 7 (1961) 161. E. GUTMANN, B. JAKONBEK, J. FISCHER AND A. BABICKY, Life Sci., 6 (1967) 2143. S. OCHS,J. Physiol., London, 227 (1972) 627. S. OCHS,Exp. Neurol., 12 (1965) 84. s. OCHS AND c. SMITH, Fed. Proc., 30 (1971) 665. s. OCHs AND N. RANISH,J. Neurobiol., 1 (1969) 247. N. KOPPANG, Ergeb. Allg. Pathol. Pathol. Anat., 47 (1966) 1. W. ZEMAN, in B. L. STREHLER (ed.), Advances in Gerontofogical Research, Vol. 3, Academic Press, New York, N.Y., 1971, pp, 147-170. N. KOPPANG, personal communication, 1972. S. OCHSAND D. HOLLINGSWORTH, J. Neurochem., 18 (1971) 107. s. OCHS AND c. SMITH, J . Newrochem., 18 (1971) 833. M. I. SABRIAND S. OCHS,Trans. Am. Soc. Neurochem., 2 (1971) 104. M. I. SABRIAND S. OCHS,J. Neurochem., 19 (1972) 2821. S. OCHS,M. I. SABRI AND J. JOHNSON, Science, 163 (1969) 686. M. I. SABRIAND S. OCHS,J. Neurobiol., 4 (1973) 145. M. A. KHANAND S. OCHS,Abstr. Am. Soc. Neurochem., 3 (1972) 93. S. PUSZKIN, S. BERL,E. PUSZKIN AND D. D. CLARKE, Science, 161 (1968) 170. S. BERLAND S. PUSZKIN, Biochemistry, 9 (1970) 2058. B. GRAFSTEIN, B. S. MCEWEN AND M. L. SHELANSKI, Nature, 227 (1970) 289. N. RANISHAND S. OCHS,J. Neurochem., 19 (1972) 2641. L. L U B I ~ S K AND A s. NIEMIERKO, Brain Res., 27 (1971) 329. R. D. TERRY, J. Neuropathol. Exp. Neurol., 22 (1963) 629. I. KLATZO,H. WISNIEWSKI AND E. STREICHER, J. Neuropathol. Exp. Neurol., 24 (1965) 187. H. WISNIEWSKI, W. KARCZEWSKI AND K. WISNIEWSKA, Acta Neuropathol., 6 (1966) 211. W. BONDAREFF, in B. J. STREHLER (ed.), Advances in Gerontological Research, Academic Press, New York, N.Y., 1964, pp. 1-22. A. N. SIAKOTES, H. H. GOEBEL, V. PATEL,I. WATANABE AND W. ZEMAN, in B. w. VOLKAND S. M. ARONSON (eds.), Sphingolipids, Sphingolipidoses and Allied Disorders, Plenum Press, New York, N.Y., 1972, pp. 53-61.
