131
J. Anat. (1990), 170, pp. 131-137 With 2 figures Printed in Great Britain
High density of nodes of Ranvier in the CNS-PNS transitional zone J. P. FRAHER AND D. C. BRISTOL
Department of Anatomy, University College, Cork, Ireland
(Accepted 21 November 1989) INTRODUCTION
Having left their parent somata in the spinal grey matter, ventral motoneuron axons are grouped into bundles (Fig. 1). Where these traverse the ventrolateral funiculus of the spinal cord they are termed intramedullary rootlets. They emerge into the peripheral nervous system (PNS) as ventral rootlets which eventually unite distally to form the ventral root. Close to its emergence into the PNS, each rootlet contains a transitional zone (TZ), which is defined as that length of rootlet which contains both peripheral and central nervous tissue (Fig. 1). Various accounts have been given of spinal nerve TZs in man (Tarlov, 1937), cat (Berthold & Carlstedt, 1977; Carlstedt, (a)
(b)
DR
+
A
VR8B.....
Fig. 1. (a-b). (a) Diagrammatic transverse section through lumbar spinal cord showing ventral motoneuron somata (arrows) and their intramedullary axon bundles sectioned obliquely (arrowheads) as they run caudally and ventrally through the ventrolateral white funiculus (VWF). AB, plane of oblique longitudinal section (b); VR ventral root; DR, dorsal root. (b) Diagrammatic longitudinal section in Plane AB indicated in (a) showing three representative myelinated ventral motoneuron axons of an intramedullary rootlet (IMR), traversing the transitional zone (TZ) and running in a ventral rootlet (VR) after emerging into the PNS. The TZ of each rootlet contains a central tissue projection (CTP) (shaded). Spinal grey matter, crosses; nodes, white.
J. P. FRAHER AND D. C. BRISTOL 132 1981) and rat (Fraher, 1978a; Fraher & Kaar, 1986). In the rat, central tissue forms a distally tapering projection into each lumbar ventral rootlet. Consequently, the TZ lies in the most proximal part of the rootlet. The rat lumbar TZ is relatively short, averaging 160 ,m or less in length. Since each myelinated fibre possesses a transitional node of Ranvier as it traverses the TZ (Fraher, 1978 a; Fraher & Kaar, 1984, 1986; Bristol & Fraher, 1987), it is possible that the density of transitional nodes within the zone is particularly high. Intramedullary rootlets contain relatively short internodes (Fraher, 1978 b) and their node densities are therefore also likely to be high. However, no quantitative data are available on these points. The aim of this study was to compare node density at the TZ with that in the central and in the peripheral parts of the nervous system. Absolute density was calculated for three different levels along ventral motoneuron axon bundles: at the TZ, in the intramedullary rootlet and in the ventral root. Values for the three levels were compared, to determine if, and to what extent, density differences existed. High node density at the TZ would raise the possibility of possible electrical interaction between myelinated fibres in vivo. MATERIALS AND METHODS
Five Wistar albino rats were anaesthetised with a 1: 3 chloroform: ether mixture and killed by intravascular perfusion of fixative (2 % glutaraldehyde and 2 5 % paraformaldehyde in an orthophosphate buffer at a pH of 7-2-74) as described previously (Fraher & Kaar, 1984). Supplementary fixation was carried out by irrigating the spinal subarachnoid space with the fixative (Kaar, O'Sullivan & Fraher, 1983). The L4 spinal cord segment and its attached dorsal and ventral roots and spinal ganglia were exposed, removed and processed for transmission electron microscopy (Fraher & Kaar, 1984). Specimen blocks of the distal ventral root, transitional zone and intramedullary rootlet (Fig. 1) were prepared from each animal. Alternating sequential series of thick (05,um) and ultrathin (100 nm) transverse sections of randomly chosen individual fibre bundles at each of these levels were cut on a previously calibrated Reichert OMU4 Ultracut-E ultramicrotome. Thick sections were stained with toluidine blue and were photographed using a Reichert Polyvar photomicroscope. They were printed at a final magnification of x 1300. Serial photomicrographs of every twelfth thick section over the entire length of TZ were used for tracing fibres, locating nodes and measuring fibre bundle cross-sectional area. Node density was calculated in each of 25 ventral rootlets. TZ cross-sectional area was measured from the photomicrographs using a Kontron Mini-IPS automatic image analyser. Cross-sectional area varied somewhat between serially adjacent TZ levels. Accordingly, the volume of the segment between each pair of adjacent levels of section photographed was estimated as that of a solid conic segment (Documenta Geigy, 1962), the height of which was the sum of the thicknesses of intervening thick and ultrathin sections. The total volume of the TZ was calculated as the sum of the volumes of all conic segments comprising it. Since each fibre traversing the TZ possesses a node of Ranvier (Fraher & Kaar, 1984) the total number of nodes in each TZ is the same as that of its constituent fibres. This was confirmed by following the fibres on serial sections (see below). Node density was calculated as the number of nodes per unit volume of the TZ and was expressed as nodes per 105,um3 (Table 1). The node density of 25 intramedullary rootlets was determined as follows. For each rootlet, the cross-sectional area was measured on photomicrographs of every sixth serial transverse section using the Kontron Mini-IPS image analyser. Intramedullary
Node density
133
Fig. 2 (a-b). Photomicrographs of sections through intramedullary rootlets (outlined) showing (a) central nervous fibres (arrows) deeply indenting the rootlet and (b) glial tissue (arrowheads) projecting between rootlet fibres. *, obliquely sectioned blood vessel in spinal cord, continuous with pial vessels. x 800.
