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Calibre and Microtubule Content of the Non-Medullated and Myelinated Domains of Optic Nerve Axons of Rats

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Calibres and microtubule contents of the non-medullated and myelinated domains of optic nerve axons of adult rats were studied with the electron microscope. The cross-sectional areas of the non-medullated domain was 0.25 μm2, and that of the
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  European Journal zyxwvusrq f Neuroscience Vol. I pp. 654-658. @European Neuroscience Association zy 953 866x189 3.00 z Calibre and Microtubule Content of the Non-Medullated and Myelinated Domains of Optic Nerve Axons of Rats Carolina Hernandez, Eileen Blackburn, and Jaime Alvarez Unidad de Neurobiologia Molecular, Facultad de Ciencias Biologicas, P. Universidad Catolica. Santiago, Chile Key words: rat, axonal cytoskeleton, slow axomplasmic transport, morphometry Abstract Calibres and microtubule contents of the non-medullated and myelinated domains of optic nerve axons of adult rats were studied with the electron microscope. The cross-sectional areas of the non-medullated domain was 0.25 pm2, and that of the myelinated domain 0.40 pm2, that is, greater by zyx 9 . The increase in size was uneven across the axonal population; it was marked in fine and medium sized axons, and modest in the largest axons. The number of microtubules increased with axonal size; the density, however, decreased from 85 mirotubules/pm2 in 0.1 pm2 axons to about 20 in 1.2 pm2 axons. In axons of equal cross sectional area, the microtubular density of the myelinated and non-medullated domains was the same. Microtubular density values of optic axons resemble those of dorsal roots more than those of peripheral nerve axons of equal calibre. The facts that optic axons increase in size and gain microtubules behind the eyeball while the microtubular packing decreases suggest a local regulation of the axonal cytoskeleton. Introduction zyxwvutsrqpo yelinated and non-medullated axons exhibit an inverse correlation between microtubular packing and calibre. This holds for cat, rat, frog, and lizard (Friede and Samorajski, 1970; Smith, 1973; Pannese et al., 1984; Fadic et al., 1985; zyxwvutsrq spejo and Alvarez, 1986; Parhad et al., 1987). In axons of equal size, microtubular density values are similar in motor, sensory, and sympathetic fibres (Alvarez et al., 1982; Alvarez and Zarour, 1983; Fadic et al., 1985; Fadic and Alvarez, 1986). Densities vary over one order of magnitude. In the cat, small non- medullated fibres (0.1 pm2) of the sural nerve have over 100 microtubules/pm* and 10 pm myelinated axons (cross-sectional area c. 80 am2) have only 12 microtubules/pm2 (Fadic et al., 1985). In dorsal root fibres, the microtubular density also exhibits an inverse correlation with the axonal calibre, but density values are one-half to one-third those of peripheral nerve fibres of equal calibre (Fddic et al., 1985). Consequently, a continuous sensory axon exhibits one pattern of microtubular packing peripheral to the ganglion and another centrally despite both parts being branches of a common stem axon. Whether the radicular pattern of microtubular packing is unique or shared by other fibres is still unknown. In a peripheral nerve, the calibre spectra of myelinated and non- medullated axons have a minimal overlap. In two instances, dorsal roots of lizards (Pannese et al., 1981) and sural nerves of developing rats (Faundez and Alvarez, 1986), the microtubular density of myelinated and non-medullated axons have been reported to be similar in the small range where calibres overlap. It remains unknown if the conspicuous low microtubular density of myelinated fibres is due to their myelinated condition or only to their large size. The aims of the present study were as follows: (i) to establish if the microtubular density-axonal calibre correlation of central axons differs from that reported for peripheral axons; (ii) to determine if myelination influences the packing of microtubules; and (iii) to determine if the microtubular packing varies along the course of a continuous unbranched fibre. The axons emerging from the ganglion cells of