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Nodal axon diameter correlates linearly with internodal axon diameter in spinal roots of the cat
Neuroscience Letters, 24 (1981) 247-250
247
Elsevier/North-Holland Scientific Publishers Ltd.
NODAL AXON DIAMETER CORRELATES LINEARLY WITH INTERNODAL A X O N D I A M E T E R IN S P I N A L ROOTS OF T H E C A T
MARTIN RYDMARK
Karolinska Institutet, Department of Anatomy, S-104 O1 Stockholm 60 (Sweden) (Received March 23rd, 1981; Revised version received April 21st, 1981; Accepted April 22nd, 1981) Nodal and internodal axon diameters of individual myelinated nerve fibres were measured electron microscopically in fibre samples from serially sectioned L7 ventral and dorsal spinal root of young adult cats. A x o n cross-sectional area at the node of Ranvier in axons more than 4 #m in diameter was reduced to less than 20% of its internodal value. Internodal and nodal axon diameters showed a rectilinear distribution and linear regression analysis gave coefficients of correlation between 0.93 and 0.99. An individual nodal diameter value could be fitted to an internodal axon diameter value in a 95°70 prediction interval of _+ 1.06-2.41 #m.
It is well known that a myelinated axon is narrower at its nodes of Ranvier than internodally [2, 3, 6, 8-16]. This poses a serious problem when dealing with reconstructive m o r p h o m e t r y of the node of Ranvier using electron microscopic examination of a series of consecutive ultrathin sections [6, 15], namely, how to obtain an estimate of the internodal axon diameter (din) o f the nerve fibre subjected to nodal analysis. However, the diameter of the nodal axon (dn) is a variable accessible from the data of a reconstructed node, that might be used for an estimate of the internodal axon diameter. It is noteworthy that no systematic electron microscopic investigations of the relationship between dn and din seems to have been performed in peripheral nerves. Light microscopic investigations in peripheral [8, 10] and in central [9] nervous systems support the idea of a rectilinear relationship between dn and din. If this relationship would prove to be useful as a standard graph (preferably rectilinear, neither too level nor too steep and of a moderate scatter), in a specimen subjected to nodal analysis, di, and fibre diameter (D) could be calculated f r o m dn and the number of myelin lamellae (nl) as estimated at the node. With this background the relationship between dn and d~, was investigated in ventral and dorsal spinal roots of 3 young adult cats (5-8 months of age). The animals were perfusion fixed with 5°7o glutaraldehyde dissolved in an isotonic phosphate buffer containing 2.7°7o low molecular Dextran (m.w. 70.000) and 50 m O s m sucrose. The roots were removed after a laminectomy, postfixed first in glutaraldehyde and then in 2°7o OsO4, dehydrated in acetone and embedded in Vestopal W [1, 7]. One series of 4000-5000 consecutive ultrathin cross-sections was made from each of the 6 different specimens using an LKB 4801 Ultrotome. The average section thickness was 100 nm [5]. The sections were placed on Formvar0 3 0 4 - 3 9 4 0 / 8 1 / 0 0 0 0 - 0 0 0 0 / $ 02.50 © Elsevier/North-Holland Scientific Publishers Ltd.
248
coated one-hole copper grids, electron-stained with uranyl acetate and lead citrate and examined in a Philips EM 300 or 301 microscope. Every 50th section o f the series was photographed at a magnification of 600 and enlarged to a final magnification of 1800. Using these survey pictures it was possible to trace the course of individual nerve fibres. Some 50 nodes o f Ranvier belonging to fibres of all different diameters were identified in sections no. 1700 to no. 2300 (the mid-part of a series) and photographed at a magnification o f 10,000. The axons equipped with these nodes were marked out on the survey pictures and the corresponding fibres identified throughout the series. Between 50 #m and 200/~m proximally and distally to each node, two internodal cross section levels separated by 2 5 - 5 0 #m were photographed at a magnification of 2000-4000. Levels containing a Schwann cell nucleus or an incisure o f Schmidt-Lantermann were F,g 1
~r~"
dl n
F,g 2
~
2O-
Fig 3
~0#
20-
din
Fig 4
~m
20-
20-
15
15
".2
%• o•
• .'.,, ~
:"I: *"**
10-
~0-
I 5
5
I
2
3
4
5
dn 0 6~rn
. 1
.
