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Journal of Physiology (2002), 541.3, pp. 769-778
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.019638

Ionic currents in isolated and in situ squid Schwann cells

Isao Inoue *, Izuo Tsutsui †, N. Joan Abbott ‡ and Euan R. Brown §

	ABSTRACT

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Ionic currents from Schwann cells isolated enzymatically from the giant axons of the squids Loligo forbesi, Loligo vulgaris and Loligo bleekeri were compared with those obtained in situ. Macroscopic and single channel ionic currents were recorded using whole-cell voltage and patch clamp. In the whole-cell configuration, depolarisation from negative holding potentials evoked two voltage-dependent currents, an inward current and a delayed outward current. The outward current resembled an outwardly rectifying K⁺ current and was activated at -40 mV after a latent period of 5-20 ms following a step depolarisation. The current was reduced by externally applied nifedipine, Co²⁺ or quinine, was not blocked by addition of apamin or charibdotoxin and was insensitive to externally applied L-glutamate or acetylcholine. The voltage-gated inward current was activated at -40 mV and was identified as an L-type calcium current sensitive to externally applied nifedipine. Schwann cells were impaled in situ in split-open axons and voltage clamped using discontinuous single electrode voltage clamp. Voltage dependent outward currents were recorded that were kinetically identical to those seen in isolated cells and that had similar current-voltage relations. Single channel currents were recorded from excised inside-out patches. A single channel type was observed with a reversal potential close to the equilibrium potential for K⁺ (E_K) and was therefore identified as a K⁺ channel. The channel conductance was 43.6 pS when both internal and external solutions contained 150 mM K⁺. Activity was weakly dependent on membrane voltage but sensitive to the internal Ca²⁺ concentration. Activity was insensitive to externally or internally applied L-glutamate or acetylcholine. The results suggest that calcium channels and calcium-activated K⁺ channels play an important role in the generation of the squid Schwann cell membrane potential, which may be controlled by the resting intracellular Ca²⁺ level.

(Resubmitted 27 February 2002; accepted after revision 11 April 2002)
Corresponding author E. R. Brown: Neurobiology Laboratory, Stazione Zoologica 'A. Dohrn', Villa Comunale, 80121 Naples, Italy. Email: brown{at}alpha.szn.it

	INTRODUCTION

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The squid giant axon has been used as a model system to study neurone-glial interactions in situ (see Evans et al. 1985; Coles & Abbott, 1996) because it is a geometrically simple arrangement where a monolayer of Schwann cells (1 µm thick, Brown & Abbott, 1993) is orientated to face a single axon. Thus uniquely, the influence of a single axon on Schwann cell physiology can be studied in relatively undisturbed conditions. Neurone-to-glial signalling was first observed using the squid giant axon preparation (Villegas, 1972), and was later demonstrated in the nervous systems of both vertebrates (e.g. Reist & Smith, 1992) and other invertebrates (Gommerat & Gola, 1994). As a result of these experiments across animal phylogeny, the existence of axon-to-glial signalling as a general phenomenon is now beyond doubt. Now it is important to establish the details of this mechanism, such as the function of neurone-glial communication, the consequences of signalling and whether it is the same in all species. As pointed out by Chiu & Kriegler (1994) we still only have a partial picture of axon-Schwann cell interactions. For example, in vertebrate systems a great deal is known about isolated or cultured Schwann cell electrical properties from patch and voltage clamp recording (e.g. Chiu et al. 1984; Shrager et al. 1985; Howe & Ritchie, 1988; Konishi, 1989, 1990; Amedee et al. 1991; Chiu, 1991) and complementary information has come from imaging techniques that have allowed direct visualisation of Ca²⁺ in vertebrate Schwann cells in situ (Grafe et al. 1999; Rochon et al. 2001). However these cells are not amenable to direct electrical recording in situ. Conversely, although signalling between axon and Schwann cells in squid is well established (Evans et al. 1995) we still lack basic information about the ion channels and electrical properties of squid Schwann cells. Because the squid axon preparation offers the possibility of achieving a more complete understanding of axon-to-Schwann cell signalling, we have attempted to address the question of the basic membrane properties of squid Schwann cells in order to understand the mechanisms of axon-to-Schwann cell signal transduction, K⁺ regulation and membrane potential change. Uniquely, we show that the currents obtained in isolated cells are virtually identical to those obtained under voltage clamp in situ and thus it is reasonable to extrapolate the data from the dissociated cells to those in situ. Parts of this study have been published in abstract form (Abbott et al. 1995a; Brown et al. 1996).