the retina suit these purposes since they are axons of the CNS and they have also a sizeable non-medullated domain followed by a myelinated one. Materials and Methods Albino Sprague-Dawley male rats weighing 200 g were used. Under pentobarbitone anaesthesia (30 mglkg-', i.p.) the heart was exposed and a cannula was inserted into the left ventricle. The descending aorta was clamped. After a short rinse with saline, warmed to 38 C, to remove the blood, fixative, 1 glutaraldehyde, 2% (p)formaldehyde, 0.05 picric acid, phosphate buffer, pH 7.4, 0.1 M, also at 38°C (cf. Ohnishi et al., 1979), was perfused for over 40 min. During this period the animal was maintained at 39°C in a stove to avoid cooling of the body. The eyeballs with their attached nerves were removed. The optic nerves including the optic disks were dissected out and left overnight in the same fixative. Optic nerves were cut lengthwise, one end being the optic disk. After a rinse in buffer, the tissue was treated with 1 osmium tetroxide for 1.5 h, 2 uranyl acetate for 1 h, dehydrated in acetone, and embedded in Epon in the standard manner. Blocks were produced to have a sample at about 2 mm from the eyeball (Fig. 1, Correspondence ro: Jaime Alvarez, Unidad de Neurobiologia Molecular, Facultad de Ciencias Biologicas, P. Universidad Catolica, Casilla 1 14-D, Santiago, Chile Received I April 1989, revised 8 June 1989, accepted 12 June 1989  Non-medullated and myelinated optic axons 655 the optic nerve (De Juan et zyx I. 1978; Reese, 1987); no attempt was made to survey systematically the whole optic nerve. Microtubules and cross-sectional areas of axons were assessed in micrographs taken at z   10 zyxwv OO and printed at zyx 20 OOO as reported elsewhere (Fadic et al., 1985). M); this we will denote the ‘myelinated sample’ despite the fact that a small group of non-medullated fibres was present. The other piece of optic nerve was oriented to produce sections from the myelinated region of the nerve toward the optic disk in order to approach the non- medullated domain of the optic fibres from the rear (Fig. zyxwvuts   NM); this we will denote the ‘non-medullated sample’. Thin sections were double stained with uranyl acetate and lead citrate, and examined under the electron microscope. A stage calibration was photographed in every session. With regard to calibre, axons are randomly distributed across EB \\ n zyxwvu ” IG. 1. Survey of the optic nerve axons. EB, eyeball; OD, optic disk; ON, optic nerve. Arrows indicate samples. Just behind the eyeball axons run parallel and are all still non-medullated (NM). At about 2 mm away from the eyeball, over 90 of axons are myelinated (M). Cohort zyxw nalysis Since optic axons are continuous, any axon at one sampling site corresponds to another at a second site (Fig. 1). If it is assumed that the rank of axons with respect to calibre does not change, the description of axons of the same rank at the two sampling sites represents the description of axons along their course. In practice, samples are sorted by size and both broken in the same way. In the present case, we found 6.5% of non-medullated axons in the myelinated sample (Fig. 1, M) (cf. Foster et al., 1982; Hunter and Bedi, 1986; Cuenca et al., 1987). These were assumed to be continuous with the smallest axons of the purely non-medullated population (Fig. 1, NM). Thus 6.5% of the non- medullated sample was removed at the left end of the distribution, and the remainder of this sample and the myelinated sample were broken down into ten cohorts each. Cohorts of the same ordinal are assumed to be axons of the same rank and hence continuous between sampling sites. Results The tissue appeared well preserved (Fig. 2A,B). The non-medullated sample had no admixture of myelinated fibres. At 2 mm from the FIG. 2. Optic nerve. A, just behind the eyeball and B, 2 mm away. Note that all axons are non-medullated at A, while only a few remain so at B, two of which are indicated *). Cal. 0 5 pm.  656 Non-medullated and myelinated optic axons eyeball, all axons were apparently myelinated but on close inspection non-medullated fibres could be recognized here and there (Fig. 2B). The mean cross-sectional area of the non-medullated fibres was 0.25 pm2 and that