Early development of voltage-dependent sodium currents in cultured mouse spinal cord neurons

DEVELOPMENTAL

BIOLOGY

113,317-326

(1986)

Early Development of Voltage-Dependent Sodium Currents in Cultured Mouse Spinal Cord Neurons A. B. MACDERMOTT* *Laboratory

AND

G. L. WESTBROOK?

of Neurophysiology, IRP, NICHD,

IRP, NINCDS, and tLaboratory of Developmental Neurobiology, National Institutes of Health, Bethesda, Maryland 20205 Received March 15, 1985

Spinal cord neurons were dissociated from 13-day embryonic mice and grown in culture for l-28 days. Sodium currents of neurons in culture for l-2 days were compared with those in culture for 2-4 weeks, using the whole-cell voltage clamp method. Rapid neurite outgrowth created space clamp limitations so that unclamped neuritic sodium action potentials prevented accurate analysis of sodium current properties. Therefore neurons were bathed in sodium-free solution and brief puffs of sodium were delivered to the cell soma so that only somatic sodium currents were recorded. Sodium currents of neurons at 1-2 days in culture had voltage-dependent activation and inactivation characteristic of these channels, both in mature cultured spinal neurons and in other preparations. However, the estimated channel density on the soma of neurons 1-2 days in culture was less than two channels per pm*. Since the available sodium conductance (as measured by action potential rise rates) increases during development of spinal cord neurons in culture (Westbrook and Brenneman, 1984), we suggest that changes in channel density and/or distribution, rather than in channel kinetics, o 1986 Academic PWS. I~C. may underlie the increase in sodium conductance. INTRODUCTION

with a tetrodotoxin (TTX)-induced depression of CAT activity in cultures of spinal cord neurons, suggesting that electrical activity may play a role in neuronal development in culture. Recently, using whole-cell patch recording in current clamp, we have found that spontaneous electrical activity as well as the properties of evoked action potentials of spinal cord neurons undergo a characteristic development during the first 21 days in culture; in particular, the rise rate (V,,,) of evoked action potentials increases from 50 to 100 V/see in the first week to 250-300 V/see after 3 weeks (Westbrook and Brenneman, 1984). These later values are similar to those reported for mature spinal cord neurons in culture (Heyer et al., 1981). The action potentials throughout this period are sodium-dependent and TTX-sensitive (Ransom and Holz, 1977; Westbrook and Brenneman, 1984). Since the sodium conductance dominates total membrane conductance during the rising phase of the action potential, the increase in action potential rise rates must be accounted for either by a change in sodium channel kinetics, channel density/distribution, or both. W e have used the patch clamp technique in the whole-cell voltage clamp mode (Hamill et ah, 1981) to examine the ionic basis and kinetic properties of the inward currents underlying action potentials during development in culture. Since the complex morphological structure of spinal cord neurons, even after l-2 days in culture, makes voltage clamp of sodium currents virtually impossible (Smith et al., 1980), we have devised a technique which restricts

Primary cultures of dissociated mammalian spinal cord neurons have been used extensively for studies of the cellular physiology and pharmacology of central neurons (Nelson et al., 1981; Macdonald and Barker, 1981). The majority of these studies have used neurons taken from embryonic tissue which is then grown in culture for several weeks before physiological study. Intracellular studies using standard microelectrodes during the first 2 weeks in such cultures have been difficult due to the small size of the neurons at this stage (soma diameter 5-10 pm). However, it is during the initial 2 weeks that cultured spinal cord neurons undergo many of the changes characteristic of development in vivo, e.g., neurite outgrowth, synapse formation, and cell death (Jackson et al., 1982); and undergo biochemical changes such as induction of choline acetyltransferase (CAT) (Brenneman and Warren, 1983) before reaching a “mature” stage at 3-4 weeks. Initial neurite outgrowth in these cultures includes both formation of new neurites and some regeneration since neurons when cultured are O-4 days postmitotic (Nornes and Carry, 1978; McConnell, 1981). The mature stage is characterized by intense synaptic activity as well as relatively constant values for total neuronal counts and biochemical parameters (see Nelson and Brenneman, 1982). Using extracellular recording with patch electrodes, Jackson et al. (1982) found that increases in spontaneous electrical activity during the second week in culture correlated 317