rootlets are generally compact on cross-section but are in some cases deeply indented by central nervous tissue (Fig. 2a). However, they are not delineated sharply from surrounding central nervous tissue by any clearly defined barrier. Bundles were followed by numbering all constituent fibres at the TZ and tracing these centrally on serial photomicrographs as far as the surface of the ventral horn grey matter. The cross-sectional area of the intramedullary rootlet was defined as the sum of the areas of all such fibres, including their myelin sheaths. Cell nuclei and perikarya apposed to the bundle were not included in cross-sectional area calculation because both astrocytes and oligodendrocytes contribute in varying degrees to both the intramedullary rootlet and the surrounding central nervous tissue (Fig. 2b). Blood vessels were also excluded in area calculation. The volume of the length of intramedullary rootlet studied was calculated as for that of the TZ, given above. The total number of nodes in each length of rootlet studied was determined by examining the serial transverse sections in the photomicroscope. The node density was calculated as the number of nodes per 105 u,m3 of the intramedullary rootlet (Table 1). Because some astrocytic and oligodendrocytic tissue which contributed to the intramedullary bundle was excluded in measuring cross-sectional area, the volume calculated for the intramedullary bundle was a minimum figure. The value obtained for node density was therefore a maximum figure which overestimated the true density.
134
J. P. FRAHER AND D. C. BRISTOL
Table 1. Mean node density (± S.E.M) at the transitional zones (TZ), in the intramedullary bundles (IMB) and ventral roots (VR) of each of 5 rats Ratios between densities are also shown. Overall mean values (±S.E.M.) are also given. Node density (±S.E.M.)
I'm3) (nodes/105 Rat no.
TZ*
IMB*
Ratios VRt
TZ/IMB TZ/VR
100 2-8 20-0+5-6 7-1 + 1 4 2-0+0-4 4-4 1-6 16-2+2-4 10-3 + 1-6 3-7+0-6 6-1 1-6 10-3+4-6 6-4+11 17+0-2 11-8 35 10-6+4-7 3 0+0 7 0-9+0-1 6-8 1*8 11-6+5 2 6-4+2 5 1-7+0 3 7-8 2-3 Overall 13-8+1 5 6-6+0-8 2-0+0-2 * Mean of 5 values for each rat. t Mean of 7 values for rats 1 to 4 and of 8 values for rat 5. 1 2 3 4 5
Nerve fibres in the distal ventral root, immediately proximal to its junction with the dorsal root ganglion, form clearly defined fascicles, each bounded by a perineurial sheath. The node density in 54-60 ,um lengths of 36 different fascicles (7 from each of 4 animals and 8 from the fifth) was calculated from the volume and the total number of nodes in each, in a manner similar to that described above, using every sixth serial transverse section (Table 1). The overall mean node density in each of the three regions examined was statistically compared with that in the other two, using a t test for comparing the means of two small samples of unknown variances (Bailey, 1959). RESULTS
Node density (Table 1) within the TZ as a whole averaged 13-8 nodes per 105 #m3. It varied considerably both within and between animals. There was a sevenfold (4 9-35 9) range between the smallest and largest values observed. Nodes were most closely packed in the smallest rootlets. In the intramedullary rootlets, node density (averaging 6-6 nodes per 105 /Zm3) was a maximum value because of the method of measurement (see Materials and Methods). Considerable variation in density (range: 1 1-1 56 nodes/ I05 ,tm3) occurred between rootlets within individual animals. In the distal ventral root, mean node density was 2-0 per 105 ,tm3. Again there was considerable variation both within and between animals (range: 0 7-6-5). Node density in the transitional zone averaged more than twice that calculated for the intramedullary rootlet and nearly eight times that for the ventral root (Table 1). These differences were statistically very significant (P < 0001). Density was very significantly greater in the intramedullary bundles than in the ventral root. DISCUSSION