INTRODUCTION
Both the fast and slow transport systems present within nerve fibers carry materials synthesized in the cell body down inside the axons in order to maintain their viability, to supply materials to the nerve terminals required for transmission, and trophic materials to the cells with which the nerve terminals synapse or to cells with which they are in ~ o n t i g u i t y l - ~ . Fast axoplasmic transport is a fundamental process which, if interrupted, either completely or partially, can have profound effects on neuronal function. It was of interest, therefore, to determine if there are changes in the rate of transport associated with maturation and aging. Several recent reports have indicated that changes in transport do occur during early life. An increase in the rate of fast transport of the optic system of the rabbit which takes place in the first few weeks of life was reported by Hendrickson and Cowan4. Injections of [3H]leucine were made into the eye for uptake by ganglion cells of the retina and following incorporation the rate of outflow in the optic nerves was estimated by the earliest time of accumulation of labeled proteins in the superior colliculus. A fast transport rate of 120 mm/day was obtained on the 6th day, rising to 150 mm/day at the end of the 3rd week. A more rapid rise occurred at the end of the 4th week, when a rate of 200 mm/day was attained which then remains fairly level at between 200 and 240 mm/day, the adult rate. Slow transport, on the other hand, declined from 5 mm/day at the end of the 1st week of life to 2 mm/day in animals over 4 weeks of age with a more rapid change at the 4th week. An increase in the rate of a fast component of transport was also found in the chick optic system by Marchisio and Sjostrand5. Rate changes for slow transport were found in the weanling kitten after injecting the dorsal root ganglion with [3H]leucine6. The slope of the activity in the sciatic nerve at 14 hours after injection was 2-3 times greater in the kitten than in the adult cat. Droz’ reported slow transport to be faster in the immature rat with a rate of 2-2.5 mm/day as compared to a rate of 0.6-0.9 mm/day found for the adult. Because fast axoplasmic transport is closely related to a variety of neuronal functions, the present studies to be described were directed to the possibility of a rate change of fast axoplasmic transport with age. In our laboratory fast axoplasmic References a. 362
350
S. OCHS
transport is measured by the position of the crest of activity found in the sciatic nerve after injection of the [3H]leucine either into the L7 dorsal root or the L7 spinal cord for uptake by motoneuron somas3* ’. This technique clearly differentiates between possible changes relating to the level of synthesis of materials in the cell bodies as caused by maturation or aging’, l o , from changes in the mechanism responsible for the transport of materials in the fibers per se. Hopefully, studies on the rate of fast axoplasmic transport in the fibers as affected by maturation and aging might give us additional information on the mechanism underlying fast axoplasmic transport. An hypothesis of a “transport filament” was recently proposed as the mechanism underlying fast axoplasmic transport3, 8 , and this mechanism and the relation of maturation and aging to it will be discussed.
REGULARITY OF RATE OF FAST AXOPLASMIC TRANSPORT
IN THE MATURE ANIMAL
To study fast axoplasmic transport of materials in mammalian nerves, the L7 dorsal root ganglia were injected with [3H]leucine, and after allowing time for downflow of labeled proteins in the nerves, the nerves were removed and cut into 5-mm sections to determine the pattern of activity in them. The rate is determined by the distance to which the crest of labeled material has moved down the nerve fibers during the time allowed, and it was found to have a regular rate close to 410 mm/day (Fig. 1)”. This same rate of fast axoplasmic transport was found in the sciatic nerve of a number of different species differing markedly in size (Table 1) and as shown in Fig. 2 for a large dog. As noted in Table 1, the rate is the same for both the sensory and in the motor fibers of the sciatic nerve. The latter was shown by injecting the L7 segment of the ventral horns of the spinal cord with [3H]leucine in the vicinity of the motoneuron cell bodies and after a period of downflow, finding a similar crest of activity which had moved down to the distance expected of fast axoplasmic transport. The fast downflow thus found for the motor fibers of the rat has the expected fast rate (Fig. 3). The same rate of fast axoplasmic transport was found present in myelinated nerve fibers ranging in diameter from 3 to 23 p. This was shown by taking small pieces of sciatic nerve at the crest of downflowing activity and preparing them for radioautography by freeze-substitution”. In such preparations the osmium tetroxide of the substitution medium stains the myelin sheaths dark and grains representing labeled proteins were found present within fibers over the whole range of diameters from 3 to 23 1.1 (ref. 11). If the large diametered fibers had a faster rate, no grains would be seen in the smaller diameter fibers and vice versa. Thus the rate is the same in all these myelinated fibers. Recently the same idea was pursued using electron microscopy and radioautography, and grains found in non-myelinated fibers indicating these also have the same fast rate close to 410 mm/day. The rate of fast axoplasmic transport is markedly affected by temperature with a Q, of 2-2.3 (ref. 13). At a Q1 of 2 this means that a change in temperature as small
