Microtubules in short and in long axons of the same caliber: Implications for the maintenance of the neuron

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Microtubules in short and in long axons of the same caliber: Implications for the maintenance of the neuron
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  EXPERIMENTAL NEUROLOGY 79,283-286 (1983) RESEARCH NOTE Microtubules in Short and in Long Axons of the Same Caliber: Implications for the Maintenance of the Neuron L&oratorio de Neurocitologia, Facultad de Ciencias Biobgicas, Universidad Cat6lica de Chile, Casilla 114-D. Santiago, Chile Received April 12, 1982; revision received June 21, I982 Microtubules are the morphological manifestation of a defined protein, tubulin, and they sustain axonal transport. Phrenic fibers are seven times longer than abducent fibers; however, in S-Cm axons of either nerve the density of the microtubules is the same (-2 1 microtubules/~m2). In addition, no difference was found in microtubule density between the cervical and juxtadiaphragmatic levels of the phrenic axons. These findings are contrasted with predictions based on the perikaryal theory of the srcin of axoplasm. It is accepted that all proteins in the nerve cell are synthesized in the perikaryon. Tubulin, the building block of microtubules, has been shown to travel along the axon in the slow flow (7, 8), and to have a half-life of about 9 days in both whole brain (6) and nerve endings (8). Because the slow flow is estimated to have a velocity of about 1 to 2 mm/day, the axonal tubulin content would be halved every 9 to 18 mm of nerve span. Con- sequently, it seems reasonable to expect a decrease in microtubule density along the axon, as this is the morphological manifestation of the assembled tubulin. Further, an axon with a large axoplasmic mass must require more material conveyed from the soma to maintain its steady state, compared with a small axon. Because microtubules are accepted to sustain transport, axons with large axoplasmic masses should contain more microtubules to ’ The assistance of Mr. Juan C. Tortes with statistical analysis and the technical a&stance of Mrs. Mdnica P6rex and Mr. Rat% Fuentes are greatly appreciated. This research was sup ported by the Direcci6n de Invest&&ones de la Universidad Cat&a. Correspondence should be addressed to Jaime Alvarez. 283 0014-4886/83/010283-04 03.00/0 Copyright Q 1983 by Academic Pres Inc. All rights of repmduction in any form rracrwd.  284 ALVAREZ AND ZAROUR TABLE 1 Microtubule Density in Axons about 5 pm in Diameter from Abducent and Phrenic Nerves” C D A 18.47 + 1.77 (11) 22.12 + 1.63 (11) 23.93 + 1.71 (12) 22.82 + 1.52 (12) 20.16 + 1.98 (8) 22.61 + 2.08 (11) 21.16 + 1.36 (10) 19.79 + 1.19 (9) 22.60 + 2.63 (10) 23.76 + 1.02 (13) 18.36 k 1.99 (7) 20.66 + 1.36 (15) 17.55 f 1.66 (13) 19.88 + 1.31 (18) 17.62 + 1.50 (11) 21.73 k 3.86 (IO) 21.67 f 2.83 (9) Average 20.59 20.06 22.45 n Each entry represents a nerve; figures indicate mean + SE of density (microtubules/rm* of axoplasm); in parentheses is the number of axons surveyed. The analysis of variance indicated that there was no significant difference between groups. C-cervical region, and D-juxtadia- phragmatic region of the phrenic nerve, 12 cm from C, A-abducent nerve. cope with the greater transport demand. To test these possibilities, we stud- ied microtubule density in the short (3 cm) abducent nerve and at two levels in the long ( 18 to 2 1 cm) phrenic nerve of the cat. In cats under pentobarbital anesthesia (36 mg kg-r), samples of the phrenic nerve were taken from both the neck and from the thorax just over the diaphragm more than 12 cm apart. The abducent nerve was taken at its exit from the brain stem. In all cases, fixation (glutaraldehyde 2.5%, cacodylate buffer 0.05 M, pH 7.4) was initiated in situ at body temperature; a few minutes later samples were taken and placed in fresh fixative at 37°C for a total of 1.5 h. Postosmication, dehydration, embedding in Epon, and staining were done routinely (1). Cross sections were observed at low power under the electron microscope+ to select axons 5 pm in diameter; they were photographed at 20,000X. The