Schwann cells to express sodium channels is discussed in relation to a recent

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1 Journal of Physiology (1988), 396, With 9 text-figures Printed in Great Britain CHANGES IN EXCITABLE MEMBRANE PROPERTIES IN SCHWANN CELLS OF ADULT RABBIT SCIATIC NERVES FOLLOWING NERVE TRANSECTION BY S. Y. CHIU From the Department of Neurophysiology, 283 Medical Sciences Building, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, U.S.A. (Received 15 April 1987) SUMMARY 1. Whole-cell patch clamp studies were carried out on Schwann cells associated with myelinated and non-myelinated axons in the distal nerve stump of transected adult rabbit sciatic nerves which had undergone in vivo degeneration for 0-13 days. 2. Voltage-gated sodium current of Schwann cells associated wth non-myelinated axons decreased in amplitude following nerve transection; the peak current density decreased to about 51 % by day 3, and to 27 % by day 6. The number of nonmyelinated axons, determined from cross-sectional electron microscopy of the whole nerve bundle, also exhibited a similar decline to 48 % (day 3) and 17 % (day 6) of the control values. 3. In contrast, both voltage-gated sodium and potassium currents of Schwann cells associated with myelinated axons showed an increase following nerve transection. These two currents, normally not detectable in these Schwann cells, first appeared at around day 4 after nerve transection and increased progressively with time thereafter as Wallerian degeneration persisted. 4. The whole-cell membrane capacity of Schwann cells also exhibited different pattern of changes, depending on whether the cell normally lacked or produced myelin. In the former, the cell capacity remained relatively constant following nerve transection. In the latter, the whole-cell capacity was reduced to 11% of control values by day 13. This capacity decline is consistent with the observed detachment of myelin membranes from these latter Schwann cells. 5. There was an apparent inverse relation between the whole-cell capacity and the density of sodium and potassium currents in Schwann cells undergoing progressive myelin loss. It is suggested that the appearance of sodium and potassium currents in these cells might be related to myelin degeneration. 6. A hypothesis is proposed that the expression of excitable ion channels (sodium and potassium) on a Schwann cell is under opposing regulation by degenerating myelin and by axons. Axonal degeneration leads to a decline of Schwann cell sodium currents. Myelin degeneration, in contrast, leads to an increase of both Schwann cell sodium and potassium currents. 7. The above hypothesis that axons normally exert a positive trophic influence on

2 174 S. Y. CHIU Schwann cells to express sodium channels is discussed in relation to a recent speculation of a Schwann-to-axon transfer of excitable ion channels. INTRODUCTION In the peripheral nervous system of mammals, ion channels of excitable membranes, like sodium and potassium channels, are present not only on the axons, but also on Schwann cells (for reviews, see Gray & Ritchie, 1985; Waxman & Ritchie, 1985). As a Schwann cell interacts with an axon and elaborates myelin around it, the ion channels on the axon are apparently altered (Ritchie & Rogart, 1977; Chiu & Ritchie, 1981, 1982). Recent observations have further revealed that ion channels on the ensheathing Schwann cell are also altered by myelination. Using the patch clamp to record from axon-associated Schwann cells from adult rabbits, it was found that neuronal-like sodium currents could only be detected in cells which did not make myelin (Chiu, 1987). Once Schwann cells elaborated compact myelin, sodium current became undetectable (Chiu, 1987). In this report, the interrelationships between Schwann cells, myelin and axons were further studied by documenting the progressive changes in the excitable membrane properties (sodium and potassium channels) of Schwann cells in adult rabbit sciatic nerves following nerve transection. Whole-cell recordings were made from Schwann cells of non-myelinated and of myelinated axons. Axonal degeneration occurred in the former type of Schwann cell, while both axonal and myelin degeneration occurred in the latter cell type. Post-transectional variations of membrane properties in these two cell types, if present, could provide insight into how Schwann cell membrane properties are regulated by myelin and/or axons. The results suggest that myelin and axons exert an opposing regulation on the expression of Schwann cell ion channels. METHODS Surgery Experiments were performed on 10-week-old rabbits of either sex anaesthetized with methoxyfluorane. The sciatic nerves in the mid-thigh were exposed and tightly ligated with nylon suture material at two sites located 3-4 mm apart. The nerve was then transected between the pair of ligatures, and the two cut ends were picked up with fine forceps, reflected away from each other by a 180 deg rotation, and inserted into the neighbouring muscles proximal and distal to the cut. This procedure prevents regenerating axons from invading the distal degenerating nerve stump (Weinberg & Spencer, 1978). The rabbits were allowed to recover from anaesthesia, and at day 0 to day 13 after nerve transection, the rabbits were killed and the distal sciatic nerve stumps removed and desheathed. Cell preparation Schwann cells from the degenerating nerve stump were prepared for patch clamp studies by a 1-2 h treatment with collagenase (3 %, type I, Cooper Biomedical) as described previously (Chiu, 1987). The collagenase was dissolved either in Locke solution as in the previous study (Chiu, 1987), or in a culture medium (Dulbecco modified Eagle's medium (DMEM) plus 10 % fetal bovine serum, GIBCO) to improve cell viability. In the latter case, the cells were incubated in the culture medium containing the collagenase at 37 C for 1-2 h in a humidified air mixture containing 95% 02 and 5% C02.