	METHODS

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Isolation of cells

Three species of squid were used in this study: Loligo forbesi and Loligo vulgaris (Plymouth, UK and Stazione Zoologica, Naples, Italy) and Loligo bleekeri (Ine-cho, Kyoto, Japan). Animals were maintained in the seawater systems of the Marine Biological Association Laboratory, Plymouth, UK, the stabulario of the Stazione Zoologica and the Marine Laboratory of the National Institute of Physiological Sciences Ine-cho, Kyoto, Japan. In all three laboratories we applied a voluntary code for the humane handling of cephalopods by applying the guidelines for vertebrates in the Animal (Scientific Procedures) Act 1986 (UK) and additional procedures for cephalopods recommended in Boyle (1991). Squid were decapitated and the third order most medial stellar nerves dissected from the mantle in artificial seawater (ASW: 450 mM NaCl, 10 mM KCl, 50 mM MgCl₂, 10 mM CaCl₂, 10 mM Hepes-Na, pH 7.8). Giant axons were cleaned by microdissection of small nerve fibres in chilled 10-14 °C Ca²⁺-free artificial seawater (Ca²⁺-free ASW: 450 mM NaCl, 10 mM KCl, 50 mM MgCl₂, 10 mM Hepes-Na, 3 mM EGTA-Na, pH 7.8). Axons were incubated for 30 min in Ca²⁺-free ASW containing 5 mg ml^-1 trypsin type III (Sigma, USA) at 26 °C. During this incubation the giant axon became detached from the sheath of connective tissue (Fig. 1A and B). The axon with attached Schwann cells was then removed from the sheath by fine dissection (Fig. 1C). The axon was then incubated in collagenase P (Boehringer Mannheim, Germany), 1 mg ml^-1 in nominally Ca²⁺-free ASW (450 mM NaCl, 10 mM KCl, 60 mM MgCl₂, 10 mM Hepes-Na, pH 7.8) for 30 min at 22 °C. Axons were then gently agitated in the experimental chambers. Single Schwann cells that were observed to attach to the glass surface of the chamber were used for electrophysiological experiments within 3 h from starting the dissection (Fig. 1D). Schwann cells were identified by morphological criteria (see Results) and subjected to whole-cell voltage clamp. For in situ recordings, isolated split-open axons were prepared as previously described (Brown et al.1991).

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Figure 1. Light micrographs under Nomarski optics showing the process of enzyme digestion of the giant axon A, giant axon preparation (scale bar = 500 µm. B, the same portion of axon 20 min after treatment with trypsin, with indications that the giant axon is shrinking and is separated from the sheath of endoneurial cells by digestion of the basal lamina. C, the giant axon with a layer of Schwann cells, isolated from the sheath using fine scissors and forceps (scale bar = 100 µm). D, single Schwann cell dissociated from the sheath (scale bar = 10 µm).

Measurement of membrane currents

Whole-cell voltage clamp was conducted with a laboratory-made amplifier which had a headstage current-voltage (I-V) converter with a feedback resistance of 1 G. Single channel currents were measured with a List L/M EPC7 amplifier (List, Germany). Pulse generation and data acquisition were conducted with a 12 bit A/D converter (Scientific Solutions Inc. Labmaster/DMA, USA) and an IBM AT-compatible computer using pCLAMP software (Axon Instruments, USA). Electrode resistance was around 2 M for whole-cell voltage clamp and 6-8 M for single channel recording. Whole-cell capacitance (C_m) was calculated from the area under the transient capacitative current produced by a 10 mV step depolarisation from the holding potential of -60 mV. Series resistance was calculated by measuring the peak deflection of the positive-going current pulse (by extrapolation) and was used to estimate the voltage error during evoked large amplitude voltage-sensitive currents. Current data were acquired either directly or with on-line leak subtraction using a P/4 pulse protocol (where five current records were added, one associated with the test pulse and four associated with negative control pulses at one-quarter the amplitude of the test pulse). For in situ recordings, single 10 M sharp microelectrodes (containing 450 mM KCl) were inserted into Schwann cells using a Narashige MHW-3 hydraulic microdrive. Cells were voltage clamped using an Axoclamp 2B in discontinuous single-electrode voltage clamp mode, optimised for the RC characteristics of the cells; typical values for the switch circuit ranged between 0.6-5 kHz.

