Quantitative study of the non-circularity of myelinated peripheral nerve fibres in the cat.

1. One hind limb of each of four cats was either chronically de-efferentated, or chronically de-afferentated, and perfused with buffered glutaraldehyde fixative. Up to three different muscle nerves were dissected from each limb, post-fixed in osmium tetroxide and embedded in Epon. Ultrathin transverse sections were mounted on Formvar-coated single-hole specimen grids so that all the fibres in each nerve could be examined individually by electron microscopy.2. Non-circularity was expressed as the ratio (ø): [Formula: see text] The degree of non-circularity of all the afferent axons, or all the efferent axons, in each muscle nerve was determined. The proportion of fibres cut through the paranodal region, or through the Schwann cell nucleus, was as expected for group I afferent and for alpha and gamma efferent fibres, but hardly any typical paranodal sections of group II or III afferent fibres were encountered which suggests that their paranodal arrangement differs from that of other groups. In a quantitative comparison of noncircularity in different functional groups, fibres cut through paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were rejected.3. All the gamma efferent fibres in one nerve were studied in a series of sections cut at 25 mum intervals. The degree of non-circularity was found to be relatively constant along the internode of most fibres when the values at paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were ignored.4. The value of ø varied widely from 1.0 (circular) to 0.5 or less from fibre to fibre within every functional group. However, the mean value of ø was less for gamma axons (0.68) than for alpha axons (0.78), and less for group III axons (0.79) than for axons in groups I and II (both 0.84). When the results for all the nerves were aggregated, these differences were statistically very highly significant, as was the difference in ø between group I and alpha fibres. If values of ø < 0.5 were rejected, the difference between the mean ø for group III and group II was then of doubtful significance whereas that between alpha and gamma fibres was still very highly significant.5. The external perimeter (S) of a non-circular fibre differs from pi times the diameter of a circle just enclosing the fibre (D). It is shown that S = 0.95 pi D for group I and II fibres, S = 0.90 piD for alpha and group III fibres, and S = 0.85 piD for gamma fibres.6. The myelin period, or interperiod repeat distance, varied from 14.1 to 15.6 nm in different cats, implying radial shrinkage of the myelin sheath from 15 to 23%. The myelin period in a particular cat was the same for several nerves, and the same for fibres in different functional groups.7. The possibility that repetitive firing of axons during fixation contributed to the varying degree of non-circularity is considered but rejected as unlikely.8. It is deduced that about 10% radial shrinkage of the myelin sheath, but little or no osmotic shrinkage of the axon, occurred during fixation and rinsing. Further radial shrinkage of about 8% in all components of the fibre probably occurred as a result of subsequent histological processing. It is concluded that the non-circularity of all axons, and the greater non-circularity of small axons, is unlikely to have been due to histological processing.9. It is concluded that axons are non-circular in vivo. The hypothesis that non-circularity allows axons to accommodate swelling during repetitive activity is discussed. Suggestions are made as to why gamma axons may be more non-circular than alpha or group III axons in an anaesthetized cat immediately prior to fixation, and why alpha axons may be more non-circular than axons in groups I and II.

Ultrastructural dimensions of myelinated peripheral nerve fibres in the cat and their relation to conduction velocity.