3 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION 175 Patch clamp and kinetic analy8i8 The procedures for tecording whole-cell ionic currents by the patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981), for kinetic analysis of the current-voltage and the h. relations of ionic currents, and for calculation of the whole-cell membrane capacity, were essentially identical to those described previously (Chiu, 1987). Unless otherwise mentioned, the linear leakage and capacitative current were always subtracted from the displayed whole-cell currents using a scaled current response elicited with a hyperpolarizing pulse. Solution3 The pipette and bath solutions were essentially similar to those used in the previous study (Chiu, 1987). In all experimen,ts, the pipette was filled with a 140 mm-kcl solution, which contained (mm): KCl, 140; CaCl2, 1; MgCl2, 2; EGTA, 10; HEPES buffer, 10; the ph of the solution was brought to 7-2 by addition of 27 mm-naoh. The Locke solution in the bath contained (mm): NaCl, 154; KCl, 5-6; CaCl2, 2-2; morpholinopropionyl sulphonate buffer (ph 7 4), 10. The junction potential that existed between the pipette solution (KCl) and the bath solution (NaCl), which would be of the order of 3-5 mv, was not corrected for. Whenever possible mean values+the standard error of the means were quoted. All experiments were done at a room temperature of around 25 C. Electron micro8copy The degree of axonal degeneration was determined from cross-sectional electron microscopy of thin sections of the whole sciatic nerve bundle. At intervals of 1, 3, 6 and 11 days after nerve transection, the rabbits were first anaesthetized with methoxyfluorane then intra-aortically perfused with 1 % paraformaldehyde and 2% glutaraldehyde for 30 min. Following aldehyde fix, the tissue was taken out, post-fixed in Dalton's chrome-osmium reagent, dehydrated in a graded series of ethanol and embedded in Durcupan. Sections nm thick were obtained at a distance of about 0-5 cm distal to the transection site. The degree of degeneration of both the nonmyelinated and myelinated axons was determined, using the criterion adopted by Donat & Wisniewski (1973) which classified axons as either 'non-degenerated' or 'degenerated' according to the presence or absence of recognizable tubules and/or filament. At day 0 (control), a total of 304 non-degenerated myelinated axons and 627 non-myelinated axons, irrespective of size, were counted over twenty randomly selected grid areas. Counts at various days after transection were then expressed as a percentage of these two control counts. RESULTS Whole-cell patch clamp recordings were made at the cell body regions of Schwann cells of myelinated and of non-myelinated axons from the distal transected nerve stump at various post-transectional periods (day 1 to day 13). Unless otherwise mentioned, the cells were located cm distal to the transection site. All wholecell recordings were made with patch pipettes containing a 140-mM-KCl solution to allow the post-transectional variations of both sodium and potassium currents to be determined in the same cell. The morphology of the two cell types is first described. Morphology Light microscopy. Figure 1 shows light microscopic pictures of Schwann cells associated with non-myelinated axons (upper panels) and with myelinated axons (lower panels) in the distal nerve stump at increasing (from left to right) posttransectional periods (0-11 days). The two cell types could be distinguished, at least during the first 13 days, by the absence (upper panels) and presence (lower panels) of myelin debris.

4 176 S. Y. CHIU Fig. 1. Light microscopy of adult rabbit sciatic nerve fibres at various days after nerve transection. Upper panel: Schwann cells associated with non-myelinated axons at day 0 (A), days 3 (B), day 6 (C) and day 11(D) after nerve transection. Lower panel: Schwann cells associated with myelinated axons at day 1 (E), day 4 (F), day 6 (G) and day 11(H) after nerve transection. Cells shown were dispersed with collagenase (see Methods) from the nerve stumps about 1 cm distal to the site of transection. All pictures are shown at the same magnification; bar in H represents 20,sm. The white arrow in E points at a Schwann cell body. Two patch clamp pipettes are shown in F and G, making contact with the Schwann cell bodies in a whole-cell recording configuration mode. Note progressive myelin degeneration with days after nerve transection (E-H). Figure 1 A shows bipolar spindle-shaped Schwann cells associated with nonmyelinated axons 2 h after isolation (with collagenase, see Methods) from the sciatic nerve of a normal unoperated 10-week-old rabbit (Chiu, 1987). Figure 1 (B-D) shows bipolar cells obtained at day 3 (B), day 6 (C) and day 11(D) after transection. The spindle-shaped cells in the transected nerves (B-D) resembled those present in normal nerves, and were assumed in this study to be Schwann cells associated with