lob 105
104
4
In\
f I\\
10 HR
103
s
102
a u lo5 104
lo3 102
ROOTI
I
24 12
-NERVE I
I
I
I
I
I
I
I
I
l
l
1
1
1
1
0 12 24 36 48 60 72 84 98 108 120132 144156 166 Elm
Fig. 1. Fast axoplasmic transport in cat nerves. Distribution of radioactivity in the dorsal root ganglia and sciatic nerves of five cats taken between 2 to 10 hours after injecting [3H]leucineinto the L7 ganglia (G). The activity present in 5-mm segments of roots, ganglia, and nerves (abscissa) is given on the ordinate in logarithmic divisions. The ordinate scale for the nerve 2 hours after injection is given at the bottom left with divisions in cpm. At the top left a scale is given for the nerve taken 10 hours after injection. Only partial scales are shown at the right for the nerves taken 4, 6 and 8 hours after injection. TABLE 1 RATES OF FAST AXOPLASMIC TRANSPORT
Rate values given as means and standard deviations. Sensory nerves after L7 dorsal root ganglia injection with [3H]leucine, motor nerves after [3H]leucineuptake by L7 motoneuron. _ _ Number of nerves
Species
Motor (sciatic)
18 5
Rat Monkey
411 -+ 50 417 38 (400 f 35)
Sensory (sciatic)
14 26
4
Monkey Cat Rabbit Goat Dog
416 409 394 389 423
3 4
Monkey Cat
397 57 391 f 59
~~
Nerve type
f f
Dorsal column spinal cord
References p . 362
f 30 i 50
(360, 415) (382, 396) f 15
*
f J
G
I
I
I
60
45
30
I
15
I
0
I
I
15
30
I
45
I
I mm
15
60
I
90
I
I
105
120
I
135
I
I
165
150
Fig. 2. Fast axoplasmic transport in dog nerves. The same procedure was used as for the cat in Fig. 1 and the usual crest and rate of transport was found. I '
- 105
- lo4
a u e: 3 0
- lo3
U
7
- 102
e:
a 0 X I
Lo
103-
102-
0
15
30
45
60
15
90
105
mm
Fig. 3. Fast axoplasmic transport in rat motor nerves. Thc L7 region of the spinal cord was injected with [3H]leucineand downflow in the nerves of two rats at 5- and 6-hour intervals is seen. The crest positions (arrows 1 and 2, respectively) are at the front of the crest at a position expected of the usual rate of fast transport. The slope of activity near the cord represents leakage.
353
AGING AND FAST TRANSPORT
as 0.5"C will change the calculated rate of transport by 15 mm/day. It is likely, therefore, that the rate of fast axoplasmic transport of close to 410 mm/day so far found may be even more constant than indicated by the experimental variability and that it is a general feature of mammalian nerve. A contribution to this concept of generality for the rate is the finding that the same fast rate is present in the dorsal roots as in the sciatic nerve. The dorsal root ga'iglia contains T-shaped neurons with one branch descending in the sciatic nerve to terminate as a receptor in the periphery and the other branch ascending via the dorsal root to enter the central nervous system (CNS) and subserve synaptic transmission. Thus, different kinds of substances are likely to be transported into the two branches of the ganglion neurons, i.e. into the sensory terminal for transduction and into the presynaptic terminals for synaptic transmission. It was possible, in the longer lengths of dorsal root present in the larger rhesus monkeys, to determine the rate from the crest of the usual form which was seen to advance to the same distance in the dorsal root as in the corresponding part of the sciatic nerve after injection of the L7 ganglia with
(3
A
. 106
i\
ROOT
ROOT ENTRY
-
DORSAL COLUMN
m
.1042 0
- 103
106-
lo5
MONKEY 6 HR
-
P
I lo4-
V
- 105
I
L
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- 102
J DORSAL ROOT
---G
NERVE
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Fig. 4. Fast axoplasmic transport in monkey nerve and in spinal cord. The L7 dorsal ganglia were injected with [3H]leucine and 6 hours later the typical outflow is seen in the two nerves (A). In addition, the long dorsal root was taken and part of the dorsal columns and arranged so that it corresponds with the nerve (B). A crest (arrow 2) is shown at the same distance from the ganglia as in the nerves (arrow 1). Refer e/ices P . 362
354
S. OCHS
[3H]leucine". These experiments indicate that the rate does not depend on the function of the nerve fiber or, incidentally, on the direction of nerve impulse conduction. The dorsal root fibers entering the spinal cord ascend in the dorsal columns of the spinal cord. After L7 ganglion injection with [3H]leucine the usual crest of activity was also found to ascend in this CNS tract and to have the same fast rate (Fig. 4). The figure is arranged so that the crest of activity in the dorsal columns can be directly compared with the crest in the sciatic nerves and the similarity in their rates shown''. The finding that the rate in nerves taken from large animals such as the goat and monkey is the same as the rate in an animal as small as the rat indicates that size per se is not a factor controlling the rate of fast axoplasmic transport. This is of prime importance in a study of the effect of immaturity on changes in rate insofar as the nerves in the weanling kitten are very much smaller than those of the adult or aged cat.
