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JOURNALOFN EUROPHYSIOLOGY

Vol. 43, No. 3, March 1980. Printed in U.S.A.

Analysis of Ionic Conductance Mechanisms in Motor Cells Mediating Inking Behavior in Aplysia californica

JOHN H. BYRNE

Department of Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

SUMMARY AND CONCLUSIONS

1. The release of ink in response to a noxious stimulus is a relatively stereotyped behavior produced by strong and long-lasting stimuli. The purpose of this series of papers is to determine the quantitative extent to which the known voltage- and time-dependent ionic conductance mechanisms and synaptic influences can account for the ink gland motor neurons’ firing pattern and, thus, the features of the behavior.

2. Four voltage- and time-dependent ionic currents have been analyzed. These include a fast transient Na+-mediated inward current, a slower Ca2+-mediated inward current, a fast transient K+-mediated outward current, and a slower delayed outward current also mediated by K+ ions.

3. The current-voltage (Z-V) relationships, equilibrium potentials, and steady-state activation and inactivation characteristics appear qualitatively similar to comparable currents observed in other gastropod neurons.

4. The recovery from inactivation of the delayed outward current has two time con- stants, one comparable to the inactivation time constant and the other more than an order of magnitude larger. The fast transient K+ current also appears to have a similar slow recovery from inactivation.

5. The synaptic current contributing to the firing pattern of the ink motor cells is a complex function of time. Initially, the synaptic conductance is high and the equilibrium potential near 0 mV. But, with time there is a gradual decrease in synaptic conductance and shift in the equilibrium potential to more depolarized levels.

INTRODUCTION

The release of ink in response to a noxious stimulus in Apfysia cafifornica is a relatively stereotyped behavior produced by strong and long-lasting stimuli (11, 33). A noxious stimulus must persist for greater than approximately 2 s to cause a significant release of ink (33). Carew and Kandel (11) found that the motor component of the behavior is mediated by three electrically coupled cells called Ll4~ B c located in the abdominal ganglion. There appears to be a good cor- respondence between the firing pattern of these cells and the features of the behavior. A train of electrical stimuli to the connectives that normally link the abdominal ganglion to the head mimics the synaptic input to the ink motor neurons produced by a noxious stimulus to the head (33). The initial synaptic input is relatively ineffective in firing the cell. As a result, there is a several- second silent period or pause before an accelerating burst of action potentials is produced, which leads to the subsequent release of ink from the ink gland. The dis- charge properties of the ink motor cells in turn appear, at least in a qualitative fashion, to result from two unique features of the L14 ink motor cells: a fast early K+ current that shunts the initial excitatory input and a late buildup of EPSPs (10).

The purpose of this series of papers is to determine the quantitative extent to which the known voltage- and time-dependent ionic conductance mechanisms and synaptic influences can account for the ink motor cells firing pattern and thus the features of the behavior. To accomplish these ends, the

630 0022-3077/80/0000-0000$01.25 Copyright 0 1980 The American Physiological Society

INK MOTOR CELL ANALYSIS 631

various conductance channels have been analyzed using the voltage-clamp technique. The first paper describes the details of the analysis, and the second paper (9) describes the development of a kinetic model where it is possible to examine to what extent the known ionic conductance mechanisms and synaptic influences can quantitatively account for the firing pattern of the cells. Pre- liminary reports of some of this material have previously been presented (6, 7).

METHODS

The methods are similar to those previously described (10). Additional details are summarized below.