. 2
. 3
. 4
. 5
dn 6fin
***
***= *
5
0
dn
1
2
3
4
5
lff/.
0
6~n
Fig 5
2
3
4
5
dn e; H m
Fig 6 dn 10'
~o~~din 08-
],n
08 ¸ et
06-
•
tl# 04-
0
,
•
o
°~.~.
° .•I , . . . ~
..:,,:.. el
02"
°°o
•
02"
....
; ....
~b . . . .
~'s . . . .
2'0/.~l °
O0
. . . .
,
5
. . . .
,
10
. . . .
,
~5
. . . . .
chn
20 Un
Figs. 1 and 2. The nodal axon diameter dn:circ, plotted against the internodal axon diameter din:circ., and the fitted linear functions in the ventral and dorsal L? spinal roots, respectively, of one young adult cat. Figs. 3 and 4. Pooled plottings of the nodal axon diameter dn:circ, versus the internodal axon diameter in the ventral and dorsal spinal roots, respectively, of 3 y o u n g adult cats. * = values from Hess and Young [8]. Figs. 5 and 6. The ratio between the nodal axon diameter (dn:circ.) and the internodal axon diameter (~). The same material as in Figs. 3 and 4.
249 omitted. In this way each of the selected fibres was documented at 4 separate internodal levels, giving a ~i~ value, and at one intervening nodal level. This was done in order to minimize the error of an internodal axon diameter that might vary as much as 30°70 [12, 13]. The axon circumference and area were measured with a map-reader and a planimeter, respectively. The values of these variables were recalculated to diameters. The calculated variables - ~ , ~ , dn:area, dn:circ. were treated separately for each sample (in all 12) and submitted to linear regression analysis and plotting, using a Nord-10 computer. As judged by eye the different plotting of d n versus din appeared similar and showed a rectilinear point distribution with a positive increment and a moderate scatter. A dn value could in a single sample be fitted to a ~ value within a range of 1.5-3 #m (see Figs. l and 2). Linear regression analysis - d n versus ~n - gave coefficients of correlation in the range of 0.93-0.99 with a mean of 0.97. The variance explained by these values was 86-98070 (mean 94°70). The slopes (C1) o f the fitted regression lines were 2.13-3.32 (mean 2.72). The 95% confidence intervals for the fitted regression lines did not coincide for large values of dn. On the average the slopes were 2207o steeper in dorsal roots as compared to ventral roots. The dcirc, values gave on the average 13°70 steeper slopes than the corresponding darea values. Difference in slope between the animals were on the average in the range of ___0-9°7o in relation to the mean values. In most samples the fitted regression line, ~ -- Co + CI. dn had a negative intercept with the ~ axis (Co: mean - 0 . 6 7 , range - 1.42 to + 0.19). As a consequence the ratio dn/~in will, when plotted against ~in (see Figs. 5 and 6), describe an inverse function similar to an exponential function with a negative exponent. The dn/~nin ratio values were found to be distributed between 0.3 and 0.4 for most axons larger than 4 #m, to decrease with increasing axon size and to slowly approach an asymtotic value between 0.30 and 0.47 (mean 0.37). In axons smaller than 4/~m, the ratio dn/¢~in was found to increase with decreasing ~n from 0.4-0.5 to 0.7-0.8. The smallest dorsal root fibres showed the highest dn/di-'-~ ratios. The curvilinear relationship between dn/~in and i ~ confirms observations claiming that, in small nerve fibres, the axon is less constricted at the node as compared to large ones [2, 3, 11, 14, 16]. The present material does not include axons small enough to give dn values as large as or larger than the ~ value of the same nerve fibres. Though, considering an axonal swelling of 20% or more in small axons during the preparatory procedure for electron microscopy [4], the ratio d~/din might well be larger than 1 in the native small nerve fibres. Furthermore, the relationship between dn and din might be different in other species, nerve fibre samples or preparations. In this connection the difficulties of observing a representative part of the nodal axon together with the internodal axon electron microscopically in longitudinally sectioned nerve fibres should also be emphasized. For up to 50 #m on both sides of the node the axon is narrower than in the internodal part and equipped with longitudinal ridges [2, 3, 6, 12]. In a r a n d o m longitudinal section a paranodal axon ridge might easily be mistaken for the axon proper. Furthermore, the barrel-shaped outbulging of the nodal axon is relatively large in small nerve fibres [6, 15].