The greatest node density found was in the TZ. This is because all fibres possess a node as they traverse its short length. Transitional nodes are therefore, to a considerable extent, in register with one another. No such restrictions on node distribution apply along fibre bundles centrally or peripherally. The greater node density in the TZ compared to that in the intramedullary rootlet
Node
density
135
is likely to be an underestimate. This is because all transitional nodes are distributed within an irregularly tapering central tissue projection (CTP) (Fraher & Kaar, 1986) which comprises about half the volume of the TZ (Bristol & Fraher, 1987). Furthermore, the method used overestimates intramedullary node density because some astrocytic and oligodendrocytic material was excluded in calculating intramedullary bundle volume. For these reasons it is likely that node density in the TZ averages at least five times that'in the intramedullary rootlet. The findings of this study suggest that, if node densities in the intramedullary rootlet and in the ventral root resemble values generally occurring in the central nervous system (CNS) and PNS, respectively, the possibility of electrical interaction between myelinated fibres is greater in the TZ than in any other part of the nervous system. Furthermore, since the highest density values were found in the smallest rootlets, any interaction is likely to be greatest in these. Stampfli (1954) suggested that individual fibres with closely related nodes may influence one another's activity. Subthreshold electrical interaction has been experimentally demonstrated between myelinated fibres in mammals (Blair & Erlanger, 1932; Rosenblueth, 1941, 1944) and amphibia (Marazzi & Lorente de No, 1944). Such interaction may be of significance in the functioning of motoneuron axon bundles as they traverse the central-peripheral nervous boundary: Waxman (1972, 1975) proposed that nerve fibres may not function solely as transmission lines as described in classical neurophysiology, but may perform a 'multiplex' role in neuronal signalling and different arrangements of fibres may function as delay lines and filters by modification of the conduction velocity and amplitude of the signals that they transmit. However, any tendency for fibre interaction at the TZ is likely to be offset to some degree by the presence of large numbers of concentrically arranged astrocyte processes surrounding the peripheral paranodal and nodal segments of motoneuron axons (Fraher, 1978a). These may serve to insulate transitional nodes more effectively than the less elaborately arranged astrocyte processes related to central nodes. The morphology of the rat L4 ventral rootlet transitional zones shows them to be very well suited for studying possible electrical interaction between fibres with closely packed nodes, for the following reasons. Firstly, node density is likely to be considerably higher than that found elsewhere, particularly in the PNS. Secondly, the TZs are readily accessible as they lie in small, discrete rootlets, superficial to the cord surface (Kaar & Fraher, 1986, 1987). Central bundles lack such delineation and their component fibres intermingle with those of other bundles having different origins, courses, distribution and functions. Cervical TZs are less accessible because they lie at, or just deep to, the cord surface (Fraher, 1978 a). Thirdly, L4 ventral rootlet TZs have the advantage over those of dorsal rootlets of containing a relatively pure population of myelinated fibres of a single functional class. They consist very largely of myelinated motoneuron axons, there being no autonomic outflow at the L4 level (Coggeshall, Emery, Ito & Maynard, 1977). Of the small number of sensory axons which they may contain, only a few are myelinated (Loeb, 1976; Coggeshall et al. 1977; Coggeshall, 1986; Kim, Shin & Chung, 1987). Furthermore, dorsal rootlet TZs (Fraher & Sheehan, 1987) are larger and less clearly delineated from one another than those of ventral rootlets. Fourthly, the muscles innervated through individual rootlets may be identified electrophysiologically or by tracer methods and so studies of transitional node function and muscle excitation may be readily combined.