Experimental data for this series of papers are based on over 200 experiments performed on the marine mollusk Aplysia californica. Animals were supplied by Pacific Bio-Marine Laboratories, Venice, CA and weighed between 100 and 150 g. To activate the L14 cells synaptically (Fig. 16), both the left and right connectives were drawn into suction electrodes and a 4.5-s train of current pulses (2 ms duration, 160-ms interpulse interval) was passed between the suction electrodes and a separate return electrode placed in the bath. Stimuli were presented once every 2-3 min to avoid decrement of the synaptic input. All experiments were performed at 15 2 1°C except those designed to examine the early inward current (Figs. 2, 3) and early outward current- reversal potential (Fig. 7). The preparation was normally perfused with artificial seawater (ASW) maintained at pH 7.6 by 10 mM of Tris buffer (Sigma). In experiments using cobalt chloride, tetraethylammonium (TEA), tetrodotoxin (TTX), or 4-aminopyridine (4-AP), concentrated volumes of these drugs were titrated directly into the experimental chamber containing a known volume of artificial seawater until the desired blocking effect was obtained. It was previously shown that TEA is an effective blocking agent for the ink motor cell delayed outward current (10) and was utilized for that purpose in the present analysis. 4-AP was shown to be effective in blocking the fast K+ current, but also partially blocked delayed K+ current. As a result, whenever 4-AP was used to isolate the fast K+ current, the delayed K+ current was always first blocked with TEA. In some experiments, blocking concentrations of TEA, 4-AP, or CoCl, were directly mixed with ASW prior to the experiment. In experiments designed to examine the delayed outward current recovery from inactivation, a solution of reduced Na+ and Ca*+ of the following composition was used (mM): 23

NaCl, 10 KCl, 55 MgC12, 0.5 CaCI,, 455 choline chloride, and 10 Tris.

Each soma with its intact axon was impaled with two electrodes, one for recording and the other for current injection. There was usually no more than a several millivolt difference in the potentials recorded from the two electrodes. Normal resting potentials were approximately -75 mV. Data were not collected from cells in which the resting potential fell below -60 mV or where there was more than a 5-mV change in potential when the second electrode penetrated the cell. Throughout this series of papers, typical responses are given from experimental runs where the most complete set of data was obtained. Each experimental ob- servation was, however, confirmed in other experiments, the results being in agreement with the data presented. In cases where it was difficult to choose an ideal data set or where considerable variability exists, responses from more than one experiment are illustrated. Due to difficulties in estimating surface area precisely, membrane currents are expressed in nanoamps rather than current density. In order to obtain comparable currents, however, cells of relatively similar diameters (approximately 175 pm) were selected for investigation.

Details of the voltage-clamp apparatus have previously been presented (10). For some experiments where large outward currents were examined (Figs. 5A and 9A), the voltage-clamp circuit was modified to compensate for up to 10 ka of series resistance. Clamp pulses were delivered at intervals ranging between 1 and 3 min so as to allow complete recovery from inactivation. Voltage and current traces were recorded on an FM tape recorder (Hewlett-Packard 3%0A) at a speed of 3.75 inches/s and displayed on a four-channel recorder (Gould 2400). Digital sam- pling (12-bit resolution) of the current waveforms was done on-line with a PDP11/34 computer (Digital Equipment Corp.) and the sampled traces were stored for later analysis on a magnetic disk. For each response, 500 samples were taken with the sample rate adjusted to be sufficiently fast to retain the details of the original current waveform. Multiple responses at each clamp level were usually obtained and then averaged. Individual ionic currents were then obtained by computer subtraction of postdrug treatment responses from predrug treatment responses. Digitized clamp waveforms were displayed on a storage display monitor (Tektronics type 603) driven by the computer digital to analog (D-A) converter. In one case (Fig. 16) the D-A converter signal was fed to the pen recorder. Best-fitting curves for the experimental data were obtained by a nonlinear least-squares regression routine using the “complex” method of Box (4) modified from Kuester and Mize (27).

632 J. H. BYRNE

mV

nA

mV

5CNImsec

FIG. 1. Block of axon-spike invasion of clamped soma. A : each of two ink gland motor neurons was impaled with two microelectrodes, one for recording and the other for current injection. Preparation was perfused with ASW to which 4-AP, 5 mM; TEA, 40 mM; and TTX had been added. Initially a small lo-mV hyperpolarizing clamp pulse was delivered to cell 1 to test the input resistance. This was followed 1.2 s later by an 80-mV step depolarization from resting level (-68 mV). The clamp pulse to cell 1 resulted in a slow multicomponent inward current (middle trace) and caused the initiation of an action potential in cell 2 (lower trace). B: an identical hyperpolarizing clamp was delivered to cell 1 but now it was followed 100 ms later by a depolarizing current pulse to cell 2, which triggered an action potential (lower trace). The depolarizing pulse to cell 2 was terminated, and 100 ms later an identical depolarizing clamp pulse as in A was again delivered to cell 1. A single inward current is observed (middle trace) and no action potential in cell 2 is initiated. The depolarizing prepulse to cell 2 presumably inactivates the slow inward conductance mechanism, preventing an additional action potential from being initiated with the test-clamp pulse.