250 T h e p r e s e n t results r e c a l l t h e classical o b s e r v a t i o n s o f H e s s a n d Y o u n g [8] in the rabbit
on
the node
of Ranvier
in p e r i p h e r a l
nerves.
Their
light m i c r o s c o p i c
o b s e r v a t i o n s in c r o s s - s e c t i o n s o n t h e r e l a t i o n s h i p b e t w e e n di, a n d d , s e e m , o n t h e w h o l e , to fit t h e p r e s e n t results (see Figs. 5 a n d 6). T h e i n c r e m e n t in t h e i r m a t e r i a l is s m a l l e r t h a n in t h e p r e s e n t o n e . T h i s is p r o b a b l y d u e to t h e s e c t i o n t h i c k n e s s used ('5 ~ m ' ) . S u c h c o m p a r a t i v e l y t h i c k s e c t i o n s m i g h t c a u s e t h e i r r e g u l a r i t i e s o f t h e m y e l i n s h e a t h to s u p e r p o s e w h i c h gives t o o s m a l l din v a l u e s . The observed relationship between d, and ~ to
estimate
din
d , = Co + C ~ . d , .
in
reconstructed
U s i n g this m e t h o d ,
supplies a useful graph or algorithm
longitudinally
sectioned
nodal
t h e 95°7o p r e d i c t i o n i n t e r v a l o f ~
regions: by a n y
single dn v a l u e will be in the r a n g e o f + 1 . 0 6 - 2 . 4 1 t~m ( m e a n 1.73 t~m). H e n c e , to e s t i m a t e din f r o m dn is a m e t h o d a p p l i c a b l e t o all f i b r e sizes. T h i s i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s f r o m t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l ( P r o j e c t 03157) a n d by f u n d s f r o m the K a r o l i n s k a I n s t i t u t e t . I a m m u c h i n d e b t e d to P r o f e s s o r C l a e s - H e n r i c B e r t h o l d f o r d i s c u s s i o n s a n d c o m m e n t s a n d to Ms. M o n i c a A n d e r s s o n , Ms. A n i t a B e r g s t r a n d . Ms. E v a B j O r k n e r a n d M s . A n e t t e Fransson for excellent technical assistance. 1 Berthold, C.-H., A study on the fixation of large mature feline mylinated ventral lumbar spinal root fibres, Acta Soc. med. Uppsala, 78, Suppl. 9 (1968) 1 36. 2 Berthold, C.-H., Ultrastructure of the node-paranode region of mature feline ventral lumbar spinal root fibres, Acta Soc. med. Uppsala, 73, Suppl. 9 (1968) 37-70. 3 Berthold, C.-H., Morphology of normal peripheral axons. In S.G. Waxman (Ed.), Physiology and Pathobiology of Axons, Raven Press, New York, 1978, pp. 3-82. 4 Berthold, C.-H., Corneliuson, O. and Rydmark, M., Tissue shrinkage and deformation in cat spinal roots during preparation for electron microscopy, in preparation. 5 Berthold, C.-H., Rydmark, M. and Corneliuson, O., Estimation of compression and thickness in ultrathin sections through Vestopal W embedded cat spinal roots, in preparation. 6 Berthold, C.