136
J. P. FRAHER AND D. C. BRISTOL SUMMARY
Node of Ranvier density was examined at three levels along rat lumbar motoneuron axon bundles: where they lie in the central nervous system, in the peripheral nervous system and in the transitional zone (TZ) between these. Density was considerably and significantly greater in the TZ than in either of the other locations. It is possible that such densely packed nodes in the TZ could interact electrically with one another. Because of its structure and position and because it contains a relatively pure fibre population, the rat L4 ventral rootlet TZ lends itself readily to electrophysiological investigation of this possibility. The work presented here was supported by grants from the Health Research Board of Ireland and the Wellcome Trust. The authors are grateful to Ms B. Rea for her technical assistance. REFERENCES BAILEY, N. T. J. (1959). Statistical Methods in Biology. London: The English Universities Press. BERTHOLD, C.-H. & CARLSTEDT, T. (1977). Observations on the morphology at the transition between the peripheral and central nervous system in the cat. II. General organisation of the transitional region in Si dorsal rootlets. Acta physiologica scandinavica, Suppl. 466, 23-42. BLAIR, E. A. & ERLANGER, J. (1932). On the effects of polarization of nerve fibres by extrinsic action potentials. American Journal of Physiology 101, 559-564. BRISTOL, D. C. & FRAHER, J. P. (1987). A morphometric study of the CNS-PNS transitional zone in rat lumbar ventral roots. Journal of Anatomy 152, 236. CARLSTEDT, T. (1981). An electron microscopic study of the developing transitional region in feline SI dorsal rootlets. Journal of the Neurological Sciences 50, 357-372. COGGESHALL, R. E. (1986). Non-classical features of dorsal root ganglion cell organisation. In Spinal Afferent Processing (ed. T. L. Yaksh), pp. 83-96. New York: Plenum Press. COGGESHALL, R. E., EMERY, D. G., ITO, H. & MAYNARD, C. W. (1977). Unmyelinated and small myelinated axons in rat ventral roots. Journal of Comparative Neurology 173, 175-184. Documenta Geigy, (1962). 6th ed. p. 143. Basle: Geigy. FRAHER, J. P. (1978a). The maturation of the ventral root-spinal cord transitional zone. Journal of the Neurological Sciences 36, 427-449. FRAHER, J. P. (1978b). Quantitative studies on the maturation of central and peripheral parts of individual ventral motoneuron axons.II. Internodal length. Journal of Anatomy 127, 1-15. FRAHER, J. P. & KAAR, G. F. (1984). The transitional node of Ranvier at the junction of the central and peripheral nervous systems: an ultrastructural study of its development and mature form. Journal of Anatomy 139, 215-238. FRAHER, J. P. & KAAR, G. F. (1986). The lumbar ventral root-spinal cord transitional zone in the rat. A morphological study during development and at maturity. Journal of Anatomy 145, 109-122. FRAHER, J. P. & SHEEHAN, M. M. (1987). The CNS-PNS transitional zone of rat cervical dorsal roots during development and at maturity. A morphological and morphometric study. Journal of Anatomy 152, 189-203. KAAR, G. F. & FRAHER, J. P. (1986). The sheaths surrounding the attachments of rat lumbar ventral roots to the spinal cord: a light and electron microscopical study. Journal of Anatomy 148, 137-146. KAAR, G. F. & FRAHER, J. P. (1987). The vascularisation of the central-peripheral transitional zone of rat lumbar ventral rootlets: a morphological and morphometric study. Journal of Anatomy 150, 145-154. KAAR, G. F., O'SULLIVAN, V. R. & FRAHER, J. P. (1983). Combined vascular perfusion and in situ fixation of rat spinal nerve roots. Irish Journal of Medical Science 152, 222. KIM, J., SHIN, H. K. & CHUNG, J. M. (1987). Many ventral root efferent fibres in the cat are third branches of dorsal root ganglion cells. Brain Research 417, 304-314. LOEB, G. E. (1976). Ventral root projections of myelinated dorsal root ganglion cells in the cat. Brain Research
106, 159-165. MARAZZI, A. S. & LORENTE DE No, R. (1944). Interaction of neighbouring fibres in myelinated nerve. Journal of Neurophysiology 7, 83-101. ROSENBLUETH, A.(1941). The stimulation of myelinated axons by nerve impulses in adjacent myelinated axons. American Journal of Physiology 132, 119-128. ROSENBLUETH, A. (1944). The interaction of myelinated nerve fibres in mammalian nerve trunks. American Journal of Physiology 140, 656-670.