As indicated above, voltage-clamp experiments were performed on the somata of ink gland motor neurons and with their axons intact. A serious complication, especially with the analysis of the inward currents, is that in some cases the clamp depolarization leads to the generation of axon spikes, which contaminate the current records (Fig. IA). To avoid these difficulties attempts were made to isolate the soma from the axon by ligature (3), but the ink motor cells seemed par- ticularly sensitive to this procedure and the original resting potentials could not be obtained after ligation. An alternative, although in principle less satisfactory technique, illustrated in Fig. 1 was utilized in cases where axon spikes were present. Recordings were made from two of the electrically coupled ink motor neurons. One cell was voltage clamped while the second cell was current clamped. To eliminate currents from axon spikes the depolarizing voltage-clamp pulse was preceded by a depolarizing current in the current-clamped cell, which was of sufficient magnitude to fire an action potential (Fig. 1B). This action potential presumably inactivates the inward conductance mechanisms in the unclamped cell and the axon

In order to determine the quantitative contribution which the ionic conductance mechanisms make to the firing pattern of the ink motor neurons, a detailed analysis of the membrane currents is necessary. It was previously shown that these cells have at least five pharmacologically identified voltage- dependent ionic conductance mechanisms (10, 32). These include a TTX-sensitive fast inward Na2+ current, a cobalt-sensitive slow Ca2+ inward current, a fast transient 4-AP- sensitive K+ outward current, a delayed TEA-sensitive K+ outward current, and an ultraslow TEA-insensitive outward current. The magnitude of the ultraslow outward current, which may represent a Ca2+-activated K+ current, appears to be small and has not been included in the present analysis.

of the clamped cell. The voltage-clamp pulse was then delivered and a membrane current relatively Fast inward current

uncontaminated by axon currents was obtained As previously described (lo), the fast (cf. current traces in Fig. 1A and B). In some inward current is selectively blocked by TTX cases axon spikes could also be blocked by and is presumably mediated by Na+ ions.

intense hyperpolarization of the current-clamped cell.

RESULTS


It i .s therefore possible to isolate this c bY subtracting voltage-clamp respon

urrent ses in

TTX from clamp responses in ASW. Due to the specificity of TTX for the fast inward current it is not necessary to block other voltage-dependent currents (see below) prior to the isolation procedure. Figure 2A illustrates an example of this approach with the temperature at 6OC. An ink motor cell is clamped from its resting potential (-60 mV) to a depolarized level of - 10 mV. There is a capacitive artifact, which is followed by a

transient inward current, which then decays and converts to an outward current. Figure 223 illustrates that this current is blocked when TTX is added to the experimental chamber. In Fig. 2C the currents from the normal seawater and TTX solutions have been subtracted, yielding a fast inward current uncontaminated by other voltage-dependent and leakage currents. All subsequent fast inward currents referred to in this study have been isolated using similar techniques.

A ASW 6 OC

B +TTX

C

50 nA

50 msec

FIG. 2. Fast inward current isolation. A: current produced as a result of a 200-ms step depolarization from a resting level of -60 mV to a depolarized value of - 10 mV. Preparation is perfused with artifical seawater (ASW) at 6°C. B: after adding 7 x low5 g/ml TTX to the perfusing solution, the clamp step is repeated. Note that the inward component is blocked. C: isolated fast inward current. The current waveform in B is subtracted from the current waveform in A, leaving an inward current uncontaminated by other cu i-rents normally present. The fast inward current has a rapid activation phase followed by a slower exponential inactivation phase.

634 J. H. BYRNE

P 1 la-

g-120-

- E -240 -

L a z

-360 -

-480 I I I I I I I I I I I 11

-55 -35 -15 5 25 45 65 HEMBRANE POTENTIAL (MVI

FIG. 3. Properties of fast inward current. A : I-V relationship. Fast inward currents are isolated as in Fig. 2 and the peak inward currents for a series of clamps from resting level to various depolarized levels are extrapolated to time of clamp onset. The estimated reversal level occurs at +67 mV. B : fast inward current inactivation. In a different cell as A membrane potential is stepped to various holding levels ranging from -96 to -6 mV. After a 200-ms period the cell is stepped to a new level of +4 mV. Points on the graph represent ratios between currents obtained when clamping from depolarized holding potentials and the maximum current obtained at a negative holding potential.