exact magnification was always assessed with a stage calibration. Micro- tubule counts were carried out blind as reported elsewhere (3). Table 1 shows microtubule density of axons from phrenic nerves at the cervical and juxtadiaphragmatic levels and from abducent nerves. The mean density, about 21 microtubules/clm*, did not differ significantly from one group to another; this value was very close to the microtubule density re- ported for 3-pm axons of the sural nerve (23 microtubule/pm*) explored with the same technique (3). The constancy of the microtubule density along the nerve is hard to  MICROTUBULES IN SHORT AND LONG AXONS 285 explain using current ideas. Based on the perikaryal srcin of proteins, the transport velocity of tubulin, and its half-life, the thoracic sample of the phrenic nerve should contain less than 1% of the tubulin present in the cervical sample; however, the microtubule density-which is the expression of the polymerized tubulin+lid not differ. It may be argued that the equi- librium microtubule G= free subunit shifts along the axon so as to exactly match the decay of tubulin; however, this would require in the initial seg- ments of long axons 10,000 or even one million free subunits for each subunit in the assembled state. Alternatively, tubulin in transit does not decay; this would imply that in fibers 1 m long (e.g., from a human sciatic nerve) proteins moving in the slow flow may survive for years without decay; however, experimental evidence indicates that proteins conveyed in the slow flow do decay at the usual rates (10). Other possible explanations include (i) a certain amount of tubulin heretofore undetected is conveyed in the fast flow to replace degraded molecules; (ii) some axoplasmic proteins may have unusually long half-lives; and (iii) a local supply of axonal proteins exists (2, 5, 9). Phrenic axons have sevenfold the axoplasm of abducent axons of the same caliber; the former must convey more material to keep the balance; notwithstanding, microtubule densities were equal. Microtubules seem to relate to axon caliber rather than to transport demand. Several alternative explanations arise: (i) the material that enters the axon is the same, no matter what the mass of the axoplasm; (ii) microtubules largely exceed those required to sustain transport; or (iii) microtubules are not involved in trans- pod (4). In conclusion, it seems that our present knowledge on the maintenance of the neuron is insufficient to account for the constancy of axonal micro- tubules. REFERENCES 1. ALVAREZ J. F. ARFUXIONDO F. ESPFIJO AND V. WILLIAMS. 1982. Regulation of axonal microtubules: effect of sympathetic hyperactivity elicited by reserpine. Neuroscience, in press. 2. ALVAREZ J., AND W. Y. CHEN. 1972. Injection of leucine into a myelinated axon: incor- poration in the axoplasm and transfer to associated cells. Acfa Physiol. Lat. Am. 22: 266- 269. 3. ALVAREZ J., AND B. U. RAMIREZ. 1979. Axonal microtubules: their regulation by the eleCtrical activity of the nerve. Neurosci. Lett. 15: 19-22. 4. BYERS, M. R. 1974. Structural correlates of rapid axonal transport: evidence that micro- tubule may not be directly involved. Brain Res. 7% 97-l 13. 5. EDSTR~M A. 1966. Amino acid incorporation in isolated Mauthner nerve fibre compo- nents. J. Neurochem. 13: 3 15-321.  286 ALVAREZ AND ZAROUR 6. FORGUE, T. S., AND J. L. DAHL. 1978. The turnover rate of tubulin in rat brain. J. Neurochem. 31: 1289-1297. 7. HOFFMAN, P. N., AND R. J. LASEK. 1975. The slow component of axonal transport. Identification of major structural polypcptides of the axon and their generality among mammalian neurones. J. Cell Biol. 66: 35 l-366. 8. KARLSON, J. O., AND J. SJ&TRAND. 197 1. Transport of microtubular protein in axons of retinal ganglion cells. J. Neurochem. 18: 975-982. 9. KOENIG, E. 1967. Synthetic mechanisms in the axon. IV. In vitro incorporation of [‘HI precursors into axonal protein and RNA. .I. Neurochem. 14: 437-446. 10. NIXON, R. A. 1980. Protein degradation in the mouse visual system. I. Degradation of axonally transported and retinal proteins. Brain Res. 200: 69-83.
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