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STAMPFLI, R. (1954). Saltatory conduction in nerve. Physiological Reviews 34, 101-112. TARLOV, I. M. (1937). Structure of the nerve root. I. Nature of the junction between the central and the peripheral nervous system. Archives of Neurology and Psychiatry 37, 555-583. WAXMAN, S. G. (1972). Regional differentiation of the axon: a review with special reference to the concept of the multiplex neuron. Brain Research 47, 269-288. WAXMAN, S. G. (1975). Integrative properties and design principles of axons. International Review of Neurobiology 18, 1-40.
J. Anat. (1990), 170, pp. 131-137 With 2 figures Printed in Great Britain
High density of nodes of Ranvier in the CNS-PNS transitional zone J. P. FRAHER AND D. C. BRISTOL
Department of Anatomy, University College, Cork, Ireland
(Accepted 21 November 1989) INTRODUCTION
Having left their parent somata in the spinal grey matter, ventral motoneuron axons are grouped into bundles (Fig. 1). Where these traverse the ventrolateral funiculus of the spinal cord they are termed intramedullary rootlets. They emerge into the peripheral nervous system (PNS) as ventral rootlets which eventually unite distally to form the ventral root. Close to its emergence into the PNS, each rootlet contains a transitional zone (TZ), which is defined as that length of rootlet which contains both peripheral and central nervous tissue (Fig. 1). Various accounts have been given of spinal nerve TZs in man (Tarlov, 1937), cat (Berthold & Carlstedt, 1977; Carlstedt, (a)
(b)
DR
+
A
VR8B.....
Fig. 1. (a-b). (a) Diagrammatic transverse section through lumbar spinal cord showing ventral motoneuron somata (arrows) and their intramedullary axon bundles sectioned obliquely (arrowheads) as they run caudally and ventrally through the ventrolateral white funiculus (VWF). AB, plane of oblique longitudinal section (b); VR ventral root; DR, dorsal root. (b) Diagrammatic longitudinal section in Plane AB indicated in (a) showing three representative myelinated ventral motoneuron axons of an intramedullary rootlet (IMR), traversing the transitional zone (TZ) and running in a ventral rootlet (VR) after emerging into the PNS. The TZ of each rootlet contains a central tissue projection (CTP) (shaded). Spinal grey matter, crosses; nodes, white.
J. P. FRAHER AND D. C. BRISTOL 132 1981) and rat (Fraher, 1978a; Fraher & Kaar, 1986). In the rat, central tissue forms a distally tapering projection into each lumbar ventral rootlet. Consequently, the TZ lies in the most proximal part of the rootlet. The rat lumbar TZ is relatively short, averaging 160 ,m or less in length. Since each myelinated fibre possesses a transitional node of Ranvier as it traverses the TZ (Fraher, 1978 a; Fraher & Kaar, 1984, 1986; Bristol & Fraher, 1987), it is possible that the density of transitional nodes within the zone is particularly high. Intramedullary rootlets contain relatively short internodes (Fraher, 1978 b) and their node densities are therefore also likely to be high. However, no quantitative data are available on these points. The aim of this study was to compare node density at the TZ with that in the central and in the peripheral parts of the nervous system. Absolute density was calculated for three different levels along ventral motoneuron axon bundles: at the TZ, in the intramedullary rootlet and in the ventral root. Values for the three levels were compared, to determine if, and to what extent, density differences existed. High node density at the TZ would raise the possibility of possible electrical interaction between myelinated fibres in vivo. MATERIALS AND METHODS