The fast inward current has a rapid rise and a slower exponential decay. At this level of depolarization (- 10 mV) the level of inactivation is almost complete (Fig. 2C). The peak inward current is voltage dependent. Figure 3A illustrates a plot of the peak inward current extrapolated to time of clamp onset, when the cell was clamped from a fixed holding potential (-60 mV) to various depolarizing levels. The fast inward current becomes strongly activated at a membrane potential of about -20 mV. The Z-V (current-voltage) data can be fit with an equation of the form:

I Na = $?Na *zNa(V) * (V - ENa) (1)

where gNa represents the maximum Na+ conductance, ZNa ( V) a voltage-dependent Na+ activation term, V the membrane potential, and ENa the Na+ equilibrium potential. The voltage-dependent Na+ activation ZNa can in turn be represented as the reciprocal of an exponential term added to unity (see Refs. 9 and 24).

where P determ ines the position of the activation curve on the voltage axis and S and II shape parameters. Using the Z-V data and the nonlinear regres sion analysis, the param-

zNa(v) = 1

(1 + e(p-v)‘s n )

eters of equations 2 and 2 can be estimated. The Z-V data are best fit by the equation:

I= 7.8

(1 + 4 (-ZR..i- L’J16.9 ) S (V - 67) (3)

where current is in nanoamps and voltage in millivolts. Raising the activation term to a power of 5 gives the best fit to the experimental data. The estimated reversal potential is +67 mV. In some cases, the equilibrium potential was also measured by observing the reversal of the inward peak with large depolarizing voltages. The results appeared similar to the curve fit estimates, but were somewhat ambiguous due to the initial capacitive artifact.

The time to peak of the fast inward current is voltage dependent, ranging from 17 ms at -20 mV to 5 ms at +30 mV (at 6°C). The peak inward current is dependent on the holding potential. Figure 3B illustrates the results of clamping another cell from various holdings potentials ranging from -96 to -6 mV to a fixed test level of +4 mV. The data points are the ratios between the currents obtained when clamping from depolarized holding potentials and the maximum current obtained when clamping from a hyperpolarized holding potential.

The inactivation data are best fit by the


equation (see Ref. 23): firing pattern of the L14 cells and the resultant

I 1 features of the behavior (10, 12). This current

- = I 1 + p+27.5”9.2

(4) is presumably mediated by K+ ions. Previous max studies (10, 35) have indicated that the fast

The half-inactivation point occurs at transient currents of this type can be rather -27 mV. selectively blocked by 4-aminopyridine (4-

AP). It is therefore possible to isolate and Fast transient outward current examine the fast transient outward current

A fast transient outward current has been by subtracting voltage-clamp responses ob- implicated as playing a critical role in the tained in 4-AP from voltage-clamp responses

A ASW + TEA + TTX + Co++

l -

B + 49AP

C

25 nA

200 msec

FIG. 4. Fast outward current isolation. A : current produced as a result of a l-s step depolarization from a resting level of -65 mV to a new value of -25 mV. Ganglion is perfused in ASW to which TEA (45 mM) TTX and Co2+ (15 mM) have been added. B: after adding approximately 12 mM 4-AP to the perfusing solution the fast transient current is blocked, leaving the leakage current. C: isolated fast outward current. Current in B is subtracted from current in A to yield a fast transient outward current uncontaminated by leakage currents. There is a rapid activation phase followed by a slower exponential inactivation phase.

J. H. BYRNE

-35 MV

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-5 MV ‘\ ii

45 E 168- - - MVj< _

j

5MV i 55 MV :.

