Axonal constriction at Ranvier's node increases during development

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Neuroscience Letters 190 (1995) 159-162

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Axonal constriction at Ranvier's node increases during development C. S w ~ d a,*, C.-H. Berthold a, I. Nilsson-Remahl b, M. R y d m a r k a aSection of Neuroanatomy, Department of Anatomy and Cell Biology, GOteborgs University, Medicinaregatan 3A, S-413 90 GOteborg, Sweden bDepartment of Anatomy, Karolinska lnstitutet, Stockholm, Sweden Received 7 November 1994; accepted 30 March 1995

Abstract

We have studied the ratio between the nodal and the intemodal diameter (the do~dinratio) of large myelinated axons in the L7 ventral spinal root of the cat during pre- and postnatal development using light and electron microscopy. A substantial nodal constriction, do~din= 0.6, was found at the beginning of myelination, about 2 weeks before birth The ratio decreased during the subsequent 10 weeks and approached the adult value of 0.47 (SE 0.01, N= 45) in the 8 weeks old kitten. The observations are discussed with respect to the maturation of the nodal region and to our earlier idea that the constricted nodal axon segments of large peripheral myelinated nerve fibres of adult cats and ki~Ltens2 months and more of age are sites capable of interacting with and perhaps even controlling the passage of axonally transported materials.

Keywords: Axon; Cat; Morphology; Nerve fibre; Ranvier's node; Ultrastructure The diameter of mature peripheral (spinal root level) myelinated axons of the cat is reduced to 50-70% to a length of 8-10/zm at the nodes of Ranvier [1]. The correlation between nodal (dn) and internodal (din) axon diameter is rectilinear with an average dn/din ratio of 0.4 for large fibres in lumbar ventral and dorsal roots, the dorsal root axons showing a more pronounced nodal constriction than the ventral ones [117]. In the living axon, substances and organdies move bidirectionally in the axoplasm [13]. Rheological principles state that flow in a tube depends on the fourth power of the radius. From this it follows that a reduction of the diameter of a tube to 50-70% will diminish the flow to 94-!99%. Although the axoplasm with its cytoskeleton, its built-in force generating machinery and its various membranous organdies is far from the ideal system proposed by the rheological postulate, the presence of nodal conslxictions should be of great importance with regard to axoplasmic transport. We have shown earlier: (i) that axoplasmic organelles under normal conditions amass in the paranode-node-paranode (pnp) regions of mature large ventral and dorsal cat spinal root nerve fibres; and (ii) that the nodal axoplasm in the peripheral (PNS) parts of alpha motor axons arrests and appears to interact with retrogradely transported horserad* Corresponding author.

ish peroxidase (HRP) in a temporally ordered fashion [4,6]. One step in this interaction seems to be the transformation of HRP-containing endosomes and prelysosomes to active lysosomes followed by degradation of arrested materials at the nodes of Ranvier [11]. During postnatal development, retrogradely transported HRP begins to accumulate at the PNS nodes of amotor axons in the 6-woek-old kitten and lysosomal aggregations appear in the 2 months old animal [12]. Since these findings might relate to the degree of nodal constriction during development, we have investigated the dnldin ratio of a-motor axons of the L7 ventral spinal root in three eat foetuses (extracted by caesarean section 47, 50 and 55 days postconceptionally), 8 kittens (newborn, 1, 2, 3, 4, 6, 8 and 16 weeks) and 3 adult cats (0.5, 1, 5 years). The animals were fixed by vascular perfusion with glutaraldehyde (5%). The left I-,7 ventral root of each animal was removed and prepared for light- or electron microscopy using Vestopal W embedding and serial sectioning. Specimens from animals less than 6 weeks of age were examined by electron microscopy and the rest by light microscopy. A detailed preparatory protocol has been presented elsewhere [7]. In each of the 14 series of cross sections one root fascicle containing 200--400 axons was selected and its transverse area photographed at even intervals (for details

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Fig. 1. Schematic drawing of two consecutive nodes of Ranvier with an interrupted internode in between, showing the abbreviations and terms used in the text. Proximal and distal refers to the cell body in the ventral horn. din, internodal diameter, calculated as the mean value of the axonal diameter measured at three well separated sites in the stereotype part of the internodal axon; dMld, axonal diameter at the distal myelin sheath attachment of the proximal pnp-region; dM1p, axonal diameter at the proximal myelin sheath attachment of the proximal pnp-region; dM2d, axonal diameter at the distal myelin sheath attachment of the distal pop-region; dM2p, axonai diameter at the proximal myelin sheath attachment of the distal pnp-region; dnl, axonal diameter at the proximal node of Ranvier; dn2, axonal diameter at the distal node of Ranvier; MySA, myelin sheath attachment; nR, node of Ranvier; pnp, paranode-node-paranode region; stin, stereotype part of the internode. see Ref. [7]). The 10% largest axons of a root fascicle were then identified, traced throughout the series and their longitudinal extension reconstructed noting the presence of nodes o f Ranvier and Schwann cell nuclei. Those axons that could be reconstructed over two consecutive

nodal regions were taken to diameter analysis. Diameter values were in all cases estimated by the use o f a series o f preformed circles of known diameters (stepwise increments of 0.2/xm) [10]. In the case of light micrographs a final magnification of × 5 0 0 0 was used. Electron micrographs were examined at a final magnification of about × 2 0 000. An internodal diameter value (din) was calculated as the mean value of three estimations performed at well separated sites along the stereotype part of an internode. Measurements were evaluated employing standard parametric statistics (2P = 95%). A schematic presentation of the nomenclature and the position of measuring points along a reconstructed axon are given in Fig. 1. The transversal appearance of the axon at the three measuring points in a pnp-region as seen in a fully myelinated axon from the 6-month-old cat and in a just myelinated axon from the 50-day-old foetus is illustrated in Fig. 2 a - c and Fig. 3a-c, respectively. In the adult stage (Fig. 2a-c), the mean value of the dnldin ratios of the three adult animals was 0.47 (SE 0.01, N = 45). The individual mean values were 0.48 (SE 0.01, N = 14), 0.47 (SE 0.01, N = 16) and 0.46 (SE 0.01, N = 15), respectively. This is in good agreement with earlier data from ventral spinal roots [17]. No significant differences were noted between the separate dn/din mean values