RATES OF FAST AXOPLASMIC TRANSPORT IN THE NEWBORN KITTEN
A number of profound neuronal changes occur within the first 4 weeks of life in the kitten, including a considerable growth of dendritic and axon processes. Kittens as young as 2 weeks had L7 ganglia large enough to be injected with C3H]leucine by the usual procedure. The shorter length of their sciatic nerves, however, precluded downflow times longer than 3-3.5 hours. Fortunately, the nerves are long enough to observe the whole of the characteristic crest of downflow. An example is shown in 106
-
5 10
-
b
5: a lo4-
2
u
lo3
-
102-
ROOT NERVE t=476 6
0
6
12
18
24
30
36
42
48
54
60
m m
Fig. 5. Fast axoplasmic transport in a 12-day-old kitten. The L7 ganglia are injected and the usual pattern of outflow is seen.
355
AGING AND FAST TRANSPORT
l0iP 1
I
°
lo5
'
1
KITTEN 1 4 IiR
-lo5
430
.lo4 n
E w
I
.103
0-AI
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I
I
I
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2
- 10
Fig. 6. Fast axoplasmic transport in sensory and motor fibers of 6-week kittens. The L7 ganglia in one animal (No. 1) were injected and the usual outflow is seen. III addition the outflow in nerves taken from another kitten (No. 2) is presented and it shows the usual crest positions indicated by the arrows.
Fig. 5 where the usual high level of activity is seen remaining in the ganglion with a fall of activity to the plateau level and a rise to a crest before a fall to base-line levels. The rate of fast axoplasmic transport is determined as usual by the distance from the intersection of front of the crest with the base-line level of activity back to the center of high activity in the ganglia and the amount of time allowed for downflow. The rate of transport while close to that of the adult cat is a little lower (Table 2). Another example taken from a 6-week kitten is shown with, in addition, the outflow pattern in the motor fibers of a sciatic nerve after injection of the L7 motoneuron region with [3H]leucine (Fig. 6). The similarity in the positions of the crests indicates that the rate of fast axoplasmic transport in the motor fibers are the same as that of the sensory fibers. The crest positions are determined by taking into account the more anterior position of the L7 motoneuron as compared to the L7 ganglion". 14. The slope of References P. 362
356
S. OCHS
TABLE 2 FAST AXOPLASMIC DOWNFLOW RATES IN NEWBORN KITTENS
Expt . No.
Downflow time (hours)
Distance
f mmi
(mmldayl
56 58 48 55
384 398
15 15
3.5 3.5 3.5 3.5
453
13
3
454 454
13 13
3 3
45 45 45
360 360 360
455 455
14 14
3 3
408 416
456 456
15 15
3 3
51 52 51 52
451 457
3 3 3
49 46 48
392 368
465
15 15 14
466 466 467 461
14 14 14 14
3 3 3 3
48 49
384 392
47 48
316 384
468 468 469 469
14 14 15 15
3 3 3 3
49 47 52 51
392 316 416 408
410
14
3
49
392
471 47 1
15 15
3 3
48 52
408 416
416 416
12 12
3 3
49 50
392 400
( A ) Two weeks 446 446 447 441
15 15
No. nerves 27
(B) Three weeks 452 452 492 492 493 493 494 494 503 503 504 504 505
Rate
330 371
408 416
384
Mean i S.D. 389 1 29 22 22 21 21 21 21
4 4
21 21 21 21
3 3 3 3 3 3 3.5 3.5
62 62 45 45 45 42 45 39 60 60
21 21
3.5 3.5
60 60
360 336 360 312 41 1 41 1 41 1 41 1
21
3.5
60
41 1
372 372 360 360
357
AGING AND FAST TRANSPORT
TABLE 2 (continued) ~~
-
~
-
~
Expt . No.