5 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION 177 degenerating non-myelinated axons. Indeed, fine cable-like processes, which are presumably remains of thin non-myelinated axons as described previously (Chiu, 1987), could sometimes be seen associated loosely with the spindle-shaped cells in the transected nerve stump (Fig. 1 D). For the myelinated fibres, the myelin was mostly intact at day 1 (Fig. IE), with the Schwann cell body (white arrow) taking its characteristic position along a normal looking internode. By day 4 (F), myelin degeneration was apparent in most fibres; myelin appeared to degenerate first at the cell body region (pointed by a patch clamp pipette), then progressively away from it. By day 6 (G), myelin had degenerated around the cell body region to such an extent that the characteristic bipolar spindle shape of a Schwann cell was beginning to be revealed. The Schwann cell in G, in particular, might even be undergoing an apparent cell division. At day 11 (H), the myelin had largely disappeared around the cell body, but myelin debris could still be seen along the extreme poles of the spindles. While it was not possible in the present studies to follow the pattern of myelin degeneration in the same Schwann cell, the pattern of degeneration extrapolated from observations on a large number of cells at various post-transectional periods (Fig. 1E-H) is very similar to that reconstructed from observations on a same cell in Wallerian degeneration in explant cultures of cat sciatic nerve (Crang & Blakemore, 1986). Electron microscopy. Cross-sections of the whole bundle of a normal sciatic nerve revealed numerous axons showing no signs of degeneration, as indicated by the presence of microtubules and neurofilaments (Fig. 2A). Nerve transection led to a decrease in the number of both myelinated and non-myelinated axons. At day 6 (Fig. 2 B), virtually all myelinated axons encountered, irrespective of their sizes, had degenerated. Extensive splitting of the myelin laminae could also be seen. These electron microscopic observations closely resembled those reported previously in transected rabbit sciatic nerves (Weinberg & Spencer, 1978). On the other hand, some non-myelinated axons showing little degeneration could still be found at day 6 (asterisk on Fig. 2B). Electrophysiology (patch clamp) Figure 3 shows whole-cell ionic currents recorded from Schwann cells of nonmyelinated axons (upper panels) and myelinated axons (lower panels) at increasing (from left to right) times (1-11 days) after nerve transection. As will be discussed later, there is considerable scatter in the current size from different cells at the same day. The cells in Fig. 3 are chosen on the basis that they reflect the general trend in the current variation obtained when all data are later averaged (see Fig. 9). Transient inward current. Whole-cell ionic currents recorded from Schwann cells of non-myelinated axons are shown in Fig. 3 at day 1 (A), day 6 (B) and day 11 (C) after nerve transection. Membrane depolarizations were applied from a holding potential of -75 mv and a hyperpolarizing pre-pulse to mv was used to remove sodium inactivation. In agreement with previous studies (Chiu, 1987) on Schwann cells from unoperated rabbits, a transient inward current, presumably a sodium current, was consistently recorded. The new observations here is that the amplitude of the sodium current decreased slowly after prolonged nerve transection (A-C). The reduction in the peak sodium current did not appear to be caused by an

6 178 S. Y. CHIU *'?,: *-.; ;%';,- '.. g. Fig. 2. Cross-sectional electron microscopy of adult rabbit sciatic nerves. A, control. B, 6 days after transection (distal nerve stump). Bar in B 1 #esm, and applies to both panels (A and B). * in B denotes an axon which shows no detectable degeneration. 10-week-old rabbit.