0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

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the area of membrane exposed to sodium to the soma, and thus allows adequate voltage clamp of fast inward currents. Our results demonstrate that within 24 hr in culture, the action potentials of spinal cord neurons from 13-day embryonic mice arise from sodium currents. We were unable to detect a change in sodium current activation or inactivation over the first 4 weeks in culture. Thus a change in channel density and/or distribution rather than in channel kinetics may be a more likely explanation for the increase in action potential rise rates. A preliminary description of these results has been presented in abstract form (MacDermott and Westbrook, 1984). MATERIALS

AND

METHODS

Cell culture. Cultures were prepared from the spinal cords of 13-day embryonic mice (C57BL/6J) similar to those previously described (Ransom et ab, 1977). In brief, the dissociated spinal cord neurons were plated on collagen-coated plastic 35-mm dishes (Falcon) at a density of 6 X lo5 cells per dish. The cells were maintained for the first 24 hr in plating medium: 80% minimal essential medium (MEM, GIBCO), 10% horse serum, and 10% fetal bovine serum. At Day 1 the cells were switched to growth medium. The growth medium consisted of 95% MEM, 5% horse serum with an added nutrient supplement (Romijn et al., 1982). A half change of medium was performed twice weekly; Fluoro-2’-deoxyuridine (13 pg/ml) with uridine (33 pg/ml) was present from Day 5 to Day 9 to suppress growth of background cells. No antibiotics were used. Electrophysiology. Experiments were performed at room temperature (25-27°C) on the stage of an inverted phase-contrast microscope. Three recording solutions were used. Sodium solution contained (mM): 140 Na, 5 K, 2 Ca, 1 Mg, 151 Cl, 10 Hepes, 10 glucose. Choline and sucrose solutions were identical except sodium chloride was replaced with 140 mh4 choline chloride or 280 mM sucrose, respectively. All solutions were adjusted to pH 7.3 with NaOH and to 325 mOsm with sucrose. Patch electrodes for whole-cell voltage clamp and current clamp were made using 1.5-mm O.D. borosilicate (“hard”) glass (TW150F, W-P Instruments) with a twostage vertical pull (Kopf 700D). Firepolished electrodes had resistances of 4-5 MR when filled with either KMeS04 or CsCl solutions (mM): 140 KMeS04 or CsCl, 2 Mg, 1.1 EGTA, 10 Hepes; pH 7.2, 310 mOsm. Voltage clamp was performed using either a discontinuous oneelectrode voltage clamp (Axoclamp, Axon Instruments) or a whole-cell voltage clamp (List EPC-7). The discontinuous clamp had the advantage of measuring membrane voltages rather than headstage voltages as was the case with the patch clamp amplifier; however, for