Age (days)
Downflow time
Distance
(hours)
(trim)
Rate (mmldayi
506 506
21 21
3.5 3.5
60 66
41 1 452
501 507
22 22
3.5 3.5
60 57
41 1 390
508 508
22 22
3.5 3.5
66 66
452 452
509 509
20 20
3.5 3.5
60 56
41 1 3 84
510 510
21 21
3.5 3.5
60 57
41 I 391
51 1 511
21 21
3.5 3.5
62 63
425 432
512 512
21 21
3.5 3.5
57 51
390 390
519 519 520 520
21 21 21 21
3.5 3.5
58 58 60 60
398 398 41 1 41 1
~-
3.5 3.5
._
~.
.
Mean & S.D. 397 t 32
No. nerves 31 ( C ) Four weeks 442 442
~~
27 21
4 4
73 13
432 432
443 443
21 27
4 4
444 444
21 21
4 4
12 72 65 65
432 432 390 390
488 488 489 489
28 28 28 28
3.5 3.5 3.5 3.5
51 51
350 350 391 370
57 54
Mean
No. nerves 10 ( D ) Six weeks 430 430
S.D. 397
42 42
4 4
70 10
420 420
433 433
42 42
4 4
68 68
408 408
413 413
42 42
4 4
74 14
444 440
474 474
42 42
4 4
72 74
432 444
415 415
42 42
4 4
10 11
420 426
No. nerves 10 References p . 362
Mean
-+ 40
S.D. 426 f 26
~~-
358
S . OCHS
activity near the cord is due to leakage as a result of the relatively high level of precursor injected (cf. Fig. 11, ref. 14). The rates of fast axoplasmic transport in kittens 2 and 3 weeks of age are just a little less than the adult rate of 410 mm/day and do not statistically differ from adult animals. TABLE 3 FAST AXOPLASMIC DOWNFLOW RATES I N AGED ANIMALS
Weight (kg)
Downflow time (hours)
8 8
4.55 4.55
5.25 5.25
361 367
9-1 0 9-10
3.7 3.7
409 409
16 16
791 791
20 20
Expt. No.
Age (years)
-
( A ) Aged cats 366 366
Distance (mm)
96 95
439 434
1 1
135 138
463 473
4.3 4.3
6 6
102 102
408 408
3.6 3.6
8 8
132 140
396 420
No. nerves 8
Mean f S.D. 430 i 21
( B ) Control dogs 478 478
6 6
1 7
122 122
490
21.5
6
120
491 491
11.5 17.5
6 6
125 125
No. nerves 5
( C ) Aged dogs 450 450
418 41 8 480 499 499
-
60 60
=
439 439
S.D. 439 k 34
Mean 13.5 13.5
20 20
7 7
152 153
521 525
464 464
12 12
21.5 21.5
6 6
108 123
432 492
479 419
12 12
11 11
7 7
149 150
51 1 514
485
12
18.5
1
130
452
486 486
8-9 8-9
5 5
6 6
100
105
400 420
495 495
> 12
23 23
1 1
140 140
496 496
> 12
31 31
6 6
117 117
18.5
6
105
487 487 No. nerves 15
12
12
Prematurely Aged 1
490 490
420 420
-
45 45
+ 90 + 90
=
445 445 468 468
= =
510 510
Mean & S.D. 469
+ 47
AGING AND FAST TRANSPORT
359
The rates found for the 4-6-week kittens were, as can be seen, close to the rate of transport seen for the adult, i.e. statistically they were not significantly different.