1. The ultrastructure of all the afferent fibres, or all the efferent fibres, was studied in selected nerves from chronically de-afferentated or de-efferentated cat hind limbs perfusion-fixed with glutaraldehyde.2. The following parameters were measured: number of lamellae in the myelin sheath (n), axon perimeter (s), external fibre perimeter (S), axon cross-sectional area (A). Fibres were allocated to afferent groups I, II, III or efferent groups alpha and gamma according to the number of lamellae in the myelin sheath.3. The thickness of the myelin sheath (m) was linearly related to axon perimeter within the range s = 4 mum to s = 20 mum (groups II, III and gamma). The relation m = 0.103 s - 0.26 provided a good fit for all afferent and efferent axons in this range in several different anatomical muscle nerves in three cats. The myelin sheaths were thinner in a fourth, presumably younger, cat.4. The myelin sheaths were relatively thinner for large fibres in groups I and alpha (s = 20-50 mum). The results are interpreted in one of three ways. Either m tends to a limit of about 2.2 mum, or m is linearly related to s such that for large fibres m = 0.032 s + 1.11.5. Alternatively, m may be considered to be proportional to log(10)s for all sizes of axon so that m = 2.58 log(10) S - 1.73. The interpretation that there are two separate linear relations for large and small fibres is favoured.6. The ratio of axon to external fibre perimeter (g) falls from about 0.70 for group III and small gamma fibres in the cat to about 0.62 for group II and large gamma fibres and then rises again to 0.70, or even 0.75 for group I and alpha axons.7. The above relations between m and s are combined with the observations of Boyd & Kalu (1979) that Theta = 5.7 D for groups I and alpha and Theta = 4.6 D for groups II, III and gamma. It is shown that Theta = 2.5 s approximately for all sizes of axon (s from material fixed for electron microscopy) in rat, cat and man. The accuracy of this equation may be improved by deducting 3 m/sec in the case of small fibres. This conclusion is compatible with experimental observations of the relation between l and D (Hursh, 1939; Lubinska, 1960; Coppin, 1973) and between l and Theta (Coppin & Jack, 1972).8. From the theoretical analyses of Rushton (1951) and others Theta should be proportional to the external dimensions of the fibre rather than to axon size. It is shown that the thinning of the myelin sheath ought to affect Theta substantially. Thus some other factors must compensate for the thinning of the sheath.9. Small fibres are significantly more non-circular than large fibres. From the quantitative data of Arbuthnott et al. (1980) it is concluded that non-circularity may contribute to the fact that Theta proportional, variant s rather than Theta proportional, variant S, but cannot wholly account for it. Other possibilities considered are that axoplasmic resistivity or specific nodal conductance may differ for large and small fibres.10. It is suggested that myelinated peripheral nerve fibres may fall into two distinct classes with different properties, one comprising groups I and alpha and the other groups II, III and gamma. The conclusion predicted from theory may apply to each of these classes separately so that Theta = 2.0 S for the large-fibre class and Theta = 1.6 S for the small-fibre class.

Scaling factor relating conduction velocity and diameter for myelinated afferent nerve fibres in the cat hind limb.

1. Compound action potentials were recorded from certain muscle and cutaneous nerves in normal and chronically de-efferentated hind limbs of cats during stimulation of the appropriate dorsal spinal roots, 2. The peaks for groups I, II and III in the compound action potential were correlated with the corresponding peaks in the fibre-diameter histograms of the same de-efferentated nerve after processing it for light microscopy. 3. The scaling factor (ratio of conduction velocity in m/sec to total diameter in micrometer) was not constant for all sizes of fibre nor did it increase progressively with fibre size. Evidence is presented that a logarithmic relation between conduction velocity and fibre diameter is not appropriate. 4. In muscle nerves the scaling factor for fibres fixed by glutaraldehyde perfusion and embedded in Epon was 5.7 for group I afferent fibres and 4.6 for myelinated fibres in both group II and group III. 5. In cutaneous nerves the scaling factor was 5.6 for large fibres (group I or Abeta) and 4.6 for small fibres (group III or Adelta). 6. The scaling factor for group I fibres is the same as was found previously for alpha-efferent fibres, and that for groups II and III is the same as for gamma-efferent fibres (Boyd & Davey, 1968). 7. The possibility that there is a clear discontinuity in scaling factor between fibres in groups I and alpha, and those in other functional groups, is discussed. 8. It is concluded that there must be some structural feature of alpha and group I fibres which differs from that of smaller myelinated fibres. It is likely that a difference in the relative thickness of the myelin sheath is involved and possibly also in the conductances responsible for generating the action potential.