. 0- I -68 -48 -20 0 20 48 68 75

MA

- \L-J ,2ze HEH8RRNE POTENTIAL (HVl

250 MSEC

FIG. 5. Fast outward current Z-V relationship. A: membrane potential is stepped from a fixed holding level of -65 mV to various depolarized levels ranging between -35 and +55 mV. Currents are isolated using pharmacologic separation. B : I-V plot. The peak current extrapolated to time of clamp onset for data of A are plotted as X'S. Additional data from 13 other experiments are plotted as stars (see text for details).

obtained in the absence of 4-AP. Since the 4-AP also acts as a partial blocker of the delayed outward currents and alters its kinetics (28), the delayed outward current was always first blocked with TEA (see below and Ref. 10). Figure 4 illustrates the general strategy utilized. Part A shows the response of one cell depolarized to -25 mV from a holding potential (resting potential) of -65 mV. In this example, the fast inward current was blocked by TTX, the delayed outward current blocked by low concentrations of TEA, and the slow inward current blocked by Co2+ (see below). Part B shows the response of the same cell after approximately 12 mM 4-AP was added. In C, the response in B was subtracted from A, yielding an uncontaminated fast outward current. Except where indicated, the subsequent analysis of the fast transient outward current utilizes a similar pharmacological separation.

The fast transient outward current has a rapid activation phase followed by a slower exponential inactivation phase. At -25 mV the inactivation is nearly complete (Fig. 4C). The magnitude of the peak outward current was highly voltage dependent. Figure 5A illustrates a series of responses obtained bv

clamping the cell from a fixed holding potential of -65 mV to various depolarized levels ranging between -35 and +55 mV. The peak current extrapolated to the time of clamp onset is plotted in Fig. 5B. In addition to the data from the experiment of Fig. 5A, Fig. 5B contains data from 13 other experiments where the fast outward current was isolated by a nonpharmacologic means. These experiments were performed to obtain an independent estimate of the fast K+ current since previous studies have indicated that concentrations of TEA utilized to first block the delayed K+ current may also partly block the fast K+ current (10,35). For weak depolarizations, the fast outward current is the only voltage-dependent current activated. Therefore, at these levels of membrane potential the fast outward current can be estimated by subtracting the extrapolated leakage current from the total peak outward current. The Z-V data were again described using equations similar to equations 2 and2 and the various parameters estimated. The Z-V data for the fast outward current are best fit by the equation:

I 7.0 =l+e t&7-fW18.4

(V + 65) (5)


200 MSFf

0

FIG. 6. Fast outward current steady-state inactivation. A : cell is stepped to various holding levels ranging from - 110 to -40 mV. After a 500-ms period, the cell is stepped to,a new fixed value of +20 mV. Traces shown are currents produced by that second step. Currents are isolated by pharmacologic means. B: points on the graph are ratios between the fast outward currents obtained when clamping from depolarized holding levels and the maximum value obtained at a hyperpolarized holding level. See text for equation best fitting the data.

In this case however, the equilibrium potential of -65 mV was estimated by direct means, as illustrated in the experiment of Fig. 7. The fast outward current has a threshold near -50 mV, well below the level of significant activation of the other identified ink motor cell currents.

As with the fast inward current, the magnitude of the fast outward current is dependent on the value of the holding potential. Figure 6A illustrates the outward currents obtained when the membrane potential is stepped from holding potentials between - 110 and -40 mV to a fixed clamp level of 0 mV. As in the Z-V studies, the early outward current is separated using 4-AP. The relationship between the holding potential and the current for each respons e divided by the maximum current is plotted in Fig. 6B. The data are best fit by the equation:

I 1 -= I 1 + ,W+54.2)/6.5 (6)

max

The half-inactivation point occurs at -54.2 mV.

The equilibrium potential of the fast outward current was investigated using the double-pulse technique of Hodgkin and Huxley (22). To obtain a more precise measurement of the tail current, this experiment

was performed at low temperature. Figure 7 illustrates these results. The fast inward and delayed outward (see below) currents are blocked and a series of pulses as shown in part Al are generated. The fast outward current is then blocked by 4-AP (Fig. 7A,) and the clamp series repeated. The currents are subtracted and the tails extrapolated to the onset of the second clamp pulse. The results are plotted in Part B. The instantaneous Z-V plot is nonlinear, but a reversal appears to occur at about -65 mV.

Delayed outward current

As the cell is depolarized beyond the threshold for the fast outward current, an additional slower outward current is activated. It was previously found (10) that this current is selectively blocked by TEA and is presumably mediated by K+ ions. Figure 8 illustrates the general strategy for its isolation. The early inward current is blocked by TTX, the slow inward current blocked by 10 mM Co2+ (see below), and a clamp series is initiated. In Fig. 8A the cell is clamped from a holding potential of -65 mV to a depolarized level of +5 mV. The delayed outward current is then blocked by TEA, leaving the fast transient outward current and the clamp procedure is repeated (Fig.