-H. and Rydmark, M., Electron microscopic serial section analysis of nodes of Ranvier in lumbosacral spinal roots of the cat. I. General organisation of nodal compartments in fibres of different sizes, in preparation. 7 Carlstedt, T., A preparative procedure useful for electron microscopy of the lumbosacral dorsal rootlets, Acta physiol, scand., Suppl. 446 (1977) 61 72. 8 Hess, A. and Young, J.Z., The nodes of Ranvier, Proc. roy. Soc. B, 140 (1952) 301 320. 9 Hildebrand, C., Uhrastructural and light-microscopic studies of the nodal region in large myelinated fibres of the adult feline spinal cord white matter, Acta physiol, scand., Suppl. 364 (1971) 43-81. 10 Kashef, R., The node of Ranvier - A Comparative and Dimensional Study, Ph.D. Thesis, Guy's Hospital, School of Medicine, London, 1966, 149 pp. 11 Landon, D.N. and Hall, S., The myelinated nerve fibre. In D.N. Landon (Ed.), The Peripheral Nerve, Chapman and Hall, London, 1976, pp. 1-105. 12 Lubinska, L. and Lukaszewska, 1., Shape of myelinated nerve fibres and proximo-distal flow of axoplasm, Acta biol. exp., 17 (1956) 115-133. 13 Ochs, S., Beading of myelinated nerve fibres, Exp. Neurol., 12 (1965) 84 95. 14 Robertson, J.D., Preliminary observations on the ultrastructure of nodes of Ranvier, Z. Zellforsch., 50 (1959) 553 560. 15 Rydmark, M. and Berthold, C.-H., Electron microscopic serial section analysis of noders of Ranvier in lumbosacral spinal roots of the cat. 11. A morphometric study of nodal compartments in fibres of different sizes, in preparation. 16 Uzman, B.G. and Nogueira-Graf, G., Electron microscope studies of the formation of nodes of Ranvier in mouse sciatic nerves, J. Biophys. Biochem. Cytol., 3 (1957) 589-613.
247
Elsevier/North-Holland Scientific Publishers Ltd.
NODAL AXON DIAMETER CORRELATES LINEARLY WITH INTERNODAL A X O N D I A M E T E R IN S P I N A L ROOTS OF T H E C A T
MARTIN RYDMARK
Karolinska Institutet, Department of Anatomy, S-104 O1 Stockholm 60 (Sweden) (Received March 23rd, 1981; Revised version received April 21st, 1981; Accepted April 22nd, 1981) Nodal and internodal axon diameters of individual myelinated nerve fibres were measured electron microscopically in fibre samples from serially sectioned L7 ventral and dorsal spinal root of young adult cats. A x o n cross-sectional area at the node of Ranvier in axons more than 4 #m in diameter was reduced to less than 20% of its internodal value. Internodal and nodal axon diameters showed a rectilinear distribution and linear regression analysis gave coefficients of correlation between 0.93 and 0.99. An individual nodal diameter value could be fitted to an internodal axon diameter value in a 95°70 prediction interval of _+ 1.06-2.41 #m.