TRANSPORT RATES IN AGED ANIMALS
Cats 8 years and older of verified age were difficult to obtain. In the small sample so far studied the unexpected finding was that some of these aged animals had a greater rate of transport seen (Table 3). Aged dogs which were easier to obtain were used to further augment the group of aged animals studied. A higher rate was also found for several of the aged dogs (Table 3). It is possible that some of the rate increase in the larger dogs was due to their higher body temperature, which, in some cases, rose to a temperature of 40°C. Considering the Q1 of 2-2.3 for fast axoplasmic transport, this could account for part of the rate difference. However, higher body temperatures may not account for all the rate increases seen. Possibly the nerves may have been more easily elongated in these aged animals. In any case, as indicated in the preceding section, size per se does not account for the higher rates found. The finding of a higher rate in the nerves of some animals and not in others indicates that the rate increase is not due to aging itself but that it may likely be caused by a process associated with aging. This interpretation suggested the use of the prematurely aged dog (the juvenile amaurotic familiar idiocy-like syndrome) which had been genetically isolated by Koppang. ' '. This genetic defect causes changes which mimic the group of ceroid-lipofuscinosis diseases, and it could be a model of agingI6. One such animal 12 months of age made available for study of fast transport showed an increased rate when compensation was made for the low body temperature at which downflow occurred, and another showed a normal rate (Table 3) (Ochs and Koppang, unpublished observations). The neurological signs of CNS or peripheral nerve involvement are not fully developed until these genetically defective animals are approximately 18 months of age, and the variability of rate change again suggest some concomitant changes and not one regularly associated with an aging process.
MECHANISM OF FAST AXOPLASMIC TRANSPORT
Leaving to one side the possible small reduction in rate seen in the 2-3-week kittens and some evidence of increased rates seen in some of the aged animals, the results show a remarkable uniformity in the rate of fast axoplasmic transport in the nerves from kittens of 2 weeks of age through the adult cats of approximately 8 years of age. The age of adult cats obtained without a known history is difficult to determine, and probably these animals are not much beyond 3-5 years of age. The regularity of the rate of fast axoplasmic transport seen within this large age range conforms to the similar rate seen in the nerves of various other species and in differing nerve types. Such a close similarity in the rate implies that there is one underlying mechanism of fast axoplasmic transport present in these nerves. A model for the mechanism of fast References P. 362
360
S. OCHS
\
-\-NF a
b
C
Fig. 7. Model for fast axoplasmic transport. Transport filament hypothesis of fast axoplasmic transport. Glucose (G) enters the fiber and after glycolysis and oxidation in the mitochondrion (Mit), the resulting ATP supplies the sodium pump (dashed arrow) controlling Na+ and K+ in the fiber. The ATP is also shown supplying energy to the “transport filament” which is indicated by a black bar to which particulates (a), soluble protein (b), and other undesignated substances (c) are bound, and in this way the various species are carried down the axon at the same fast rate. The crossbridges shown between the transporting filament and the microtubules (M) (or neurofilaments (NF)), effect this downward movement and require ATP as a source of energy in analogy to cross-bridge action in muscle.