Five Wistar albino rats were anaesthetised with a 1: 3 chloroform: ether mixture and killed by intravascular perfusion of fixative (2 % glutaraldehyde and 2 5 % paraformaldehyde in an orthophosphate buffer at a pH of 7-2-74) as described previously (Fraher & Kaar, 1984). Supplementary fixation was carried out by irrigating the spinal subarachnoid space with the fixative (Kaar, O'Sullivan & Fraher, 1983). The L4 spinal cord segment and its attached dorsal and ventral roots and spinal ganglia were exposed, removed and processed for transmission electron microscopy (Fraher & Kaar, 1984). Specimen blocks of the distal ventral root, transitional zone and intramedullary rootlet (Fig. 1) were prepared from each animal. Alternating sequential series of thick (05,um) and ultrathin (100 nm) transverse sections of randomly chosen individual fibre bundles at each of these levels were cut on a previously calibrated Reichert OMU4 Ultracut-E ultramicrotome. Thick sections were stained with toluidine blue and were photographed using a Reichert Polyvar photomicroscope. They were printed at a final magnification of x 1300. Serial photomicrographs of every twelfth thick section over the entire length of TZ were used for tracing fibres, locating nodes and measuring fibre bundle cross-sectional area. Node density was calculated in each of 25 ventral rootlets. TZ cross-sectional area was measured from the photomicrographs using a Kontron Mini-IPS automatic image analyser. Cross-sectional area varied somewhat between serially adjacent TZ levels. Accordingly, the volume of the segment between each pair of adjacent levels of section photographed was estimated as that of a solid conic segment (Documenta Geigy, 1962), the height of which was the sum of the thicknesses of intervening thick and ultrathin sections. The total volume of the TZ was calculated as the sum of the volumes of all conic segments comprising it. Since each fibre traversing the TZ possesses a node of Ranvier (Fraher & Kaar, 1984) the total number of nodes in each TZ is the same as that of its constituent fibres. This was confirmed by following the fibres on serial sections (see below). Node density was calculated as the number of nodes per unit volume of the TZ and was expressed as nodes per 105,um3 (Table 1). The node density of 25 intramedullary rootlets was determined as follows. For each rootlet, the cross-sectional area was measured on photomicrographs of every sixth serial transverse section using the Kontron Mini-IPS image analyser. Intramedullary
Node density
133
Fig. 2 (a-b). Photomicrographs of sections through intramedullary rootlets (outlined) showing (a) central nervous fibres (arrows) deeply indenting the rootlet and (b) glial tissue (arrowheads) projecting between rootlet fibres. *, obliquely sectioned blood vessel in spinal cord, continuous with pial vessels. x 800.
rootlets are generally compact on cross-section but are in some cases deeply indented by central nervous tissue (Fig. 2a). However, they are not delineated sharply from surrounding central nervous tissue by any clearly defined barrier. Bundles were followed by numbering all constituent fibres at the TZ and tracing these centrally on serial photomicrographs as far as the surface of the ventral horn grey matter. The cross-sectional area of the intramedullary rootlet was defined as the sum of the areas of all such fibres, including their myelin sheaths. Cell nuclei and perikarya apposed to the bundle were not included in cross-sectional area calculation because both astrocytes and oligodendrocytes contribute in varying degrees to both the intramedullary rootlet and the surrounding central nervous tissue (Fig. 2b). Blood vessels were also excluded in area calculation. The volume of the length of intramedullary rootlet studied was calculated as for that of the TZ, given above. The total number of nodes in each length of rootlet studied was determined by examining the serial transverse sections in the photomicroscope. The node density was calculated as the number of nodes per 105 u,m3 of the intramedullary rootlet (Table 1). Because some astrocytic and oligodendrocytic tissue which contributed to the intramedullary bundle was excluded in measuring cross-sectional area, the volume calculated for the intramedullary bundle was a minimum figure. The value obtained for node density was therefore a maximum figure which overestimated the true density.
134
J. P. FRAHER AND D. C. BRISTOL
Table 1. Mean node density (± S.E.M) at the transitional zones (TZ), in the intramedullary bundles (IMB) and ventral roots (VR) of each of 5 rats Ratios between densities are also shown. Overall mean values (±S.E.M.) are also given. Node density (±S.E.M.)
I'm3) (nodes/105 Rat no.