7 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION A 200 B 200 C 100 _ 100 _ 100 _ 0 pa L -300 L -300 L 5 ms 5 ms 5 ms D E _ PA _ L L 5 ms 5 ms 5 ms Fig. 3. Whole-cell ionic currents in Schwann cells associated with non-myelinated axons (upper panels) and with myelinated axons (lower panels) at various days after nerve transection. Upper panels: A, day 1; B, day 6; C, day 11. Lower panels: D, day 1; E, day 4; F, day 6. Holding potential was -75 mv and a pre-pulse to -120 mv was used to remove sodium inactivation. All current families were generated by the same sequence of depolarizations to -35, - 15, 5, 25, 45, 65 and 85 mv. The duration of the pre-pulse was 100 ms (A, C, E and F), 200 ms (D) and 800 ms (B). The currents in F were from the Schwann cell shown in Fig. 1 (G). A linear leakage current was subtracted. Pipettes filled with a 140 mm-kcl solution (see Methods). increase in resting inactivation since the h. curves from different post-transectional days were similar (see Fig. 6B, discussed later). Increasing the duration of the hyperpolarizing pre-pulse to ms, as compared to that of ms used normally (Fig. 3), did not produce any significant increase in the sodium current. The simplest interpretation is that there is a reduction in the sodium channel density, at least in the cell body regions. This reduction in sodium current did not occur in Schwann cells located proximal to the transection site. In two experiments the distal current density, expressed relative to whole-cell capacity, of pa/pf (n = 24) at day 6 was significantly lower than the corresponding proximal current density of pa/pf (n = 16), which remained virtually undiminished at pa/pf (n = 7) up to day 20. The peak sodium current density for the distal cells at days 0, 1, 3, 6, 11 and 13 was, in pa/pf, respectively 29±6 (n = 10; from Chiu 1987), 22-9±6 (n= 11), (n= 10), 7'9±0-9 (n= 27), 3±0-5 (n= 8) and (n= 6). A completely opposite change in the inward currents occurred in Schwann cells of

8 180 S. Y. CHIU A L 5 ms 5 ms C D _ 1000 _ ms 50ms Fig. 4. Effects of TTX (upper panels) and TEA (lower panels) on ionic currents of Schwann cells associated with myelinated axons. Upper panels: currents were recorded before (A) and 2 min after (B) 40,1 100 /LM-TTX was introduced into the bath to achieve a final TTX concentration of about 1,UM. Lower panels: currents were recorded before (C) and 7 min after (D) TEA were introduced to the bath to achieve a final external concentration of about 2-5 mm. All current families were generated by the same sequence of depolarizations from -45 to 95 mv in 20 mv increments. Holding potential was -75 mv. A linear leakage current was substracted. Pipettes were filled with a 140 mm- KCl solution. myelinated axons. Transient inward sodium currents, normally not detectable from these cells (Chiu, 1987), remained undetectable in virtually all cells examined during the first 3 days after transection. Figure 3 D shows a typical family of currents at day 1. However, at day 4, one (E) out of twenty-three cells examined yielded a small transient inward current. At day 6 the number of cells yielding this transient inward current increased drastically, to forty out of fifty-seven examined. Figure 3F shows ionic currents recorded from one such cell at day 6. A light microscopic picture of this particular cell is shown in Fig. 10 in which the actual site of the whole-cell recording was shown by the patch pipette. This transient inward current (Fig. 3F) resembled the sodium current seen in Schwann cells associated with non-myelinated axons (Fig. 3A), was completely eliminated by 1 4uM-tetrodotoxin (TTX) (Fig. 4A and B),

9 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION 181 and was thus a voltage-gated sodium current similar to that present in excitable membranes. The peak current-voltage relation for the transient inward current is shown in Fig. 5A (*). It exhibited a reversal potential of +50 mv, which was close to the theoretical Nernstian potential of about + 45 mv for sodium ions (Chiu, 1987). A 85 mv B PA PA pal...pa ms mv mv Fig. 5. Current-voltage relations for ionic currents in Schwann cells associated with myelinated axons. A, late outward current (U) and early current (*). B, tail currents for the late outward current. The inset shows the tail current records and the pulse protocol used. Briefly, the membrane was depolarized to 85 mv for 64 ms to elicit a large late outward current (not shown). At the end of this fixed depolarization the membrane was repolarized to various potential levels and the tail currents (inset) measured. However, this close agreement is an exception, rather than a rule. Indeed, in most experiments the transient inward currents did not reverse even at large depolarizations; for example, the highest depolarization for the family of currents in Fig. 4A was + 95 mv, yet the early transient current at this potential was still inward. A likely explanation for this discrepancy, as discussed in previous studies to account for a similar phenomenon (Shrager, Chiu & Ritchie, 1985; Chiu, 1987) is that sodium currents recorded at the cell body (as in Fig. 10) were distorted by currents arising from the distant spindles. How do sodium currents in the two cell types compare kinetically? Figure 6 shows a comparison of the activation limb of the peak current-voltage relation and of inactivation (h.) of Na+ currents between the myelin-forming cells (A) and nonmyelin forming ones (B). The different symbols in each plot represent data from different post-transectional days (day 1 to day 13). Compared this way, activation and inactivation of the sodium currents are quite similar between the two cells (A and B). Nevertheless, a small but consistent difference existed between the h. curves from the two cells when all data from various post-transectional days were combined (C). Here, an empirical equation of the same form as that used for the rabbit nodal sodium current (Chiu, Ritchie, Rogart & Stagg, 1979) was fitted to the data points (continuous lines in C; see legend for the equation used). The potential for half-maximal inactivation (Eh) occurred at -65 mv for the myelin-forming Schwann cells, but at a more hyperpolarized potential of -76 mv for the non-myelin forming ones. The value for kh (a measure of the steepness of the curve) was 9 mv for both