Intracellular solutions

For the study of whole-cell K⁺ currents and outside-out patches, the internal stock solution was 450 mM potassium D-aspartate, 15 mM MgCl₂, 6 mM EGTA-K and 30 mM Mops-K (pH 7.2). For Ca²⁺ currents, the internal and external stock solutions consisted of 450 mM caesium D-aspartate, 15 mM MgCl₂, 6 mM EGTA-Cs, 30 mM Mops-Cs (pH 7.2). The external solution was ASW.

To prevent disruption of intracellular structures by perfusion with solutions of high ionic strength, all intracellular and extracellular solutions were diluted to one-third of their original concentration with a solution of 12 % glycerol in distilled water. CaCl₂ was added to the internal solution to give a range of free Ca²⁺ concentrations (~0, 16, 300, 1000 nM). Free calcium concentration was calculated using the program WinMaxc (Bers et al. 1994; http://www.stanford.edu/~cpatton/maxc.html).

For inside-out patch clamp experiments the bathing solution was one-third potassium D-aspartate 'internal' solution with 1 mM ATP added (referred to as 150 mM K-asp). CaCl₂ was added to obtain different Ca²⁺ concentrations as described above. The pipette solution was either 150 mM KCl, 20 mM MgCl₂, 5 mM Hepes-K buffer (pH 7.8) and 8 % glycerol (referred to as 150 K) or 50 mM KCl, 100 mM NaCl, 20 mM MgCl₂, 5 mM Hepes-K buffer (pH 7.8) and 8 % glycerol (referred to as 50 K-100 Na).

Additions of drugs were made by adding microlitre volumes of stock solution to the chamber. Stocks were (compounds from Sigma, USA unless otherwise specified): tetrodotoxin (TTX) 1 mM in distilled water (DW), nifedipine 5 mM in ethanol, apamin 1 mM in DW, charybdotoxin (Latoxan) 0.1 mM in DW, tetraethylammonium chloride (TEA-Cl) 0.5 M in DW, 4-aminopyridine (4-AP) 100 mM in DW, quinidine 0.1 M in DW, L-glutamate 1 M in DW and acetylcholine 0.5 M in DW. Results are given as means ± standard error of the mean (excepting Fig. 7 where standard deviation is given) and tested for significance at the 0.05 level using Student's t test.

	RESULTS

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Identification of isolated Schwann cells

After dissociation of the micro-dissected axons, only elongate cells 50-150 µm in length with short processes and oval nuclei were seen from Loligo bleekeri (Fig. 1D). In Loligo forbesi the elongate cells were 135 ± 18.6 µm 25 ± 2.9 µm (n = 11). The basic electrical properties of the isolated elongate cells were compatible with previous electrical measurements made from Schwann cells in situ (see below and Discussion).

Cell capacitance and zero current potential

With the K-asp internal solution, the cell-attached seal resistance was > 2 G. In Loligo forbesi, the cell capacitance was 237 ± 19.2 pF (n = 27) and the input resistance 254 ± 63.8 M (n = 27). Within 30 s of whole-cell seal formation the zero current potential (measured under current clamp) was -36 ± 2.05 mV (n = 27). The series resistance was 8.5 M (n = 15) and so the maximum voltage error for the largest amplitude currents (1 nA) was 9 mV. In Loligo bleekeri the cell capacitance was 156 ± 8.8 pF (n = 48). Ionic currents recorded from Schwann cells of the two species of squid were quite similar.

Outwardly rectifying currents with internal K⁺ solution

Under internal perfusion with potassium aspartate solution, a voltage-activated outward current was observed at pulse potentials positive to -40 mV. Figure 2A shows typical current records in response to voltage steps from -90 to +60 mV in 10 mV steps. The records show outwardly directed rectification. The current amplitudes are plotted vs. voltage in Fig. 2C. Figure 2A and B shows the current before and after P/4 leak subtraction, respectively. The linear leakage current conductance obtained from the I-V relation was 42.7 pS pF^-1 (93.1 ± 11.42 pS pF^-1, n = 19). These non-linear outward currents appeared after a latent period of 5-10 ms following the onset of depolarisation (shown by the arrow in Fig. 2B). Both the amplitude and the activation rate were enhanced as the membrane depolarisation was increased (filled circles in Fig. 2C). The ratio of the outward-rectifying current to the linear leakage current increased with depolarisation, being 0.1 at -40 mV, 0.2 at -30 mV and 0.4 at -20 mV.