TZ*
IMB*
Ratios VRt
TZ/IMB TZ/VR
100 2-8 20-0+5-6 7-1 + 1 4 2-0+0-4 4-4 1-6 16-2+2-4 10-3 + 1-6 3-7+0-6 6-1 1-6 10-3+4-6 6-4+11 17+0-2 11-8 35 10-6+4-7 3 0+0 7 0-9+0-1 6-8 1*8 11-6+5 2 6-4+2 5 1-7+0 3 7-8 2-3 Overall 13-8+1 5 6-6+0-8 2-0+0-2 * Mean of 5 values for each rat. t Mean of 7 values for rats 1 to 4 and of 8 values for rat 5. 1 2 3 4 5
Nerve fibres in the distal ventral root, immediately proximal to its junction with the dorsal root ganglion, form clearly defined fascicles, each bounded by a perineurial sheath. The node density in 54-60 ,um lengths of 36 different fascicles (7 from each of 4 animals and 8 from the fifth) was calculated from the volume and the total number of nodes in each, in a manner similar to that described above, using every sixth serial transverse section (Table 1). The overall mean node density in each of the three regions examined was statistically compared with that in the other two, using a t test for comparing the means of two small samples of unknown variances (Bailey, 1959). RESULTS
Node density (Table 1) within the TZ as a whole averaged 13-8 nodes per 105 #m3. It varied considerably both within and between animals. There was a sevenfold (4 9-35 9) range between the smallest and largest values observed. Nodes were most closely packed in the smallest rootlets. In the intramedullary rootlets, node density (averaging 6-6 nodes per 105 /Zm3) was a maximum value because of the method of measurement (see Materials and Methods). Considerable variation in density (range: 1 1-1 56 nodes/ I05 ,tm3) occurred between rootlets within individual animals. In the distal ventral root, mean node density was 2-0 per 105 ,tm3. Again there was considerable variation both within and between animals (range: 0 7-6-5). Node density in the transitional zone averaged more than twice that calculated for the intramedullary rootlet and nearly eight times that for the ventral root (Table 1). These differences were statistically very significant (P < 0001). Density was very significantly greater in the intramedullary bundles than in the ventral root. DISCUSSION
The greatest node density found was in the TZ. This is because all fibres possess a node as they traverse its short length. Transitional nodes are therefore, to a considerable extent, in register with one another. No such restrictions on node distribution apply along fibre bundles centrally or peripherally. The greater node density in the TZ compared to that in the intramedullary rootlet
Node
density
135
is likely to be an underestimate. This is because all transitional nodes are distributed within an irregularly tapering central tissue projection (CTP) (Fraher & Kaar, 1986) which comprises about half the volume of the TZ (Bristol & Fraher, 1987). Furthermore, the method used overestimates intramedullary node density because some astrocytic and oligodendrocytic material was excluded in calculating intramedullary bundle volume. For these reasons it is likely that node density in the TZ averages at least five times that'in the intramedullary rootlet. The findings of this study suggest that, if node densities in the intramedullary rootlet and in the ventral root resemble values generally occurring in the central nervous system (CNS) and PNS, respectively, the possibility of electrical interaction between myelinated fibres is greater in the TZ than in any other part of the nervous system. Furthermore, since the highest density values were found in the smallest rootlets, any interaction is likely to be greatest in these. Stampfli (1954) suggested that individual fibres with closely related nodes may influence one another's activity. Subthreshold electrical interaction has been experimentally demonstrated between myelinated fibres in mammals (Blair & Erlanger, 1932; Rosenblueth, 1941, 1944) and amphibia (Marazzi & Lorente de No, 1944). Such interaction may be of significance in the functioning of motoneuron axon bundles as they traverse the central-peripheral nervous boundary: Waxman (1972, 1975) proposed that nerve fibres may not function solely as transmission lines as described in classical neurophysiology, but may perform a 'multiplex' role in neuronal signalling and different arrangements of fibres may function as delay lines and filters by modification of the conduction velocity and amplitude of the signals that they transmit. However, any tendency for fibre interaction at the TZ is likely to be offset to some degree by the presence of large numbers of concentrically arranged astrocyte processes surrounding the peripheral paranodal and nodal segments of motoneuron axons (Fraher, 1978a). These may serve to insulate transitional nodes more effectively than the less elaborately arranged astrocyte processes related to central nodes. The morphology of the rat L4 ventral rootlet transitional zones shows them to be very well suited for studying possible electrical interaction between fibres with closely packed nodes, for the following reasons. Firstly, node density is likely to be considerably higher than that found elsewhere, particularly in the PNS. Secondly, the TZs are readily accessible as they lie in small, discrete rootlets, superficial to the cord surface (Kaar & Fraher, 1986, 1987). Central bundles lack such delineation and their component fibres intermingle with those of other bundles having different origins, courses, distribution and functions. Cervical TZs are less accessible because they lie at, or just deep to, the cord surface (Fraher, 1978 a). Thirdly, L4 ventral rootlet TZs have the advantage over those of dorsal rootlets of containing a relatively pure population of myelinated fibres of a single functional class. They consist very largely of myelinated motoneuron axons, there being no autonomic outflow at the L4 level (Coggeshall, Emery, Ito & Maynard, 1977). Of the small number of sensory axons which they may contain, only a few are myelinated (Loeb, 1976; Coggeshall et al. 1977; Coggeshall, 1986; Kim, Shin & Chung, 1987). Furthermore, dorsal rootlet TZs (Fraher & Sheehan, 1987) are larger and less clearly delineated from one another than those of ventral rootlets. Fourthly, the muscles innervated through individual rootlets may be identified electrophysiologically or by tracer methods and so studies of transitional node function and muscle excitation may be readily combined.