It is well known that a myelinated axon is narrower at its nodes of Ranvier than internodally [2, 3, 6, 8-16]. This poses a serious problem when dealing with reconstructive m o r p h o m e t r y of the node of Ranvier using electron microscopic examination of a series of consecutive ultrathin sections [6, 15], namely, how to obtain an estimate of the internodal axon diameter (din) o f the nerve fibre subjected to nodal analysis. However, the diameter of the nodal axon (dn) is a variable accessible from the data of a reconstructed node, that might be used for an estimate of the internodal axon diameter. It is noteworthy that no systematic electron microscopic investigations of the relationship between dn and din seems to have been performed in peripheral nerves. Light microscopic investigations in peripheral [8, 10] and in central [9] nervous systems support the idea of a rectilinear relationship between dn and din. If this relationship would prove to be useful as a standard graph (preferably rectilinear, neither too level nor too steep and of a moderate scatter), in a specimen subjected to nodal analysis, di, and fibre diameter (D) could be calculated f r o m dn and the number of myelin lamellae (nl) as estimated at the node. With this background the relationship between dn and d~, was investigated in ventral and dorsal spinal roots of 3 young adult cats (5-8 months of age). The animals were perfusion fixed with 5°7o glutaraldehyde dissolved in an isotonic phosphate buffer containing 2.7°7o low molecular Dextran (m.w. 70.000) and 50 m O s m sucrose. The roots were removed after a laminectomy, postfixed first in glutaraldehyde and then in 2°7o OsO4, dehydrated in acetone and embedded in Vestopal W [1, 7]. One series of 4000-5000 consecutive ultrathin cross-sections was made from each of the 6 different specimens using an LKB 4801 Ultrotome. The average section thickness was 100 nm [5]. The sections were placed on Formvar0 3 0 4 - 3 9 4 0 / 8 1 / 0 0 0 0 - 0 0 0 0 / $ 02.50 © Elsevier/North-Holland Scientific Publishers Ltd.
248
coated one-hole copper grids, electron-stained with uranyl acetate and lead citrate and examined in a Philips EM 300 or 301 microscope. Every 50th section o f the series was photographed at a magnification of 600 and enlarged to a final magnification of 1800. Using these survey pictures it was possible to trace the course of individual nerve fibres. Some 50 nodes o f Ranvier belonging to fibres of all different diameters were identified in sections no. 1700 to no. 2300 (the mid-part of a series) and photographed at a magnification o f 10,000. The axons equipped with these nodes were marked out on the survey pictures and the corresponding fibres identified throughout the series. Between 50 #m and 200/~m proximally and distally to each node, two internodal cross section levels separated by 2 5 - 5 0 #m were photographed at a magnification of 2000-4000. Levels containing a Schwann cell nucleus or an incisure o f Schmidt-Lantermann were F,g 1
~r~"
dl n
F,g 2
~
2O-
Fig 3
~0#
20-
din
Fig 4
~m
20-
20-
15
15
".2
%• o•
• .'.,, ~
:"I: *"**
10-
~0-
I 5
5
I
2
3
4
5
dn 0 6~rn
. 1
.
. 2
. 3
. 4
. 5
dn 6fin
***
***= *
5
0
dn
1
2
3
4
5
lff/.
0
6~n
Fig 5
2
3
4
5
dn e; H m
Fig 6 dn 10'
~o~~din 08-
],n
08 ¸ et
06-
•
tl# 04-
0
,
•
o
°~.~.
° .•I , . . . ~
..:,,:.. el
02"
°°o
•
02"
....
; ....
~b . . . .
~'s . . . .
2'0/.~l °
O0
. . . .
,
5
. . . .
,
10
. . . .
,
~5
. . . . .
chn
20 Un
Figs. 1 and 2. The nodal axon diameter dn:circ, plotted against the internodal axon diameter din:circ., and the fitted linear functions in the ventral and dorsal L? spinal roots, respectively, of one young adult cat. Figs. 3 and 4. Pooled plottings of the nodal axon diameter dn:circ, versus the internodal axon diameter in the ventral and dorsal spinal roots, respectively, of 3 y o u n g adult cats. * = values from Hess and Young [8]. Figs. 5 and 6. The ratio between the nodal axon diameter (dn:circ.) and the internodal axon diameter (~). The same material as in Figs. 3 and 4.