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Figure 2. Whole-cell currents recorded from a cell internally dialysed with 150 mM K-asp and bathed in one-third strength ASW A, currents associated with voltage clamp pulses from -90 to +60 mV at 10 mV steps from the holding potential of -50 mV. B, non-linear currents associated with depolarisation after linear current subtraction using the P/4 protocol. The arrow indicates the onset of depolarisation. C, I-V relations at the end of pulses for the records in A () and for the records in B (). Cell capacitance, 131.7 pF.

The effects of K⁺ channel blockers were investigated, and results are summarised in Fig. 3. Externally applied K⁺ current blockers TEA⁺ (20 mM) and 4-AP (5 mM), had no suppressing effect on the outward current (not shown, n = 5) but quinidine (250-500 µM) produced a dose-dependent block when added to the bath. Application of the Ca²⁺-dependent K⁺ channel blockers apamin (40 nM) or charybdotoxin (100-200 nM) had no significant effect on the outward currents. When caesium aspartate was used as the internal solution, no outward current was observed (not even the basal levels of current observed when no Ca²⁺ was added to the K⁺ internal solution), and no outward current was seen in either TEA-MeSO₄ (low Cl^-) or the ASW external solution (containing 207 mM Cl^-).

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Figure 3. Pharmacological properties of the Ca2+-dependent outward current Change in outward current after addition of compounds is shown normalized to the initial value. Drugs were applied directly to the recording chamber and allowed to act for at least 5 min. The bars represent the normalised peak outward current (mean ± S.E.M.) generated by a 130 mV depolarising pulse from a holding potential of -60 mV. The internal solution was K-aspartate plus 80 nM free Ca2+, external solution was ASW. Cells were monitored for changes in leakage current during application of drugs (n = not less than 5).

Tail current analysis was attempted to establish the reversal potential for the outward current (presumed I_K), but interpretation of the results was complicated by uncertainties about the settling time of the voltage step because of the large cell capacity transients.

Inwardly directed currents with Cs⁺ internal solution

With caesium aspartate internal solution, and external ASW, the zero current was around -10 mV and the outwardly rectifying current did not appear during depolarisation from the holding potential of -50 mV. Instead, as shown in Fig. 4A, currents were observed that decreased during the maintained depolarisation. Open circles in Fig. 4D show the voltage relation of the currents just before the end of the pulse. The currents became smaller than the linear leakage level above -40 mV. These non-linear currents were greatly suppressed by the addition of 10 µM nifedipine (see Fig. 4B and D filled circles). The linear leakage current in the voltage range -80 to -50 mV was unchanged during nifedipine application. The traces in Fig. 4C show the nifedipine-sensitive currents derived by subtraction of the records before and after nifedipine at the corresponding voltages. The nifedipine-sensitive currents were inwardly directed and activated at voltages above -40 mV, peaking at +50 mV (Fig. 4D filled triangles), consistent with I_Ca.

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Figure 4. Whole-cell membrane currents demonstrating the presence of L-type Ca2+ currents recorded from a cell dialysed with 150 mM Cs-asp internally and with one-third strength ASW externally A, currents associated with voltage pulses from -80 to +100 mV in 10 mV steps from the holding potential of -50 mV. B, currents recorded at 10 min after external application of 10 µM nifedipine. Voltage pulses from -80 to +60 mV were applied from the holding potential of -50 mV. C, nifedipine-sensitive currents obtained by subtracting the records in B from the records in A at corresponding voltages. D, I-V relations at the end of pulses for the records in A (), B () and C (). Cell capacitance, 351.2 pF.

Activation of I_K and I_Ca

It was noted that activation of both the (presumed) I_K and I_Ca showed a similar voltage dependence, although I_K developed after a latent period that probably represents more complex activation kinetics. It appeared likely that K⁺ channel activation was the result of Ca²⁺ channel activation. To test this idea, the Ca²⁺ current blockers Co²⁺ and nifedipine were added, and the outwardly rectifying I_K was found to be suppressed within 10 min (Fig. 5, n = 10 for each compound). Hence Ca²⁺ entry through voltage-gated Ca²⁺ channels is essential to activate I_K. To establish whether Ca²⁺ stores play a role in activating the K⁺ channels we examined the effects of externally applied ryanodine and caffeine on the currents, using the 150 mM K-asp internal solution. Ryanodine (20 µM) or caffeine (5 mM) did not produce any change in either the outwardly rectifying I_K or the leakage current over 10 min (data not shown). Hence it appears that under whole-cell clamp conditions, the K⁺ channel is only activated by Ca²⁺ entry.