10 182 S. Y. CHIU A B h_ 0O mv mv 1.0w, h mv Fig. 6. Comparison of inactivation (hct3) of sodium channel and activation limb of peak current-voltage relation for sodium current from Schwann cells associated with myelinated axons (A) and from Schwann cells associated with non-myelinated axons (B). Data in A were obtained at day 6 (n = 17, O), day 8 (n = 8, x ) and day 13 (n = 4, *) after nerve transection. Data in B were obtained at day 1 (n = 9, O), days 3 (n = 9, x ) and day 6 (n = 15, *). The ho: curves were measured with a standard procedure as described in the methods. Activation of sodium current was compared using the peak current-voltage relation (normalized to a common maximal peak inward current, then inverted by multiplication by -1). Only the activation limb of the normalized peak current-voltage relation is shown. This method of comparing activation is similar to that used previously (Shrager et al. 1985) and was used here because in most experiments the sodium currents in Schwann cells associated with myelinated axons did not reverse even at large depolarizations. C compares in more detail the h,, curves from the two types of Schwann cells by combining the corresponding curves from different days into a single curve. *, Schwann cells associated with non-myelinated axons; Ol, Schwann cells associated with myelinated axons. The continuous lines in C were calculated from hcj = 1/(l+exp(E-Eh)/kh)) with Eh=-76mV (*) and Eh=-65mV (Ol). kh=9mv in both curves. Schwann cells. For comparison with axonal channels, the corresponding kh values are, respectively, 5-7 and 6-3 mv for sodium currents from the nodal (Chiu et al. 1979) and internodal (Chiu & Schwarz, 1987) regions of rabbit sciatic nerves. An uncertainty which cannot be resolved here is whether a change in the Schwann cell morphology following nerve transection could account for some of the kinetic variations. Delayed outward current. Schwann cells of non-myelinated axons occasionally showed an outward, presumably potassium, current (Chiu, 1987) which, unlike the sodium current, did not show any systematic post-transectional variation.

11 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION A 20 L 20 -L 20 D z h[0 oi-0 L pf pf pf pf Fig. 7. Relations between current density and whole-cell membrane capacity for Schwann cells associated with myelinated axons at various times (days) after nerve transection. Upper row: sodium current (pa/pf). Lower row of panels potassium current (pa/pf). Time after nerve transection: day 4 (A and E); day 6 (B and F); day 8 (C and G); day 13 (D and H). Number of cells: n = 23 (day 4), n = 57 (day 6), n = 19 (day 8) and n = 9 (day 13). Each vertical bar represents one cell. Note that one data bar (200 pa/pf for IK at day 6) was omitted in F because it was too large. Both I,, and IK (at the same day) were obtained from the same cell. Sodium current (INa was determined at the peak of the current-voltage relation. Potassium current 'K was determined as the largest outward current elicited by depolarizations from 35 to 105 mv (see Fig. 5A). The average values for INa and IK and cell capacity shown in Fig. 9 E, G, and H at various days were computed from all cells at the corresponding day in this Figure. Note logarithmic scale for capacity axis. On the contrary, there was a marked increase of a delayed outward current in the Schwann cells of the myelinated axons. This delayed outward current, normally not detectable in these Schwann cells (Fig. 3D), first appeared together with the sodium current at day 4(E), then increased dramatically 2 days later, at day 6 (F). This outward current (Fig. 4C) was greatly reduced (by about 86%) when tetraethylammonium (TEA) ions (2-5 mm) were applied to the bath (Fig. 4D), suggesting that a significant portion of it was a delayed rectifying potassium current similar to that found in excitable membranes. Figure 5A (M) shows the current-voltage relation for the late outward currents, and that for the tail currents (B). The tail currents for this experiment showed a reversal potential of about -75 mv, as compared with the theoretical Nernstian potential of -83 mv expected for the K+ concentrations used in this study. Whole-cell capacity. Schwann cells of non-myelinated axons showed little change in the whole-cell membrane capacity after transection; the capacity values in pf were