249 omitted. In this way each of the selected fibres was documented at 4 separate internodal levels, giving a ~i~ value, and at one intervening nodal level. This was done in order to minimize the error of an internodal axon diameter that might vary as much as 30°70 [12, 13]. The axon circumference and area were measured with a map-reader and a planimeter, respectively. The values of these variables were recalculated to diameters. The calculated variables - ~ , ~ , dn:area, dn:circ. were treated separately for each sample (in all 12) and submitted to linear regression analysis and plotting, using a Nord-10 computer. As judged by eye the different plotting of d n versus din appeared similar and showed a rectilinear point distribution with a positive increment and a moderate scatter. A dn value could in a single sample be fitted to a ~ value within a range of 1.5-3 #m (see Figs. l and 2). Linear regression analysis - d n versus ~n - gave coefficients of correlation in the range of 0.93-0.99 with a mean of 0.97. The variance explained by these values was 86-98070 (mean 94°70). The slopes (C1) o f the fitted regression lines were 2.13-3.32 (mean 2.72). The 95% confidence intervals for the fitted regression lines did not coincide for large values of dn. On the average the slopes were 2207o steeper in dorsal roots as compared to ventral roots. The dcirc, values gave on the average 13°70 steeper slopes than the corresponding darea values. Difference in slope between the animals were on the average in the range of ___0-9°7o in relation to the mean values. In most samples the fitted regression line, ~ -- Co + CI. dn had a negative intercept with the ~ axis (Co: mean - 0 . 6 7 , range - 1.42 to + 0.19). As a consequence the ratio dn/~in will, when plotted against ~in (see Figs. 5 and 6), describe an inverse function similar to an exponential function with a negative exponent. The dn/~nin ratio values were found to be distributed between 0.3 and 0.4 for most axons larger than 4 #m, to decrease with increasing axon size and to slowly approach an asymtotic value between 0.30 and 0.47 (mean 0.37). In axons smaller than 4/~m, the ratio dn/¢~in was found to increase with decreasing ~n from 0.4-0.5 to 0.7-0.8. The smallest dorsal root fibres showed the highest dn/di-'-~ ratios. The curvilinear relationship between dn/~in and i ~ confirms observations claiming that, in small nerve fibres, the axon is less constricted at the node as compared to large ones [2, 3, 11, 14, 16]. The present material does not include axons small enough to give dn values as large as or larger than the ~ value of the same nerve fibres. Though, considering an axonal swelling of 20% or more in small axons during the preparatory procedure for electron microscopy [4], the ratio d~/din might well be larger than 1 in the native small nerve fibres. Furthermore, the relationship between dn and din might be different in other species, nerve fibre samples or preparations. In this connection the difficulties of observing a representative part of the nodal axon together with the internodal axon electron microscopically in longitudinally sectioned nerve fibres should also be emphasized. For up to 50 #m on both sides of the node the axon is narrower than in the internodal part and equipped with longitudinal ridges [2, 3, 6, 12]. In a r a n d o m longitudinal section a paranodal axon ridge might easily be mistaken for the axon proper. Furthermore, the barrel-shaped outbulging of the nodal axon is relatively large in small nerve fibres [6, 15].
250 T h e p r e s e n t results r e c a l l t h e classical o b s e r v a t i o n s o f H e s s a n d Y o u n g [8] in the rabbit
on
the node
of Ranvier
in p e r i p h e r a l
nerves.
Their
light m i c r o s c o p i c
o b s e r v a t i o n s in c r o s s - s e c t i o n s o n t h e r e l a t i o n s h i p b e t w e e n di, a n d d , s e e m , o n t h e w h o l e , to fit t h e p r e s e n t results (see Figs. 5 a n d 6). T h e i n c r e m e n t in t h e i r m a t e r i a l is s m a l l e r t h a n in t h e p r e s e n t o n e . T h i s is p r o b a b l y d u e to t h e s e c t i o n t h i c k n e s s used ('5 ~ m ' ) . S u c h c o m p a r a t i v e l y t h i c k s e c t i o n s m i g h t c a u s e t h e i r r e g u l a r i t i e s o f t h e m y e l i n s h e a t h to s u p e r p o s e w h i c h gives t o o s m a l l din v a l u e s . The observed relationship between d, and ~ to
estimate
din
d , = Co + C ~ . d , .