of myelinated axons was 59% greater (Table 1). The calibre spectrum of non-medullated and myelinated axons are shown in Figure 3. Distributions are similar although, as expected, fine axons are more frequent in the non-medullated population. The number of microtubules per axon increased by 36 when the optic axon became myelinated behind the eyeball. zyxwvut n spite of this, the average microtubular density of the myelinated domain was smaller than that of the non-medullated domain (Table zyxwvut  . When number of microtubules and density are referred to axons of equal calibre, values of both domains are similar (Fig. zyxwvutsrq ; Table 2). Densities range from about 85 microtubules/pm2 for small axons (0.1 zyxwvuts m2 to about 20 for large ones (1.2 pm2) (Fig. 4B; able 2). The description of individual axons along their course cannot be done at the present time. Nevertheless, a cohort analysis is satisfactory for such purpose (see Materials and Methods). The cross-sectional area of all non-medullated axons increases to the same extent c. 7 1 ) as TABLE . Cross-sectional area, number of microtubules and microtubular density of the non-medullated and myelinated domains of optic nerve axons. (xfSEM). Domain of axon Non-medullated Myelinated ~~~ ~~ Area zyxwvutsrqp pm2) 0.25 zyxwvut .04 0.40 0.03* 59 Diameter (pm) 0.52 0.03 0.68 0.03 31 Microtubules 9.60 1.10 13 10 0.60 36 Density 57.60 8.60 39.30 2.90 -31 Group of four rats. In each animal, about 200 axons were measured at each domain. , percentage of variation of the myelinated domain referred tn the non-meddlated domain. Diameter was computed using the cross-sectional area of each axon as datum. Density is in microtubules/pm2. *p < 0.05. 40 20 al zyxwvutsrqpon   0 Y al Q. 10 cross sectional area (pm*l FIG. 3. Histograms of optic nerve axons. Thin line, non-medullated domain. Thick line, myelinated domain. Note that the distribution of calibres shifts to the right upon myelination of axons. Each sample includes over 800 axons. they become myelinated, except for the largest ones that increase only 18% (Fig. 5A; Table 3). Upon melination, the microtubular content increases but the microtubular packing decreases in all but the largest axons, where virtually no change is observed (Fig. 5B,C; Table 3). Discussion The axons emerging from the ganglion cells of the retina at first have a non-medullated trajectory and become myelinated only behind the eyeball. Their calibres increase substantially upon myelination. The converse has been observed in dorsal root fibres, that is, the calibre decreases when the fibre loses its myelin (Pannese et al., 1988). In culture, axons of sensory neurons do not grow beyond a diameter of 1.25 pm unless glial or Schwann cells wrap the axons with myelin (Windebank et al., 1985). These observations support the notion that in zyxwvutsr  vo the axonal calibre is locally specified, partially, by the associated glial cells. Calibres of the non-medullated and myelinated domains of optic axons overlap over the whole range, and the microtubular density-axonal A 2 301 h Z 30- E c 20- L V . 2 10- E 111111111 2 10 E2 1 .2 A .6 .8 1.0 1.2 1.4 cross sectional B 1111111111111 .2 .4 .6 .8 1.0 1.2 1.4 area pm2) FIG. 4. Microtubular content of optic axons. A number of microtubules and B, microtubular density. Open circles, non-medullated domain and filled circles, myelinated domain. Note that the correlation of the microtubular content with the size of axons is not affected by myelination. For additional information, see Table 3.  Non-rnedullated and rnyelinated optic axons 657 zyx   zyxwvut   zyxwvutsr   zyxwvu u 0 VI VI . zyxwvutsr 4 VI .2- 2 U 1.0 N E zyxwvut   a b a, + IIIIItII TABLE . Microtubules and microtubular density of the non-medullated and myelinated domains of optic nerve axons, referred to defined axonal sizes. Area 0.0-0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 o -1.1 -1.2 -1.3 -1.4 ~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA   .4- Non-medullated domain n Microt. Density n 153 6.1 zyxwvuts   .2 83.9 3.0 17 329 8 5 0.2 59.3 1.5 162 189 11.2 0.4 45.8 1.6 265 68 11.4 .6 33.5 1.9 179 44 12.7 .9 28.9 2.1 127 24 12.2 .1 22.5 2.1 97 9 19.7 4.3 30.5 6.6 71 1 12.0 15.6 23 7 27.3 6.2 31.8 7.2 16 7 22.4 6.6 23.6 7.2 9 6 26.0 .5 24.8 7.1 9 3 17.0 2.5 14.8 2.0 7 5 20.2 .6 16.2 3.7 5 0 3 6 8 ~ ~~~ Myelinated domain Microt. Density ~~ 7.2 .5 89.4 8.7 9.3 0.2 59.7 1.5 10.6 0.2 42.9 0.9 12.5 0.3 36.1 0.9 14.5 0.5 32.4 1.0 15.2 0.6 28.1 1.0 17.2 0.7 26.8 1.1 17.3 1.2 23.1 1.6 19.5 2.4 23.2 2.9 20.7 2.3 21.7 2.5 22.0 2.9 21.1 2.8 30.3 4.9 26.0 4.3 26.6 4.4 21.0 3.6 23.7 1.4 17.6 0.8 Area is in pm2; n, axons of the interval; density is in microtubuleslpm2. Values are x*SEM. Last line, axons greater than 1.4 pm2. A B 4 6 8 10 cohort C E a 0- 40 . 