12 184 S. Y. CHIU (n = 10), (n = 11), ±4 (n = 10), (n = 27), (n = 8) and 13±2 (n = 6) at days 0, 1, 3, 6, 11 and 13 respectively. In contrast, the cell capacity in Schwann cells of myelinated axons showed a significant posttransectional decline. The values for the whole-cell capacity in pf are (n = 12), (n = 23), (n = 57), (n = 19) and (n = 9) A B < z 0L0L pf pf Fig. 8. Cumulative histograms of current density versus whole-cell capacity for Schwann cells associated with myelinated axons. Data from day 4 to day 13 (from Fig. 7) were combined into a single histogram plot for sodium current (A) and potassium current (B). A total of 108 different cells. INa and IK data obtained from the same cell. at days 0,4, 6, 8 and 13 respectively. Thus, a 9-fold reduction in cell capacity occurred by day 13. An explanation for this reduction in capacity is that myelin membranes, which were made by the Schwann cell, were gradually being detached from the cell during degeneration (Fig. 1E-H). How is it possible that layers of seemingly compact myelin in a myelinated fibre could contribute to cell capacity? An explanation might be that myelin membranes, according to morphological studies, are normally only partially compacted. In fact, discrete non-compacted cytoplasmic regions, called Schmidt-Lanterman incisures, run both longitudinally and transversely throughout the multi-myelin-laminae in a normal myelinated fibre. These incisures could allow the myelin membranes to contribute to the whole-cell capacity measured at the cell body region. Granted that this explanation is a correct one, a measure of myelin degeneration became possible for each cell simply by virtue of its associated whole-cell capacity. As discussed later, this becomes useful when the relation between ionic currents and myelin degeneration was explored. Whole-cell resting potential. In both cells the resting potentials became more negative after transection. For the non-myelin forming cells the resting potential was mv (n = 9) at day 1, and increased to (n = 25) and mv (n = 6) at days 6 and 11 respectively. The corresponding values for the myelinforming cells were (n = 16), -43±2 (n = 56), (n = 18) and mv (n = 9) at days 4, 6, 8 and 13 respectively.

13 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION A 1000 _ E pf 50 - ol ~~~- I I I pf I I O x 100_ 50_- O _ B I I I -I _ OR OX 50 - x F 0 \ I I I 30 - U- aq 20 - a 10_ z c I i I cl z 15 r G 10 F I 20 - OLL D 20 H a~ < 10 _ a i _ U-. O _ 0 L I I I Time (days) Time (days) Fig. 9. Plots of Schwann cell current density, whole-cell membrane capacity and percentage of axons as a function of time (days) after nerve transection. Left column: Schwann cells associated with non-myelinated axons. Right column: Schwann cells associated with myelinated axons. Whole-cell membrane capacity (A and E); percentage of axons (B and F); sodium current density (C and G); potassium current density (D and H). The delayed outward current was determined as the maximal current elicited by depolarizations between 35 and 105 mv. The number of non-myelinated (B) and myelinated (F) axons were determined as described in the methods. Current density was expressed as pa/pf. Number of cells for A, C and D: day 0 (n = 10), day 1 (n = 11), day 3 (n = 10), day 6 (n = 27), day 11 (n = 27) day 11 (n = 8), day 13 (n = 6). Number of cells for E, G and H: day 0 (n = 12), day 4 (n = 23), day 6 (n = 57), day 8 (n = 19), day 13 (n = 9). Note all cells (including those yielding no currents) were included in the calculations. Relation of current density to myelin degeneration in Schwann cells of myelinated axons. As mentioned earlier, there is considerable scatter in the current density from different cells measured at the same day after transection. Figure 7 illustrates this scatter by plotting the histograms of the current density versus whole-cell capacity. Histograms for sodium currents are shown in the upper panels, and those for potassium currents, lower panels. The post-transectional periods increased from left to right, being equalled to day 4 (Fig. 7 A and E), day 6 (B and F), day 8 (C and 0) and day 13 (D and H) respectively. At day 4 both sodium (A) and potassium (E) currents were absent. Both currents appeared at day 6 (B and F). Most interestingly, the distribution of current density follows a definite pattern: larger currents tended to occur in cells with smaller capacity. This pattern could still be seen later at day 8