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Figure 5. Effects of externally applied 3 mM Co2+ (A), and 10 µM nifedipine (B) on the outwardly rectifying IK Cells were dialysed with 150 K-asp and bathed in one-third strength ASW. Depolarising pulses were applied from the holding potential of -50 mV, and linear currents were subtracted using the P/4 protocol. The lower records in A and B show the currents at 10 min after the Co2+, and nifedipine applications, respectively. Cell capacitance: A, 138 pF; B, 242 pF.

Effect of L-glutamate on I_K

Isolated cells were subjected to whole-cell voltage clamp using Cs⁺ (n = 7) and K⁺ (n = 8) internal solutions and the holding potential stepped to a range of potentials between -70 and +50 mV. The agent implicated in axon-Schwann cell signalling, L-glutamate was iontophoretically applied by delivering 1 s 5-70 nA current steps to microelectrodes (50 M) containing 1 M sodium glutamate (pH 7.8). The delivery electrode was placed within 2-5 µm of the cell membrane. No inward or outward currents were evoked by glutamate application. Crushing the glutamate-filled microelectrode tips next to the cell or simply adding 100 µM L-glutamate, or 100 µm ACh to the bath (data not shown) had no effect on the voltage-activated currents.

Voltage clamp of Schwann cells in situ

We were able to make stable recordings for several minutes from Schwann cells in situ with sharp microelectrodes. In total 54 recordings were made from Schwann cells in 10 split-open axons. Optimised sampling rates, set to allow a full relaxation of the membrane voltage under discontinuous single electrode voltage clamp (dSEVC), varied between 0.5 and 5 kHz. Typical examples of currents evoked by step depolarisations of the membrane voltage are shown in Fig. 6A and B, which shows the membrane current in the upper panel and the actual membrane voltage in the lower panel. Figure 6B shows voltage dependent currents from the cell shown in A after subtraction of linear leakage (using P/4). The current- voltage relations of A and B are given in Fig. 6C which shows that the voltage-dependent outward current is not activated until the membrane voltage is positive to -40 mV. As in dissociated cells, the activation of the voltage dependent current is slow. In Fig. 6A and B it can be clearly seen that the current has still not fully activated 50-70 ms after the step depolarisation. This relation is shown in detail in Fig. 6D where the actual membrane voltage and the rise time of the membrane current are compared. The membrane voltage settles well within 4-5 ms but the time constant of activation of the membrane current (which was fitted with a single exponential function) is around 40 ms, similar to the values obtained from the isolated cells (compare Fig. 6 and Fig. 2).

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Figure 6. Discontinuous single electrode voltage clamp recording (dSEVC) from Schwann cells in situ A, currents in response to membrane depolarisations from the holding potential of -66 mV (10 mV steps, below). B, leak subtracted currents (P/4) from the same cell as A. C, current-voltage relations of voltage-dependent currents in A () and B (). D, detail of current and voltage from B showing a single exponential fit to the data and the rise-time of the membrane voltage (lower trace). Duty cycle was 2.5 kHz.

Single channel currents

The dependence of the potassium channel opening on Ca²⁺ was examined with the excised inside-out patch configuration. The pipette solution was 50 K-100 Na or 150 K-asp. With the cell-attached configuration, single channel activity was observed in 20 out of 61 giga-seal patches. The 20 'active' patches were examined under the inside-out configuration. When inside-out patches were made in the bathing solution containing 16 nM Ca²⁺, channel activity was very low, of short duration and infrequent (n = 5). The open channel probability (P_o) obtained with a two-Gaussian fit was 0.00048 ± 0.00054 (S.D.). When inside-out patches were made with 300 nM Ca²⁺, P_o was 0.048 ± 0.005 (S.D.) (n = 15). When the Ca²⁺ level was elevated to 1 µM, the channel activity became very high and then ran down gradually. Figure 7 shows an example of single-channel currents recorded in 300 nM Ca²⁺ (A) and just after elevation of Ca²⁺ to 1 µM (B) obtained with the pipette solution of 50 K-100 Na. The voltage of the pipette was +30 mV and the upward deflection indicates outwardly directed current through the membrane. Figure 7C and D shows the open-time histograms for the records of A and B, respectively (60 s acquisition), which were fitted with a single exponential function. The time constant was 1.71 ms at 300 nM Ca²⁺ (Fig. 7C), and it increased to 7.76 ms at 1 µM Ca²⁺. The amplitude histograms for the same records show one peak at +1.7 pA. The P_o at 300 nM Ca²⁺ was 0.054, and that at 1 µM Ca²⁺ was 0.39 (not shown). These experiments revealed that Ca²⁺ increased the open time of the channel without changing the current amplitude.