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J. P. FRAHER AND D. C. BRISTOL SUMMARY
Node of Ranvier density was examined at three levels along rat lumbar motoneuron axon bundles: where they lie in the central nervous system, in the peripheral nervous system and in the transitional zone (TZ) between these. Density was considerably and significantly greater in the TZ than in either of the other locations. It is possible that such densely packed nodes in the TZ could interact electrically with one another. Because of its structure and position and because it contains a relatively pure fibre population, the rat L4 ventral rootlet TZ lends itself readily to electrophysiological investigation of this possibility. The work presented here was supported by grants from the Health Research Board of Ireland and the Wellcome Trust. The authors are grateful to Ms B. Rea for her technical assistance. REFERENCES BAILEY, N. T. J. (1959). Statistical Methods in Biology. London: The English Universities Press. BERTHOLD, C.-H. & CARLSTEDT, T. (1977). Observations on the morphology at the transition between the peripheral and central nervous system in the cat. II. General organisation of the transitional region in Si dorsal rootlets. Acta physiologica scandinavica, Suppl. 466, 23-42. BLAIR, E. A. & ERLANGER, J. (1932). On the effects of polarization of nerve fibres by extrinsic action potentials. American Journal of Physiology 101, 559-564. BRISTOL, D. C. & FRAHER, J. P. (1987). A morphometric study of the CNS-PNS transitional zone in rat lumbar ventral roots. Journal of Anatomy 152, 236. CARLSTEDT, T. (1981). An electron microscopic study of the developing transitional region in feline SI dorsal rootlets. Journal of the Neurological Sciences 50, 357-372. COGGESHALL, R. E. (1986). Non-classical features of dorsal root ganglion cell organisation. In Spinal Afferent Processing (ed. T. L. Yaksh), pp. 83-96. New York: Plenum Press. COGGESHALL, R. E., EMERY, D. G., ITO, H. & MAYNARD, C. W. (1977). Unmyelinated and small myelinated axons in rat ventral roots. Journal of Comparative Neurology 173, 175-184. Documenta Geigy, (1962). 6th ed. p. 143. Basle: Geigy. FRAHER, J. P. (1978a). The maturation of the ventral root-spinal cord transitional zone. Journal of the Neurological Sciences 36, 427-449. FRAHER, J. P. (1978b). Quantitative studies on the maturation of central and peripheral parts of individual ventral motoneuron axons.II. Internodal length. Journal of Anatomy 127, 1-15. FRAHER, J. P. & KAAR, G. F. (1984). The transitional node of Ranvier at the junction of the central and peripheral nervous systems: an ultrastructural study of its development and mature form. Journal of Anatomy 139, 215-238. FRAHER, J. P. & KAAR, G. F. (1986). The lumbar ventral root-spinal cord transitional zone in the rat. A morphological study during development and at maturity. Journal of Anatomy 145, 109-122. FRAHER, J. P. & SHEEHAN, M. M. (1987). The CNS-PNS transitional zone of rat cervical dorsal roots during development and at maturity. A morphological and morphometric study. Journal of Anatomy 152, 189-203. KAAR, G. F. & FRAHER, J. P. (1986). The sheaths surrounding the attachments of rat lumbar ventral roots to the spinal cord: a light and electron microscopical study. Journal of Anatomy 148, 137-146. KAAR, G. F. & FRAHER, J. P. (1987). The vascularisation of the central-peripheral transitional zone of rat lumbar ventral rootlets: a morphological and morphometric study. Journal of Anatomy 150, 145-154. KAAR, G. F., O'SULLIVAN, V. R. & FRAHER, J. P. (1983). Combined vascular perfusion and in situ fixation of rat spinal nerve roots. Irish Journal of Medical Science 152, 222. KIM, J., SHIN, H. K. & CHUNG, J. M. (1987). Many ventral root efferent fibres in the cat are third branches of dorsal root ganglion cells. Brain Research 417, 304-314. LOEB, G. E. (1976). Ventral root projections of myelinated dorsal root ganglion cells in the cat. Brain Research
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