30- E \ VI aJ L I,; 2 zyxwvut   6 8 10 cohort FIG. . Cohort analysis of optic axon. A, calibre; B, microtubules; and C, microtubular density. Thin line, non-medullated domain; thick line, myelinated domain. Cohorts of the same ordinal represents the same axons with and without myelin (see Materials and Methods). Upon myelination, calibre and microtubular content of axons increase but the packing of microtubules decreases.  658 Non-medullated and myelinated optic axons TABLE zyxwvutsrqpo . Modification of area, number of microtubules, and microtubular density in the myelined domain of optic nerve axons. Cohort zyxwvutsrqp   2 3 Area 60.2 78.1 80.2 77.9 74.1 75.0 73.0 72.3 56.2 17.9 zyxwvutsrqp ~ Microtubules 40.6 38.3 24.0 26.1 24.3 29.8 29.8 24.5 40.7 19.4 Density -9.0 -22.3 -31.1 -29.1 -28.7 -25.7 -28.4 -27.8 0.6 -3.6 For construction of cohorts, see Materials and Methods. Size of axons increases with the ordinal of the cohort. Figures represent the change of morphometric values for the cohort in the myelinated domain expressed as percentage of the corresponding value in the non- medullated domain. calibre correlation is the same for both domains. This fact strongly suggests that the myelin sheath plays no role in the specification of the microtubular density of axons. Nevertheless, when an optic nerve axon becomes myelinated, the number of microtubules does increase while their packing decreases to accord with the characteristic values for its new calibre. Therefore, number and packing of microtubules vary along the course of a given axon. This suggests that the microtubular content and, by extension, the axoplasmic matrix are locally regulated. The same has been proposed for peripheral axons (Serra and Alvarez, 1989). It is currently accepted that the cell body is the sole source of neuronal proteins and that the cytoskeletal components move coherently along the axon (Lasek et al., 1983). This view has been challenged (Alvarez and Zarour, 1983; Bamburg, 1988; von Bernhardi and Alvarez, 1989), and even the reality of the slow transport has been questioned (Alvarez and Torres, 1985). The mild increase of microtubules in the myelinated domain of optic axons and the 10-fold increase observed when a stem motor axon develops its terminal arborization (Zenker and Hohberg, 1973) cannot be explained using the slow transport hypothesis (unless unsupported assumptions are made) since it does not provide for en route increase of transported proteins. Finally, the pattern of the microtubular packing of optic nerve axons deserves a comment. In the cat, central processes of sensory neurons have density values one-half to one-third that of peripheral fibres of equal calibre (Fadic et al., 1985; Serra and Alvarez, 1989). In the rat, microtubular densities of optic nerve axons resemble more the pattern of dorsal root axons (Lbpez and Alvarez, unpublished) than that of sural nerve axons (Espejo and Alvarez, 1986; Saitua and Alvarez, 1988). These obervations suggest that radicular axons and some axons of the CNS share a common pattern of microtubular packing. Other central axons must be examined to determine whether this microtubular pattern prevails in the CNS. In conclusion, present findings support the notion that myelination does influence calibre of axons and number, but not packing, of microtubules. We propose that the axoskeleton is locally regulated. Acknowledgements C. H. and E. B. were recipients of a research scholarship of the Escuela de Medicina, Universidad Catdlica. We thank Mrs M6nica PCrez, Mrs Gloria Mkndez, and Mr Raul Fuentes for technical assistance. This research was supported by Direccidn de Investigaciones de la Universidad Cat6lica and FONDECYT. References Alvarez, J., Arrendondo, F., Espejo, F., and Williams, V. 1982) Regulation of axonal microtubules: effect of sympathetic hyperactivity elicited by reserpine. Neuroscience 7: 2551 -2559. Alvarez, J. and Torres, J. C. 1985) Slow axoplasmic transport: a fiction? J. Theor. Biol. 112: 627-651. Alvarez, J. and Zarour, J. 1983) Microtubules in short and in long axons of the same caliber: implications for the maintenance of the neuron. Exp. Neurol. 