14 186 S. Y. CHIU (C and G) and day 13 (D and H), even though cells with large capacity were becoming difficult to encounter, consistent with the extensive detachment of myelin membranes from Schwann cells at these later days (see Fig. 10 and H). Summary graphs of post-transectional parameters (Figs 8 and 9) Figure 8 summarizes the inverse relation between current density and cell capacity for Schwann cells of myelinated axons by combining data from various posttransection periods into a single histogram plot for sodium current (A) and potassium current (B). Figure 9 summarizes the temporal variations in post-transectional parameters for the two cell types (non-myelin forming type, left column; myelin forming type, right column). All cells were included in the calculation, including those which did not give detectable currents. Two key observations are recapitulated here. The first is the reduction in cell capacity in the myelin forming cells (Fig. 9E), but not in the nonmyelin forming ones (A). The second is that ionic currents in the two cell types change in an opposite direction (Fig. 3). In Schwann cells of the non-myelinated axons in which only axonal degeneration occurred, the sodium current density (Fig. 9B) and the number of axons (C) decline in parallel. For example, they were both reduced to about half of their respective control values at day 3 (48% for axons; 51 % for sodium current), and further to (17 %, 27 %) at day 6 and to (4%, 10%) at day 11 respectively. In contrast, in Schwann cells of the myelinated axons in which myelin (Fig. 9E) and axonal (F) degeneration both occurred, the density of sodium current (G) and of potassium current (H) progressively increased. DISCUSSION The new observation made in this study is that nerve transection in adult rabbit sciatic nerves led to a gradual change in the excitable membrane properties of Schwann cells located distal to the transection site. The key feature of this new observation is intriguing, as illustrated in Fig. 3. The post-transectional changes in the density of Schwann cell ion channels are opposite, depending on whether the cells normally lacked (Fig. 3, upper panels) or produced myelin (lower panels). A hypothesis A simple hypothesis that would explain the opposing post-transectional changes in the currents is that the expression of excitable ion channels on a Schwann cell is under opposing regulation by myelin and by axons. Axonal degeneration alone. The sole effect of axonal degeneration on the Schwann cell currents could be assessed in cells which normally did not produce myelin. The parallel decline in axons and Schwann cell sodium current (Fig. 9B and C) suggests that axons normally influence Schwann cells to express sodium channels. This suggestion is of considerable interest since it has recently been speculated that Schwann cells made sodium channels for axons (Gray & Ritchie, 1985). The parallel decline in both Schwann cell sodium currents and axons might reflect a negative feed-back system in which supply (Schwann cell sodium channels) follows demand (axons). Axonal plus myelin degeneration. If one could generalize from the non-myelin-

15 SCHWANN CELL CURRENTS AFTER NERVE TRANSECTION 187 forming cells that axonal degeneration alone led to a decline of Schwann cell sodium currents, then the post-transectional increase of these currents when both axons and myelin degenerated simultaneously must reflect the effect of the latter. This suggestion is further supported by Fig. 8 which shows that higher current density (both sodium and potassium) was associated with smaller cell capacity (more myelin loss). Degenerated myelin fragments released after nerve transection could mitogenically stimulate the Schwann cells to express ion channels. Recent biochemical studies also suggest that there is a differential proliferative response of cultured Schwann cells to axolemma and myelin-enriched fractions. (Meador- Woodruff, Yoshino, Bigbee, Lewis & DeVries, 1985). Schwann cells in culture Why do Schwann cells cultured from newborn rabbits in the absence of axons exhibit a sodium current (Chiu, Shrager & Ritchie, 1984)? Since both nonmyelinated and myelinated fibres are already present at birth in rabbits, one explanation is that Schwann cells from newborn animals might not have yet developed the response to axons as they do when the animal matures. Alternatively, the release of myelin fragments from the myelin-forming Schwann cells in culture might cross-react with the non-myelin-forming cells, stimulating them to express sodium and potassium currents. Interestingly, this cross-reaction appeared to be minimal in the present study; the post-transectional decline in the sodium currents in the non-myelin-forming Schwann cells persisted for the first 11 days (Fig. 9C), presumably in the face of myelin fragments released from neighbouring myelinated fibres. There is, however, a slight increase in the currents at day 13 (Fig. 9C), suggesting a cross-reaction with myelin debris might be possible. Do Schwann cells of normal myelinated axons have sodium channels? If the hypothesis is correct that axons induce Schwann cells to express sodium channels, why are these channels normally not detectable in cells associated with myelinated axons (Chiu, 1987)? An explanation might be that sodium channels are expressed only at cell regions which are in the most intimate contact with an axon, like the morphologically complex paranodal Schwann cell membranes and the innermost cytoplasmic turns of a Schwann cell tongue. Such locations would effectively 'electrically' conceal sodium channels from being detected by patch clamp recordings at the cell body region (Chiu, 1987). Indeed, in binding studies the size of the saxitoxin uptake in normal rabbit sciatic nerve fibres (composed of both myelinated and non-myelinated fibres) was too large to be accounted for by axonal sodium channels alone (Ritchie & Rogart, 1977; Ritchie & Rang, 1983). Furthermore, in recent voltage clamp experiments, an internodal sodium current was revealed from adult rabbit internodes when lysolecithin was used to induce acute demyelination (Chiu & Schwarz, 1987). The lysolecithin-induced increase in the internodal membrane capacity, however, was so large that a Schwann-axon membrane fusion was suggested (Chiu & Schwarz, 1987). The open question has been raised as to whether the observed internodal currents were not axonal in origin, but rather reflected sodium channels originally present on the innermost tongues of a Schwann cell, and somehow incorporated onto the axon induced by the detergentlike lysolecithin (Chiu & Schwarz, 1987).