VOLUME 113, 1986

the small currents (20-500 PA) measured in these experiments, both amplifiers gave similar results and thus data was pooled for analysis. When using the patch clamp amplifier, series resistance compensation was employed (usually 5-10 MR). Input capacitance to the headstage was minimized (~1 pF) by lowering the bath level, using “hard” electrode glass, and plugging the electrode holder directly into the headstage. This allowed switching frequencies of lo-15 kHz when using the discontinuous clamp. Membrane current and voltage from the voltage clamp were further amplified, filtered (onepole, Fe = 5 kHz), digitized at lo-15 kHz, and collected on a PDP 11123 computer system for further analysis. Voltage-clamp protocol. Due to rapid neurite outgrowth within hours of plating, space clamp limitations prevented adequate voltage clamp of sodium currents in these neurons in normal, i.e., sodium-containing, medium (see below). Therefore a method (puff/jump) was devised to restrict the area of membrane exposed to sodium. Neurons were bathed in either choline or sucrose recording medium. Isotonic sodium was then delivered to the cell soma by brief pressure application (lo-100 msec) from a pipet positioned within 10 pm of the soma (Fig. 1A). Pressure pulses were triggered by solenoidcoupled pulse generator and were adjusted to precede the membrane voltage command at a fixed interval. Puffer pipets had tips of l-2 pm and were quite reliable in their release characteristics such that the area exposed to sodium could be limited to IO- to 20-pm zones during the period immediately following ejection. The area of membrane exposed to sodium could be monitored visually under phase-contrast microscopy since spread of the sodium solution could be seen as a phase-dark shadow. Thus delivery from individual pipets could be adjusted prior to each experiment. Movement of the sodium pipet 20-30 pm away from the soma resulted in loss of a measurable sodium current. Membrane potential was initially held at -80 mV and a series of 40- to 50-msec depolarizations to the same command voltage were applied at 0.5 Hz and averaged. This procedure was performed in the sodium-free bath and then during application of sodium solution. The membrane current records from the control and sodium puff series were then digitally subtracted to obtain the evoked sodium current. An example of the membrane current evoked by a voltage jump from -80 mV to -25 mV immediately prior to, and 50 msec after, a puff of sodium is shown in Fig. 1B. Separate controls were obtained for each voltage jump. Slow inward currents were occasionally seen in the sodium-free solution ([Ca”‘], = 2 mM), but were usually small and were removed by the subtraction procedure. This subtraction method was valid only at membrane potentials more negative than the sodium reversal po-

MAC DERMOTT AND WESTBROOK

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FIG. 1. (A) Hoffman modulation contrast photomicrograph of spinal cord neuron after 36 hr in culture. Recording electrode is on the soma while pipet for delivery of sodium solution is positioned near the soma. Calibration bar 10 pm. (B) Membrane currents before and 50 msec after the beginning of sodium puff (50 msec, 1.5 psi) are shown at left during a 50-msec voltage jump from -80 mV to -25 mV. No inward current is present in the absence of sodium, Each current is average of 12. The evoked sodium current after the subtraction procedure is shown at right. Downward deflection represents inward current. Preparation 32004 using switching one-electrode clamp at 11 kHz.

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DEVELOPMENTAL BIOLOGY

tential (ENa), i.e., when sodium current flow was net inward. Control records should contain small outward sodium currents since the sodium-free external solution provides a large driving force for the efflux of sodium. However, this effect was quite small in our experiments since [Na+]i was presumably reduced below normal levels due to perfusion with the sodium-free patch pipet solution. A slow, inward tail current sometimes followed the transient sodium current in the puff-subtracted records. This current must be either sodium or pressure dependent since it did not subtract away as would be expected for ionic currents present both before and during the puff, such as calcium or potassium currents. We did not analyze this current further. In experiments designed to estimate the equilibrium potential of the sodium current, 14 or 28 mM Na+ was added to the internal patch pipet solution. For these experiments, a standard method of leak subtraction using a negative voltage jump was employed. In some experiments with mature cultured spinal cord neurons, tetrodotoxin (5-20 r&f, Sigma) was added to the bath (and the sodium puffer pipet) to reduce the total sodium conductance. Stationarity of sodium channel kinetics was monitored by repeating a standard voltage jump (usually to a command voltage of -20 mV) throughout the experiment. On occasion, shifts in activation and inactivation kinetics were seen with longer periods of whole-cell recording (Fernandez et al., 1984); however, only periods with stable kinetics were used for analysis.

VOLUME 113, 1986

resting membrane potential fell within seconds to near 0 mV suggesting rapid perfusion of the soma (Fenwick et ah, 1982a). Following loading of the neuron with Cs+, unclamped inward currents were evoked in normal sodium-containing bath solutions. These could be identified

A ---+---

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L

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RESULTS

Sodium-Dependent

Inward Current

Experiments were performed on more than 100 spinal neurons during the first 28 days in culture. The majority of results reported here were obtained either within 48 hr of plating, or after 14-28 days in culture. Cells were identified as spinal cord neurons on the basis of their phase bright soma and presence of neurites. At the earliest time studied in these experiments (18 hr in culture) rapidly rising action potentials could be evoked with intracellular current pulses from the resting membrane potential (Fig. 2A). Early in culture, evoked action potentials had relatively high thresholds in normal medium. These action potentials are sodium-dependent and are totally blocked by 0.6 PM TTX (Westbrook and Brenneman, 1984). Under voltage clamp, a series of depolarizing voltage commands from -80 mV demonstrated a fast net inward current followed by net outward current (Fig. 2B). In order to record sodium currents in relative isolation, outward potassium currents were blocked with cesiumcontaining patch electrodes. Under these conditions the