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Figure 7. Single-channel currents recorded from inside-out patches demonstrating that the currents are activated by Ca2+ on the cytoplasmic side The pipette solution was 50 K-100 Na, and the bath solution 150 K-asp and 300 nM Ca2+ buffered with EGTA. The upward deflection of the records indicates outward current. A, part of a continuous recording in 300 nM [Ca2+] at a membrane potential of +30 mV. B, part of a continuous recording after elevation of [Ca2+] to 1 µM. The time constant obtained by a single exponential fit to the histogram is 1.17 ms. C, open time histogram for 60 s recording in 300 nM [Ca2+]. D, open-time histogram for 60 s recording after elevation of Ca2+ to 1 µM. The time constant obtained by a single exponential fit is 7.76 ms.

Figure 8 shows the I-V relations of the Ca²⁺-activated channel. Each point indicates an averaged value obtained from five to seven experiments, and error bars the magnitude of S.D. Filled circles represent the current amplitude obtained with the 50 K-100 Na externally and with 150 K-asp internally. The reversal potential is -27.1 mV. This value is close to the theoretical value of the K⁺ equilibrium potential (-30.3 mV). Filled squares show the relations obtained with 150 mM K⁺ on both sides. The reversal potential is -1.9 mV. From these two I-V relations it is concluded that this channel is a K⁺ channel. The single channel conductance calculated with linear regression to the data shown by the filled squares is 43.6 pS.

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Figure 8. I-V relations of single channel currents recorded from excised inside-out patches demonstrate that the Ca2+-activated channel is a K+-selective channel Filled circles indicate the current amplitudes obtained with the pipette solution containing 50 K-100 Na and a bathing solution of 150 K-asp, and filled squares with 150 K-asp on both sides. Each point was obtained by averaging the peak values of two Gaussian fits obtained from 5-7 experiments, and the error bars show the magnitude of S.D.

We tried to record inwardly directed single channel Ca²⁺ currents in inside-out patches with a Ca²⁺ solution externally (inside the pipettes) and with a Cs⁺ solution internally (outside the pipette), but failed to record such currents in six patches examined.

Effects of glutamate and ACh on single channel activity

L-Glutamate (100 µM) or ACh (100 µM) applied to the bath either with the inside-out configuration or with the outside-out configuration produced no detectable changes in the activity or amplitude of the single channel potassium currents (n = 4 for each experiment).

	DISCUSSION

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In the present study, we found that the squid giant axon together with its covering of Schwann cells could be separated from the surrounding sheath, the Schwann cells dissociated enzymatically and subjected to whole cell patch clamp and single channel recording. The main novel feature of this study is that voltage-dependent channels are described in squid Schwann cells for the first time. In addition we were able to exploit the advantage of the squid system by establishing that the K⁺ channels seen in isolated cells are also present on Schwann cells in situ.

It seems likely on the basis of morphology and electrical properties that the isolated elongate cells are Schwann cells. Morphologically they compare well with in situ Schwann cell dimensions estimated by morphometric methods (Brown & Abbott, 1993: 100 µm 20 µm) and direct visualisation with dye injection (Brown et al. 1991; Brown & Kukita, 1996: 600 µm 20 µm). The differences in length could be due to loss of fine processes or 'rounding up' during dissociation. The cell capacitance of between 150 and 250 pF predicts a membrane area (assuming a membrane capacitance of 1 µF cm^-2) of around 2 10^-4 cm². However, the membrane area estimated from light microscopical dimensions was 0.7 10^-4 cm². This discrepancy could represent the contribution of additional surface-connected membrane, possibly the glial tubular system (Villegas & Villegas, 1984; Zwahlen et al. 1988; Brown & Abbott, 1993). Even so, the estimate of cell membrane area is less than that obtained by electron microscopy and dye injection (4 10^-4 cm², Villegas, 1972; Brown et al. 1991) and may again reflect the loss of processes during dissociation. The fact that the K⁺ current behaviour was identical in in situ and dissociated cells also supports this identification.