Fig. 2. At 6 months: light micrographs from the proximal MySA level (a), the midnodal level (b) and the distal MySA level (c). Magnification x 5000. Fig. 3. At 50 days after conception. Electron micrographs from the corresponding levels (a, b and c) as in Fig. 2. Magnification x 18 725.

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Fig. 4. Axonal constriction, diagram showing the dnldin ratio versus age. At each age an average number of 15 (10-19) fibres/nodecouples have been analyzed. Diamonds, dn/din; circles, dnldin. A mature relationship between nodal and intemodal diameter appears at about 8 weeks (120 days after conception). Note, all animals younger than 8 weeks are significantly above the 95% confidence interval of the mean for adult animals. or when comparing the dn]ldin ratio with the dn2/din ratio pair,vise for individual fibres, separately for each animal. There were no significant differences between the axonal diameters of: (1) the two MySA segments of a pnp-region (dMlpldMld = 1.02, SE 0.02, N = 44; dM2pldM2a = 1.03, SE 0.02, N = 32); (2) the two MySA segments of two consecutive pnp-regions (dMlaldM2 p = 1.03, SE 0.03, N = 32); and (3) two consecutive nodes (dnlldn2 = 1.02, SE 0.03, N = 30). The observation that the dr/dMySA ratios are a little above one is explained by the barrel-like outbulging of the nodal axon segment compared to the more straight cylinder-like MySA-segments [18] (dn/dMysA=l.06, SE 0.01, N = 45). In the developing stage (Fig. 3a--c), the mean dr~din ratio of the 47-, 50- and 55-day-old foetuses were 0.63 (SE 0.02, N = 13), 0.55 (SE 0.01, N = 17) and 0.65 (SE 0.02, N = 1 0 ) , respectively. These values are significantly higher than the value noted in the mature animals. The mean value of the pooled dr/di, ratios of the foetuses and the newborn was 0.62 (SE 0.01, N = 53). The progression of the dr~din ratio with age is illustrated in Fig. 4. The adult dn/din ratio of 0.4.7 is first found at the end. of the second month. Comparisons between dn and dNySA values of consecutive nodes showed almost identical results as for the adult animals. These results demonstrate that: (i) the axon transverse area of future large myelinated ventral root nerve fibres is locally reduced to 50% at the nodes of Ranvier at the very beginning of myelination 2-3 weeks before birth; (ii) this degree of constriction then remains fairly constant for about a month, which is during the period when din increases x2, myelin sheath thickness ×5 and internodal length x 3 [16]; and (iii) the nodal constriction during the subsequent 6 weeks increases gradually and gives the

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mature dr~din ratio of about 0.47 at the end of the second postnatal month, i.e. the transversal area of the nodal axon has been reduced to 22% of its internodal value. The establishment of the adult dr/din ratio coincides with the final steps in the maturation of the pnp-region and should hence be included as a part of the so-called nodalization process. The nodalization process is the rather complex structural remodelling which, in prospective large feline spinal root nerve fibers, begins when myelination starts and ends in the second postnatal month as the fibres have developed their final numbers of nodes of Ranvier equipped with mature node-paranode apparatus [2,3,5,7,21] and show adult electrophysiological properties [8,19,20]. Observations during postnatal development [9] have shown that intramuscularly administered HRP can be found aggregated at nodes of Ranvier first in the 6-weekold kitten and that the adult ordered response to retrograde HRP transport through the pnp-regions appears between the 4th and 6th postnatal months (unpublished observations). The ability to accumulate lysosomes at the nodes during retrograde HRP transport in a way similar to that seen in adult animals is found at the end of the second month [12]. Our present observations thus support the idea that there is a link between the postnatally occurring accentuation of the nodal constriction and the ability of the mature pnp-region to interact with axoplasmic organelles. Some additional and so far unknown factors that enable the fully mature response seem, however, to develop later on. In effect the immature pnp-region appears to be transparent and more or less non-discriminative to retrogradely transported materials, qualities that during the second postnatal month change into their opposites. This assumed early postnatal disability of the pnp-regions of large peripheral axons to interfere with the passage of axonally transported materials, may have both positive and negative effects. For instance trophic factors which are endocytotically imbibed at the axon terminals and transported retrogradely in the axon to the soma can pass freely through the nodal regions during development (for a review, see Ref. [22]). On the other hand, retrograde axonal transport of harmful agents like herpes simplex virus (HSV) from a primary site in the PNS to the CNS would be facilitated by the immature state at the pnpregions [15]. Thus in the mouse the spreading of virus in peripheral nerves seems to some extent to be a function of age, resistance increasing rapidly with maturation. Especially severe, disseminated infection does appear in newborn and premature children when infected with HSV [14]. This investigation was supported by the Swedish Medical Research Council (Project No. 3157). [1] Berthold, C.-H., Ultrastructure of the node-paranode region of

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