50 mV 250 PA

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10 ms FIG. 2. (A, B) Comparison of current clamp and voltage clamp behavior of a spinal cord neuron after 2 days in culture using a KMeSO,containing patch electrode. An overshooting action potential was elicited by a lOO-pA lo-msec current pulse under current clamp (A). A steady hyperpolarizing current was used to hold the membrane potential at -80 mV (resting potential -50 mV). Dotted line indicates 0 mV. (B) Headstage voltage (above) and membrane current (below) recorded during voltage clamp of same neuron at -80 mV. Depolarizing voltage steps were to -70, -60, -50, -40, -30, -25, and -20 mV. Voltage sag on step to -20 mV denotes unclamped event as seen by inverting input of headstage. Current records are leak subtracted. Preparation 22308. (C) Typical voltage clamp recording of another neuron using a CsCl-containing patch electrode. Voltage step to -40 mV (holding potential -80 mV) elicits essentially no current while step to -20 mV elicits fast inward current which is preceded by a 5 msec delay and followed by an unclamped “afterhyperpolarization” as well as repetitive firing, consistent with unclamped action potentials generated in neurites (see text). Preparation 42708. Recordings in (B) and (C) obtained with patch clamp amplifier.

MAC DERMOTT AND WESTBROOK

Sodium

as all-or-none events at a variable delay after the beginning of the voltage jump. Small increases of l-5 mV in the amplitude of the voltage step decreased the delay without changing the shape of the evoked current. The evoked current also had a multiphasic shape suggesting invasion of current from unclamped regions of the neuron (Fig. 2C). Bathing neurons in either low sodium (35 mM) or low concentrations of TTX to reduce total sodium conductance still resulted in unclamped inward currents. This behavior has been previously demonstrated for sodium currents in mature cultured spinal neurons due to action potential generation in unclamped neurites (Smith, 1983). Since invasion of unclamped action potentials was apparent in neurons within 24 hr after plating when only a few thin neurites were present, it is likely that these newly formed neurites contain TTXsensitive sodium channels, as has been described in developing grasshopper neurons (Goodman and Spitzer, 1981). Using brief puffs of sodium solution, we were able to limit the access of sodium to areas of membrane near the soma (see Methods). Although the exact concentration at the membrane surface was unknown, the exposure was sufficient to evoke a regenerative sodium action potential when a current pulse was applied under current clamp (Fig. 3). The sodium solution diffused rapidly away from the neuron such that little or no sodium current was present 300 msec following application (Fig. 4). Maximal current amplitudes usually occurred 20-50 msec after onset of sodium puff, and were always absent when the next stimuli was applied at intervals of 2 sec. Current-voltage relationships were plotted at the sodium puff-voltage jump interval which resulted in the largest current amplitude for each neuron (e.g., 43 msec in Fig. 4). A spatial

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FIG. 3. Regenerative sodium-dependent event elicited during puff of sodium solution to soma of neuron bathed in isotonic choline. Voltage records before and during application of sodium (100 msec, 1.5 psi) are superimposed. Preparation 20511.

No

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FIG. 4. Time course of exposure to sodium solution. A depolarizing voltage step to -20 mV from a holding potential of -80 mV (third trace from top) was used to elicit an inward sodium current (bottom trace). The time between the onset of a 30-msec puff of sodium solution (upper trace) and the beginning of the voltage step was then varied and sodium currents measured to obtain a relative concentration profile for external sodium (second trace from top). Sodium currents at the peak (43 msec) and after a delay of 200 msec had similar time courses (43 msec: decay time constant = 4.2 msec; 200 msec: decay time constant = 4.0 msec). Preparation 10709.