Two types of ionic channels are present in isolated Schwann cells, a Ca²⁺-activated potassium channel and a voltage-gated L-type calcium channel. The K⁺ current resembles voltage-activated K⁺ currents described in glial and other inexcitable cells (see e.g. Kolb, 1990) and differs from squid giant axon K⁺ channels in Ca²⁺ sensitivity, insensitivity to 4-AP and single channel conductance. Schwann cell K⁺ channels are sensitive to Ca²⁺ in the nanomolar range, while previous work has shown that axonal K⁺ channels are relatively insensitive to internal Ca²⁺ (1 nM to 1 µm, summarised in Kolb, 1990). Giant axon delayed-rectifying K⁺ currents are blocked by both internal and externally applied 4-aminopyridine (Yeh et al. 1976; Meves & Pichon, 1977) while the Schwann cell channel was insensitive to this manipulation. We considered the possibility that the large capacitance of the cells and/or the effects of enzyme treatment, could distort these currents or in the case of any rapidly activating transient currents, be RC (resistance-capacitance) filtered in such a way as to be undetectable. These questions were directly addressed by carrying out voltage clamp of Schwann cells in situ using dSEVC. The results showed identical slowly activating outward currents, appearing with a similar delay and activation kinetics, indicating that the enzyme treatment did not substantially modify the channels.

The single channel conductance of the Schwann cell potassium current is 43.6 pS. This differs from the value of 10 pS obtained by Noceti et al. (1995) from presumed Schwann cell membranes of the squid. However, the single channel conductance of the Schwann cell channel is similar to one of the three values for K⁺ channels obtained in membrane patches from cut-open squid axons (10, 20 and 40 pS, Llano et al. 1988). The insensitivity of the currents to application of 4-AP in the present study mirrors the pharmacology observed in neonatal myelinating Schwann cells from mice (Konishi, 1990). Internal Cs⁺ or external application of quinine blocked the K⁺ current in a dose-dependent manner, resembling previously reported K⁺ currents in vertebrate Schwann cells (Konishi, 1994) and other inexcitable cells (Kolb, 1990). However despite the Ca²⁺ sensitivity of the single channel current, charibdotoxin and apamin, blockers of vertebrate Ca²⁺ dependent K⁺ channels (Gauldie et al. 1976; Miller et al. 1985), had no effect when applied externally. As ryanodine and caffeine had no effect on the outwardly rectifying I_K it is concluded that ryanodine receptor controlled intracellular Ca²⁺ stores are unlikely to be involved in the modulation of I_K. Under the present experimental conditions, potassium channels appear to be activated solely by Ca²⁺ entry into the cell through L-type calcium channels; hence the current sensitivity to nifedipine and cobalt.

The inwardly directed current appeared at pulse potentials more positive than -40 mV and was identified as an L-type Ca²⁺ current. It is therefore interesting to note that L-type Ca²⁺ currents have also been identified in vertebrate Schwann cells (Amedee et al. 1991). The L-type current in squid Schwann cells is not inactivated at relatively depolarised holding potentials (e.g. -40 mV), is completely blocked by externally applied Co²⁺, and is suppressed by nifedipine.