transport, a “transport filament” hypothesis based closely on the sliding filament theory of muscle was recently proposed3, *. The transport filament is considered to be synthesized in the soma and to move out into the axons along the microtubules and/or neurofilaments by means of cross-bridges (Fig. 7). A source of energy is required at the cross-bridges for chemomechanical transduction. The close dependence of fast axoplasmic transport on oxidative metabolism and an ATP involvement was shown in in vitro experiments18 - 2 1 . The heterogenous range of small particulates, proteins and polypeptides synthesized in the soma along with some free amino acids is considered to be bound to a transport filament and, thus, all these components are carried down the nerve fibers all at the same fast rate22*2 3 . In further analogy to the sliding filament in muscle, a Mg2+-Ca2+-activated ATPase with the characteristics of an actomyosin was found present in cat sciatic nerve24. It has many of the properties reported for the actomyosin-like material which had been found present in the brain2532 6 . This ATPase can use ATP for the movement of the transport filament down the nerve fiber. Cross-bridges between the transport filament and the microtubules and/or neurotubules are postulated. Most likely the microtubules are the stationary element. The composition of the protein monomers and dimers which combine to form the long length of the microtubules likely undergoes a turnover, and it is composed of colchicine-binding protein. There is evidence that the colchicine-binding protein is carried from the cell body by the slow transport system27. This protein is presumed to form the microtubules in the nerve which undergo a turnover in the axon and if there is a disassembly of microtubules in mammalian nerve at low temperatures, a fairly rapid reassembly must occur (Ochs, unpublished). The possibly slight low rate of transport in the newborn kitten may have a counterpart in the lower rate of fast axoplasmic transport found for retrograde transport. Acetylcholinesterase transport has a fast forward rate28 (cf. ref. 29). In our studies, acetylcholinesterase was shown to be carried down the fibers at a rate close to that of the usual fast axoplasmic
AGING AND FAST TRANSPORT
361
transport rate (a mean of 431 mm/day). In addition, as Lubiliska and N i e m i e r k ~ ~ ~ found, there is a retrograde transport of acetylcholinesterase at half this rate, at 230 mm/day. This suggests that transport filaments originating in the nerve terminal and ascending in the fiber to the soma can, in some cases, have different properties, possibly in their cross-bridge spacing3. It would suggest that possible changes in cross-bridge properties could occur as a function of maturation or aging. These remarks are admittedly speculative and reflect the limited structural knowledge we have so far of the linearly organized elements in the axon and their exact relationship to transport. The difference in rates found for kittens 2 weeks old is not significantly large to encourage a search for such possible differences. If there is a real increase in rate associated with aged animals, this would represent more of a challenge to the present concept of the underlying mechanism of transport. As already noted, the significant variable is not likely to be age per se because of the normal rate found in several of the older animals. If age were a variable we might also expect to see a progressive increase in the rate of transport with age. Possibly some condition associated with age is responsible for the few cases of increased rate seen. Possibly the overproduction of neurofibrillatory elements in the neurons such as seen in Alzheimer’s disease, the senile or presenile dementias of the aged3’ or in other degenerative diseases could contribute to increases in rate. Or, an overproduction of neurofibrillatory material elicited by local injections of aluminum salts3” 3 2 might be a model for such studies. Another possibility could relate to the accumulation of lipofuscin in neurons, a pigment found in increased amounts in aged animals33. In electron micrograph preparations the lipofuscin granules are seen as a peculiar lamellated organelle possibly related to l y s o ~ o m e s In ~ ~ the . two prematurely aged dogs so far studied a defect related to ceroid lipofuscin accumulation was not consistent with regard to an effect on the rate of transport. Admittedly, these few examples make it difficult to draw conclusions and a more extensive experimental evaluation is required to make a proper analysis. It is, however, especially noteworthy that the few rate changes seen were of an increase rather than a decrease. Every other variable so far studied in this laboratory (except for elevated temperature) produced a decrease in the rate of transport3. Further study along these lines could, if rate changes are found, lead to a better understanding of the fast axoplasmic transport mechanism. So far, studies indicate that for the most part, the rate from the young kitten through aged animals remains remarkably close to 410 mm/day.
SUMMARY
The effect of maturation and aging on the rate of fast axoplasmic transport was studied in the cat and a group of dogs used to augment the age studies. The most significant finding was of a similarity in the rate found over a wide span of age. This is in conformity to recent studies made in the nerves of a variety of species when the References P. 362
362
S. OCHS
regularity of the rate of fast axoplasmic transport at close to 410 k 50 mm/day was found. A possible slightly lower rate was seen in the newborn kitten and in some aged animals a somewhat higher rate was encountered. These were too few to be significant and the results further emphasize the regularity of the fast axoplasmic transport mechanism.
ACKNOWLEDGMENTS
This work was supported by N.S.F. GB 28664 X, P.H.S. R01-8706 and John A. Hartford Foundation, Inc. REFERENCES
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