gradient of external sodium concentration inevitably occurred across the membrane exposed to sodium. This would affect the ionic driving force and single channel conductance, but the shape of the current (i.e., activation and inactivation) should remain constant. As shown in Fig. 4 for currents evoked 50 and 200 msec after the sodium puff, this appeared to be true. Sodium

Current

Kinetics

of Neurons

Early

in Culture

An example of sodium currents evoked from a neuron after 2 days in culture is shown in Fig. 5A. The evoked current rose rapidly to a peak value then decayed back to the baseline for the remainder of the 40-msec command pulse. The peak inward current reached a maximum at -15 mV for this neuron (mean f SD = -10 k 4 mV, n = 6) and then decreased with further depolarization (Fig. 5B). Using a sphere of 8pm diameter to estimate the surface area exposed to sodium for this neuron, the peak inward current obtained was 87 pA/cm2. The rate of decay of the sodium current during sustained depolarization could be reasonably fit with a single exponential function. The decay time constants (TV) were voltage dependent over the range of depolarizations tested (Fig. 5C), decreasing e-fold per 26 f 8.7 mV depolarization (n = 6). A characteristic feature of sodium channels is voltagedependent inactivation as defined by Hodgkin and Huxley (1952a,b). Such behavior was apparent for the sodium

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FIG. 5. (A, B) Current-voltage relationship of the sodium current in a spinal cord neuron after 2 days in culture. Inward sodium currents evoked by depolarizing voltage steps to -30, -25, -15, and 0 mV from a holding potential of -80 mV are shown in (A). Peak inward current as a function of command voltage is shown in (B). Internal patch solution contained was sodium-free. (C) The decay time constant of the sodium current (T,,) during the voltage step decreased with larger depolarizations over the range of voltages examined. Time constants are plotted on a linear scale. q, decreased e-fold/39 mV depolarization for this neuron. Preparation 32004.

currents evoked in spinal cord neurons at l-2 days in culture. Figure 6A shows currents evoked by a voltage jump to -20 mV from a series of different holding potentials. A voltage jump from -80 mV evoked nearly maximal inward current while essentially no inward current was seen from a holding potential of -40 mV. The normalized peak currents as a function of voltage could be fit with an equation of the form

where Vh is the half-inactivation value (h = 0.5) and Kh is a constant. The half-inactivation voltage for three neurons was -71 -+ 8 mV where Kh was -7 + 1. Steadystate inactivation curves obtained by altering the holding potential would be expected to include effects of fast inactivation (as measured by a short prepulse) as well as ultraslow inactivation (Fox, 1976). However, we have not attempted to separate these two processes (e.g., see Huang et al., 1982).

gave an estimated [Naflo = 42 m&f during the sodium application. Nonetheless the absolute value of the external sodium concentration during the puff of sodium solution was unknown (see Fig. 4). In addition, outward currents above 0 mV interfered with measurement of sodium current amplitudes (see Fig. 7A). Thus we could not directly compare the reversal potential with the equilibrium potential for sodium. However, sodium currents in the presence of external Na+ (“with Na,” Fig. 7C) had a reversal potential which was more positive than the extrapolated reversal potential with [Na’l, = 0 mM and [Naili = 28 mM (“no Na,” Fig. 7C). This shift of reversal potential, as well as our inability to reverse sodium currents in the absence of Na+ in the patch solution, is consistent with an ion selectivity of the channel that is greater for sodium than other internal cations (primarily cesium, in our experiments) as has been demonstrated in both squid axon and frog myelinated nerve (see Hille, 1984, for review).