The presence of these Ca²⁺-activated potassium and L-type Ca²⁺ currents helps to explain the nature of changes observed in Schwann cell membrane potential. The dependence of the Schwann cell membrane potential on the external K⁺ concentration has been studied in Sepioteuthis axons by transient recordings with microelectrodes, and the intracellular K⁺ concentration was estimated to be 220 mM (Villegas et al. 1968). The fact that the zero current potential was ~-10 mV when cells were dialysed with the 150 Cs-asp solution indicates that the potassium channels play a leading role in generation of the negative membrane potential. It has also been shown that a Ca²⁺-dependent hyperpolarisation of Schwann cell membrane potential lasting several hours occurs after splitting open axons, probably due to physiological stress of the Schwann cells (Brown et al. 1991; Brown & Kukita, 1996) and similar changes have been observed in intact axons (Abbott et al. 1990). As our experiments show that K⁺ channel activity is low below 300 nM [Ca²⁺] it seems unlikely that under normal conditions of stimulation, the [Ca²⁺] would reach these levels for prolonged periods of time, unless there was considerable cellular damage. This may indicate that microelectrode penetration and physical damage due to dissection may account for some of the observed changes in Schwann cell membrane potential reported in previous studies. This raises the possibility that the Ca²⁺-activated K⁺ channel may be activated as a result of physiological stress. As potassium channel activity is low under the normal (resting) condition of the Schwann cell where the membrane potential is ~-40 mV, a possible explanation for the 'low' resting potential of the Schwann cell is that (as in Fig. 2) the ratio of I_K to the leak current at -40 mV is low (0.1). The stable value of the membrane potential of -40 mV may be maintained by the negative feedback mechanism that links the activities of the Schwann cell calcium and potassium channels.

It is likely that the Schwann cell K⁺ and Ca²⁺ channels are involved in the previously demonstrated axon-to-Schwann cell signalling (Abbott et al. 1995b). Transient recordings of the Schwann cell membrane potential with microelectrodes have revealed that the Schwann cell undergoes a hyperpolarisation by 10-20 mV after high frequency axonal firing, and that this can be mimicked by extracellular application of L-glutamate (Villegas, 1972; Evans et al. 1995) and blocked by externally applied Ca²⁺-free solution (Villegas, 1984). These reports suggest that signalling is related to intracellular Ca²⁺ dynamics. Our experiments indicate that Schwann cell depolarisation followed by activation of the Ca²⁺-dependent I_K by entry of Ca²⁺ through the L-type Ca²⁺ channels could be involved in this modest hyperpolarisation. However this cannot be the only step in signalling, as depolarisation with raised extracellular K⁺ did not evoke a subsequent hyperpolarisation (Villegas, 1972).

It has also been suggested that Schwann cells are involved in the regulation of axonally released K⁺ (Pichon et al. 1995). Although [K⁺] was reported to rise in the intercellular clefts by up to 20 mM (Frankenhaeuser & Hodgkin, 1956), more recent work using microelectrodes (Abbott et al. 1988; Astion et al. 1988; Brown, 1993) and voltage clamp (Inoue et al. 1997) established that under normal physiological conditions the steady-state rise in extracellular [K⁺] during nerve stimulation was as little as 1 mM. Thus the consequential Schwann cell depolarisation would be less than 1 mV and insufficient to activate the Ca²⁺ currents. In current- and voltage-clamp experiments, Clay (1998) has shown that during the squid axon action potential, K⁺ in the periaxonal space may transiently reach around 15-20 mM. However, measurements of Schwann cell cytoplasmic [Ca²⁺] (Brown, 1998) indicate that an increase in external [K⁺] of more than 40 mM would be necessary to evoke an increase in Schwann cell intracellular [Ca²⁺].

These considerations imply that the hyperpolarisation observed as a result of axonal firing must be mediated by chemical signalling, since the K⁺-mediated depolarisation alone would be insufficient to cause a depolarisation-induced Ca²⁺ entry. Indeed, L-glutamate has been implicated as a candidate substance in axon-Schwann cell signalling (Lieberman et al. 1989; Lieberman & Sanzenbacher, 1992). However, in our experiments externally applied L-glutamate or ACh did not activate the potassium or Ca²⁺ channels or evoke an ionotropic current.

The present study demonstrates Schwann cell K⁺ and Ca²⁺ channels that could in theory contribute to changes observed in Schwann cell membrane potential. Previously, application of agents known to block these currents did not influence K⁺ clearance following axonal stimulation (Inoue et al. 1997). Taken together, these observations suggest that axon-Schwann cell signalling, involving a cascade of neurotransmitter and membrane/intracellular events in the Schwann cells, may play other roles in controlling Schwann cell mechanisms for axonal support.

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Acknowledgements

This work was supported by the BBSRC, Wellcome Trust and the Monbusho International Research Programs (03044106, 03044152, 06044163). I.I. was supported by a Ray Lankaster Investigatorship and E.R.B. by a BBSRC Advanced Fellowship. The authors are grateful to the Director and staff of the MBA and SZN laboratories and Mr Y. Nitani for the supply of squid.

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