79: 283-286. Bamburg, J. R. 1988) The axonal cytoskeleton: stationary or moving matrix? TINS 11: 248-249. von Bernhardi, R. and Alvarez, J. 1989) Is the supply of axoplasmic proteins a burden for the cell body? Morphometry of sensory neurons and amino acid incorporation into their cell bodies. Brain Res. 478: 301 -308. Cuenca, N., Fernindez, E., De Juan, J., Carreres, J., and Iiiiguez, C. 1987) Postnatal development of microtubules and neurofilaments in the rat optic nerve: a quantitative study. J. Comp. Neurol. 263: 613-617. De Juan, J., Iiiiguez, C., and Carreres, J. 1978) Number, diameters and distribution of the rat optic nerve fibers. Acta Anat. 102: 294-299. Espejo, F. and Alvarez, J. 1986) Microtubules and calibers in normal and regenerating axons of the sural nerve of the rat. J. Comp. Neurol. 250: 65 -72. Fadic, R. and Alvarez, J. 1985) Caliber and microtubules of sympathetic axons are not subject to trophic control by the preganglionic nerve. Exp. Neurol. Fadic R., Vergara, J., and Alvarez, J. 1985) Microtubules and caliber of central and peripheral processes of sensory axons. J. Comp. Neurol. 236: 258-264. Faundez, V. and Alvarez, J. 1986) Microtubules and calibers in developing axons. J. Comp. Neurol. 250: 73-80. Foster, R. E., Connors, B. W., and Waxman, S. zyx   1982) Rat optic nerve: electrophysiological, pharmacological and anatomical studies during development. Dev. Brain. Res. 3: 371 -386. Hunter, A. and Bedi, K. S. 1986) A quantitative morphological study of interstrain variation in the developing rat optic nerve. J. Comp. Neurol. 245: Lasek, R. J., Oblinger, M. M., and Drake, P. F. 1983) Molecular biology of neuronal geometry: Expression of neurofilament genes influences axonal diameter. Cold Spring Harbor Symp. Quant. Biol. 48: 731 -744. Ohnishi, A,, O’Brien, P. C., and Dyck, P. J. 1976) Studies to improve fixation of human nerves. V. Effect of temperature, fixative and CaClz on density of microtubules and neurofilaments. J. Neuropathol. Exp. Neurol. 35: Pannese, E., Ledda, M., Arcidiacono, zyx ., igamonti, L., and Procacci, P. 1984) Density and distribution of microtubules in the axons of the lizard dorsal root. J. Submicrosc. Cytol. 13: 169-181. Pannese, E., Ledda, M., Arcidiacono, G., igamonti, L., and Procacci, P. 1984) A comparison of the density of microtubules in the central and peripheral axonal branches of the pseudounipolar neurons of lizard spinal ganglia. Anat. Rec. 208: 595-605. Pannese, E., Ledda, M., and Matsuda, S. 1988) Nerve fibres with myelinated and unmyelinated portions in dorsal spinal roots. J. Neurocytol. 17: 693-700. Parhad, 1. M., Clark, A. W., and Griffin, J. W. 1987) Effect of changes in neurofilament content on caliber of small axons: The zy  /3’-iminodipropionitrile model. J. Neurosci. 7: 2256-2263. Reese. B. E. 1987) The distribution of axons according to diameter in the optic nerve and optic tract of the rat. Neuroscience 22: 1015- 1024. Saitua, F. and Alvarez, J 1988) Do axons grow during adulthood? A study of caliber and microtubules of sural nerve axons in young, mature, and aging rats. J. Comp. Neurol. 269: 203-209. Serra. M. and Alvarez, J. 1989) On the asymmetry of the primary branching of vagal sensory axons: possible role of the supporting tissue. J. Comp. Neurol. 284: 108- 118. Smith, R. 1973) Microtubule and neurofilament densities in amphibian spinal root nerve fibers: Relationship to axoplasmic transport. Can. J. Physiol. Pharmacol 5 I : 798 06. Windebank, A. J., Wood, P., Bunge, R. P., and Dyck, P. J. 1985) Myelination determines the caliber of dorsal root ganglion neurons in culture. J. Neurosci. Zenker. W. and Hohberg, E. 1973) A-a-nerve-fibre: number of neurotubules in the stem fibre and in the terminal branches. J. 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