in
reconstructed
U s i n g this m e t h o d ,
supplies a useful graph or algorithm
longitudinally
sectioned
nodal
t h e 95°7o p r e d i c t i o n i n t e r v a l o f ~
regions: by a n y
single dn v a l u e will be in the r a n g e o f + 1 . 0 6 - 2 . 4 1 t~m ( m e a n 1.73 t~m). H e n c e , to e s t i m a t e din f r o m dn is a m e t h o d a p p l i c a b l e t o all f i b r e sizes. T h i s i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s f r o m t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l ( P r o j e c t 03157) a n d by f u n d s f r o m the K a r o l i n s k a I n s t i t u t e t . I a m m u c h i n d e b t e d to P r o f e s s o r C l a e s - H e n r i c B e r t h o l d f o r d i s c u s s i o n s a n d c o m m e n t s a n d to Ms. M o n i c a A n d e r s s o n , Ms. A n i t a B e r g s t r a n d . Ms. E v a B j O r k n e r a n d M s . A n e t t e Fransson for excellent technical assistance. 1 Berthold, C.-H., A study on the fixation of large mature feline mylinated ventral lumbar spinal root fibres, Acta Soc. med. Uppsala, 78, Suppl. 9 (1968) 1 36. 2 Berthold, C.-H., Ultrastructure of the node-paranode region of mature feline ventral lumbar spinal root fibres, Acta Soc. med. Uppsala, 73, Suppl. 9 (1968) 37-70. 3 Berthold, C.-H., Morphology of normal peripheral axons. In S.G. Waxman (Ed.), Physiology and Pathobiology of Axons, Raven Press, New York, 1978, pp. 3-82. 4 Berthold, C.-H., Corneliuson, O. and Rydmark, M., Tissue shrinkage and deformation in cat spinal roots during preparation for electron microscopy, in preparation. 5 Berthold, C.-H., Rydmark, M. and Corneliuson, O., Estimation of compression and thickness in ultrathin sections through Vestopal W embedded cat spinal roots, in preparation. 6 Berthold, C.-H. and Rydmark, M., Electron microscopic serial section analysis of nodes of Ranvier in lumbosacral spinal roots of the cat. I. General organisation of nodal compartments in fibres of different sizes, in preparation. 7 Carlstedt, T., A preparative procedure useful for electron microscopy of the lumbosacral dorsal rootlets, Acta physiol, scand., Suppl. 446 (1977) 61 72. 8 Hess, A. and Young, J.Z., The nodes of Ranvier, Proc. roy. Soc. B, 140 (1952) 301 320. 9 Hildebrand, C., Uhrastructural and light-microscopic studies of the nodal region in large myelinated fibres of the adult feline spinal cord white matter, Acta physiol, scand., Suppl. 364 (1971) 43-81. 10 Kashef, R., The node of Ranvier - A Comparative and Dimensional Study, Ph.D. Thesis, Guy's Hospital, School of Medicine, London, 1966, 149 pp. 11 Landon, D.N. and Hall, S., The myelinated nerve fibre. In D.N. Landon (Ed.), The Peripheral Nerve, Chapman and Hall, London, 1976, pp. 1-105. 12 Lubinska, L. and Lukaszewska, 1., Shape of myelinated nerve fibres and proximo-distal flow of axoplasm, Acta biol. exp., 17 (1956) 115-133. 13 Ochs, S., Beading of myelinated nerve fibres, Exp. Neurol., 12 (1965) 84 95. 14 Robertson, J.D., Preliminary observations on the ultrastructure of nodes of Ranvier, Z. Zellforsch., 50 (1959) 553 560. 15 Rydmark, M. and Berthold, C.-H., Electron microscopic serial section analysis of noders of Ranvier in lumbosacral spinal roots of the cat. 11. A morphometric study of nodal compartments in fibres of different sizes, in preparation. 16 Uzman, B.G. and Nogueira-Graf, G., Electron microscope studies of the formation of nodes of Ranvier in mouse sciatic nerves, J. Biophys. Biochem. Cytol., 3 (1957) 589-613.