16 188 S. Y. CHIU The hypothesis presented here also casts new light on the nature of the posttransectional changes in the saxitoxin uptake in an adult rabbit sciatic nerve (Ritchie & Rang, 1983). According to the hypothesis, the large initial posttransectional decline in saxitoxin uptake was caused not only by degenerating axons but also (presumably to a larger extent if the bulk of the normal uptake was to Schwann cells) by a decreased expression of sodium channels in Schwann cells as the trophic influence of axons was removed. The subsequent post-transectional increase in saxitoxin uptake in the rabbit (Ritchie & Rang, 1983) would be due presumably to the myelin-induced re-expression of these channels in Schwann cells. I thank Grayson Scott, Sherry Feig, Tom Pienkowski and Paul Lewandoski for help in the electron microscopy studies, and Carol Dizack and Terry Stewart for help in illustration and photography. This work was supported in part by grants NS from the U.S.P.H.S., RG-1839 from the U.S. National Multiple Sclerosis Society, and a General Research Support Grant to the University of Wisconsin Medical School from the NIH, Division of Research Facilities and Resources. REFERENCES CHIu, S. Y. (1987). Sodium currents in axon-associated Schwann cells from adult rabbits. Journal of Physiology 386, CHIU, S. Y. & RITCHIE, J. M. (1981). Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres. Journal of Physiology 313, CHIu, S. Y. & RITCHIE, J. M. (1982). Evidence for the presence of potassium channels in the internode of frog myelinated fibres. Journal of Physiology 322, CHIU, S. Y., RITCHIE, J. M., ROGART, R. B. & STAGG, D. (1979). A quantitative description of membrane currents in rabbit myelinated nerve. Journal of Physiology 292, CHIU, S. Y. & SCHWARZ, W. (1987). Sodium and potassium currents in acutely demyelinated internodes of rabbit sciatic nerves. Journal of Physiology 391, CHIU, S. Y., SHRAGIER, P. & RITCHIE, J. M. (1984). Neuronal-type Na and K channels in rabbit cultured Schwann cells. Nature 311, CRANG, A. J. & BLAKEMORE, W. F. (1986). Observations on Wallerian degeneration in explant cultures of cat sciatic nerve. Journal of Neurocytology 15, DONAT, J. R. & WISNIEWSKI, H. M. (1973). The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain Research 53, GRAY, P. T. A. & RITCHIE, J. M. (1985). Ion channels in Schwann and glial cells. Trends in Neurosciences 8, HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Archiv 391, MEADOR-WOODRUFF, J. H., YosHINo, J. E., BIGBEE, J. W., LEWIS, B. L. & DEVRIES, H. G. (1985). Differential proliferative responses of cultured Schwann cells to axolemma and myelin-enriched fractions. II. Morphological studies. Journal of Neurocytology 14, RITCHIE, J. M. & RANG, H. P. (1983). Extraneuronal saxitoxin binding sites in rabbit myelinated nerve. Proceedings of the National Academy of Sciences of the U.S.A. 80, RITCHIE, J. M. & ROGART, R. B. (1977). The density of sodium channels in mammalian myelinated nerve fibers and the nature of the axonal membrane under the myelin sheath. Proceedings of the National Academy of Sciences of the U.S.A. 74, SHRAGER, P., CHIu, S. Y. & RITCHIE, J. M. (1985). Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells. Proceedings of the National Academy of Sciences of the U.S.A. 82, WAXMAN, S. G. & RITCHIE, J. M. (1985). Organization of ion channels in the myelinated nerve fibre. Science 228, WEINBERG, H. J. & SPENCER, P. S. (1978). The fate of Schwann cells isolated from axonal contact. Journal of Neurocytology 7,