Reversal Potential

Sodium Currents

h = l/[l

+ exp( V-

Vh)/Kh

of the Sodium Current

We were unable to reverse the sodium current in neurons dialyzed with sodium-free patch solution. However, when 28 mM Na+ was added to the patch solution, a clear reversal of the sodium current could be obtained. The sodium currents and current-voltage relationship from one such experiment are shown in Fig. 7A and B. The interpolated reversal potential was +lO mV. This

in Mature

Spinal Cord Neurons

To compare the sodium currents of developing neurons in culture with mature cultured spinal cord neurons, we used the puff/jump method to record sodium currents in neurons after 2-4 weeks in culture. After a puff of sodium solution to the soma, the peak amplitude of the sodium current was much larger (approximately l-2 nA) than at l-2 days in culture and the voltage control was

MAC DERMOTT AND WESTBROOK

dent decreasing e-fold per 15 f 5 mV depolarization (n = 6). The steady-state inactivation curve, obtained from a series of voltage steps to -20 mV from the same neuron as in Figs. 8A and B, is shown in Fig. 8C. The halfinactivation voltage was -65 mV for this neuron (V = -64 k 2 mV, n = 4).

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DISCUSSION 1

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msec

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I

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0 .,.,.,.,.,.I=, -105 -95 -85

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MV

FIG. 6. (A, B) Steady-state inactivation of the sodium current in a Z-day neuron. Voltage steps to ~20 mV were made from a series of holding potentials between -100 and -40. Sodium currents at holding potentials of -80, -70, -60, and -50 mV are shown in (A). Peak inward currents were plotted as a function of holding potential in (B). Peak inward current at a holding potential of -100 was -144 pA. Curve was fitted by nonlinear regression to the function described in the text. The half-inactivation voltage (V,) was -62 mV and Kh = -5.8 for this neuron. Preparation 32004.

poor. To reduce this large sodium conductance and thus improve the voltage control, low concentrations of TTX were added to the bath and to the sodium-containing pipets. TTX reduces the maximum sodium conductance without affecting the time course of the remaining sodium currents (Hille, 1968); and, as expected, action potentials had reduced rates of rise when the bath contained 140 mM sodium and 5 nM TTX. Thus both restriction of the membrane exposed to sodium and a lowered sodium conductance were necessary to voltageclamp mature neurons. A family of sodium currents obtained from a spinal cord neuron after 25 days in culture is shown in Fig. 8A. As with the neurons at l-2 days in culture, the amplitude and time course of the currents were voltage-dependent. The current-voltage relationship reached a peak near -15 mV (Fig. 8B). For four neurons, the peak inward current occurred at -16 -t 5 mV. The decay time constant during sustained depolarization, rh, was voltage depen-

Protocol

Although neurons early in culture had only a few fine neurites, the resulting voltage gradients, i.e., spaceclamp errors, seriously limited the analysis of the fast kinetics of the current underlying action potentials. As has been demonstrated with the use of neuron models this effect is frequency-dependent such that distortion is severe for rapid conductance changes (Johnston and Brown, 1983; Rall and Segev, 1985). The method of puff/ jump allowed us to analyze sodium currents within regions of voltage control. This was substantiated by increasing the duration of the puff which led to unclamped events of the type seen in Fig. 2B. This strategy may be particularly useful to characterize ionic channels when channel density is low, or to compare macroscopic currents with single channel behavior on cells where adequate space clamp is difficult. A similar approach has recently been used at the squid giant synapse to activate only a limited portion of the presynaptic membrane by controlling the area exposed to calcium (Smith et al., 1985). Sodium

Channel Properties

Voltage-dependent activation and inactivation of the Hodgkin-Huxley (H-H) type, and ionic selectivity comprise three major properties for comparison of sodium channels in different cells and tissues. Although recent observations of single sodium channels require reevaluation of sodium channel gating (Aldrich et al., 1983), we have used the standard H-H definition of inactivation as one basis for comparison of sodium currents. The sodium currents of spinal cord neurons at l-2 days in culture display these three basic characteristics. However, large capacitative currents flowed into neurites following a voltage jump and partially obscured the rising phase of the sodium current at depolarizations beyond about -30 mV. Therefore we did not attempt a complete H-H kinetic analysis. However, the voltage of maximum inward current on the I-V relationship (-10 mV) in the 1-2 day cells was similar to mature spinal cord neurons (-16 mV) and to other preparations (Hille, 1984). The voltage dependence of Th (25’C) between -40 and 0 mV was e-fold/26 mV and e-fold/l5 mV for l-2 day and mature spinal cord cells, respectively. These

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VOLUME 113, 1986

A

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MEMBRANE POTENTIAL

( 90

MV

FIG. ‘7. Reversal potential of the sodium current. (A) Sodium currents were evoked by a series of voltage jumps to command potentials from -50 mV to +70 mV from a holding potential of -80 mV. Currents illustrated are from jumps to -30, -20, -10,20,40, and 60 mV. Capacitative current transient during initial 500 psec not shown. (B) Peak inward currents from (A) are plotted as a function of command potential. Dotted line shows interpolated reversal potential (+5 to +lO mV). (C) I n another neuron, currents in the absence of [Na+& (diamonds) were compared to those during the sodium puff (dots). Holding potential was -80 mV; command voltages shown are from -20 to +80 mV. Presence of external Na+ caused a depolarizing shift in the estimated reversal for the sodium-dependent current. Sodium concentration in patch electrode was 28 mMfor both neurons. Preparation 50611 in (A) and (B) (7 days in culture); 40111 in (C) (24 hr in culture).

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FIG. 8. (A, B) Sodium current kinetics in a spinal cord cell after 25 days in culture. Current-voltage relationship of the sodium current. Inward sodium currents evoked by depolarizing voltage steps to -35, -30, -20, -10, and 0 mV from a holding potential of -80 mV are shown in (A). Peak inward current as a function of command voltage is shown in (B). Internal patch solution was sodium-free. (C) The steady-state inactivation curve is shown for the same cell. Preparation 10906.

MAC DERMOTT AND WESTBROOK

Sodium

values are within the range reported for squid axon (Bezanilla and Armstrong, 197’7) frog skeletal muscle (Campbell and Hille, 19’76), mouse neuroblastoma cells (Moolenaar and Spector, 1978), and rat clonal pituitary (GH3) cells (Vandenberg and Horn, 1984). The half-inactivation voltages (1-2 day neuron: Vh = -71 mV; mature neuron Vh = -64) compare with values of near -60 mV for squid axon (Hodgkin and Huxley, 1952) -75 to -85 mV in frog skeletal muscle (Campbell and Hille, 1976), -65 mV in neuroblastoma cells (Moolenaar and Spector, 1977), and -66 mV in GH3 cells (Vandenberg and Horn, 1984). Action

Potential

Mechanism

Action potential rise rates of these neurons increase approximately 3~ during the first three weeks in culture (Westbrook and Brenneman, 1984). Several possible explanations could account for this change. During development, some neurons, e.g., Rohon-Beard cells, progress through a stage of calcium-dependent action potentials before reaching a stage when sodium is the major charge carrier for the action potential (for review, see Spitzer, 1983). Calcium currents can be seen in spinal cord neurons at 2 days in culture when [Ca2+lo is raised to 5 mM and potassium channels are blocked (Westbrook, unpublished observation); however, the total calcium conductance in physiological solutions is not sufficient to produce an action potential. These results were obtained from spinal cord neurons which were between 1 and 5 days after final mitosis (Nornes and Carry, 1978; McConnell, 1981), thus an earlier period with Ca2+-dependent action potentials might occur. On the other hand, patterns of action potential development may vary between neurons, e.g., action potentials of cortical neurons from chick embryos at an early stage show a mixed Nat/ Ca2+ mechanism (Mori-Okamoto et ab, 1983) whereas the initial action potential current in quail neural crest cells is sodium-dependent (Bader et al., 1983a). The kinetics of the sodium currents in spinal cord cells at different times in culture were within the range of values found for other preparations. We are unsure if the small differences between the inactivation parameters (voltage dependence of Th and h) reflect true differences between developing and mature cultured spinal cord cells. Thus we cannot exclude the possibility of small kinetic changes during development but it is unlikely that such differences account for the markedly slower action potential rise rates at 2 days. However, increases in 3H-Saxitoxin binding in spinal cord cultures do parallel the changes in action potential rise rates, suggesting that increases in channel density may underlie this effect (Westbrook and Brenneman, unpublished observation). The neuron in Fig. 5 had an estimated peak inward cur-

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rent density at -20 mV of 87 pA/cm2, similar to estimates on chromaffin cells (Fenwick et al., 1982b) and on developing chick ciliary ganglion neurons in culture (Bader et al., 1983b). Using this current density we could obtain an approximate channel density. Single channel currents of up to 2 pA have been seen with sodium-free patch solutions in chromaffin cells (Fenwick et al, 198213). Assuming [Nalo = 35-70 mM the single channel current would be 0.5-l pA at -20 mV. Combined with the fraction of available conductance at -20 mV (estimated at 0.9 from data of Fig. 7B) the number of active sodium channels would be only l-Z/pm2 of somal membrane. Catteral (1984) using 3H-Saxitoxin binding has found a density of 50-75 sodium channels per square micrometer on the cell bodies of rat spinal cord neurons after 3 weeks in culture. We have observed larger sodium currents after 2-4 weeks in culture. Thus it seems likely that sodium channels do increase in density during the development of spinal cord neurons in culture. In addition, Catteral (1981) has shown that about 40% of rat spinal cord neurons after 2-4 weeks in culture have sodium channel “hot spots” on proximal neurites as determined by 1251labeled scorpion toxin autoradiography. The role of nonhomogeneous distributions of sodium channels in the development of excitability remains to be determined. We thank Dr. P. G. Nelson and Dr. J. L. Barker for their support, Dr. P. Guthrie and Dr. D. Lange for use of computer programs, S. Fitzgerald for preparation of cultures, and Dr. T. G. Smith and Dr. M. L. Mayer for reading earlier versions of this manuscript.

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MOOLENAAR, W. H., and SPECTOR,I. (1978). Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions. J. Physiol. (Londcm) 278, 265-286. MORI-OKAMOTO, J., ASHIDA, H., MARU, E., and TATSUNO, J. (1983). The development of action potentials in cultures of explanted cortical neurons from chick embryos. De% Biol. 97,408-416. NELSON, P. G., NEALE, E. A., and MACDONALD, R. L. (1981). Electrophysiological and structural studies of neurons in dissociated cell cultures of the central nervous system. In “Excitable Cells in Tissue Culture” (P. G. Nelson and M. Lieberman, eds.), pp. 39-80. Plenum, New York/London. NELSON, P. G., and BRENNEMAN, D. E. (1982). Electrical activity of neurons and development of the brain. Trends Neurosci. 5,229-232. NORNES, H. O., and CARRY, M. (1978). Neurogenesis in spinal cord of mouse: An autoradiographic analysis. Brain Res. 159, l-16. RALL, W., and SEGEV, I. (1985). Space-clamp problems when voltage clamping branched neurons with intracellular microelectrodes. In “Voltage and Patch Clamping with Microelectrodes” (T. G. Smith Jr., H. Lecar, S. J. Redman, and P. W. Gage, eds.), pp. 191-215. Amer. Physiol. Sot., Bethesda, Md. RANSOM, B. R., NEALE, E., HENKART, M., BULLOCK, P. N., and NELSON, P. G. (1977). Mouse spinal cord in cell culture. I. Morphology and intrinsic electrophysiologic properties, J. Neurophysiol. 40, 11321140. RANSOM, B. R., and HOLZ, R. W. (1977). Ionic determinants of excitability in cultured mouse dorsal root ganglion and spinal cord cells. Brain Res. 136,445-453. ROMIJN, H. J., HABETS, A. M. M. C., MUD, M. T., and WOLTERS, P. S. (1982). Nerve outgrowth, synaptogenesis and bioelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium. Dev. Brain Res. 2, 583-589. SMITH, S. J.. AUGUSTINE, G. J., and CHARLTON, M. P. (1985). Transmission at voltage-clamped giant synapse of the squid: Evidence for cooperativity of presynaptic calcium action. Proc. NatL Acad. Sci. USA 82, 622-625. SMITH, T. G., JR. (1983). Sites of action potential generation in cultured vertebrate neurons. Bruin Res. 288,381-383. SMITH, T. G., JR., BARKER, J. L., SMITH, B. M., and COLBURN, T. R. (1980). Voltage clamping with microelectrodes. J. Neurosci. Methods 3,105-128.

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