638

A I

J. H. BYRNE

2TTX tTEA

3TTX tTEA t4-AP

__---_--se ___-m--e---

_ - - - - - -se- - - - - - - - - - - -

- - - - - - _ - - - - - - - - - - - - - ~

- - - - - - - - - - - - - - -______

-60 _ --------------- ___--- ~---~__--------_____ -----_----------____-

VI-, I 20mV

/ -. lr

I 50nA I

200mSec MEMBRANE POTENTIAL (mV)

FIG. 7. Fast outward current equilibrium potential estimation. A : clamp protocol and pharmacologic separation. A,: with TTX in the bathing medium (ASW), TEA is titrated into the experimental chamber until the delayed outward current is blocked (final TEA concentration, 21 mM). Cell is then stepped from a resting level of -60 mV to a fixed depolarization of + 10 mV. The membrane potential is then stepped back to various less depolarized levels. A,: typical response to above protocol when membrane potential is stepped back to -20 mV. A,: after adding 3.6 mM 4-AP, the fast transient outward current is abolished. B : tail current magnitude versus membrane potential. For each clamp level the tail current extrapolated to time of second clamp pulse in presence of 4-AP is subtracted from the tail current obtained in TTX and TEA solutions. The smooth curve was drawn by eye. Temperature, less than 8°C.

8B). The uncontaminated delayed outward current is then obtained by computer subtraction (Fig. 8C).

Like the fast inward and fast outward currents, the delayed outward current shows a rapid activation phase followed by a slower exponential inactivation phase. With the level of depolarization in Fig. 8C (+5 mV), the inactivation is incomplete. The delayed outward current is also highly voltage dependent (Fig. 9A). The cell was clamped from a fixed holding level of -65 mV to various test levels ranging from -45 to 45 mV. The peaks have been extrapolated to the time of the clamp onset. These results, in addition to data from three additional experiments, are plotted in Fig. 9B. The Z-V data are best fit by the equation:

cell is clamped to a fixed depolarized level of -3 mV from various holding levels ranging from -93 to -13 mV. The ratio between the current produced by the second clamp and the maximum current obtained with the holding potential at a hyperpolarized level was then calculated. These results in addition to the results from a similar experiment performed on another cell are plotted in Fig. 1OB. The pooled data are best fit by the equation:

I 1.0 -= I (1 +e (v+8.0)/12.8 3

max ) (8)

Raising the inactivation equation to the third power gave the best fit to the experimental data. The half-inactivation point occurs at about -25 mV.

The recovery from inactivation of the de- I= 19.8

( 1 + e’-“.6- W/7.6)2 (V + 75) (7) layed outward current was also examined.

A series of double-clamp pulses are first The equilibrium potential of -75 mV was delivered while the cell was perfused in a estimated independently by the experiment 5% Na+ and 5% Ca2+ solution to attenuate of Fig. 12. The time to peak ranged from the early and slow inward currents. The cell 428 ms at -25 mV to 38 ms at +45 mV. is clamped to +20 mV for 5 s to inactivate The current is also dependent on the holding the delayed outward current, and then fol- potential, as illustrated in Fig. 1OA. Here a lowed at intervals ranging between 0.05 and


100 s by a second test pulse 500 ms in duration, also to +20 mV (Fig. HA,). The procedure is repeated in TEA (Fig. 1 lAz) and the responses at each test time are subtracted yielding a series of responses illustrated in Fig. 1 IA3. Complete recovery occurred by 100 s (zero inactivation). The small current produced with the 0.05-s pulse was arbitrarily defined as 100% inactivation. The responses at the other times fell between these two values and are plotted in Fig. 11B. It was

typically found that there is a rapid recovery period with a time constant of about 1 s, followed by a slower recovery period with a time constant of about 30 s. The data of Fig. 1lB are best fit by the sum of two exponentials:

% inactivation = 73.4 e+l*ls + 26.6 e-t/32.g (9)

The equilibrium potential of the delayed outward current was obtained with a procedure similar to that used for the fast transient

A ASW + TTX + Co++

.

+5 mV.

B + TEA

.

C

250 nA

500 msec

FIG. 8. Delayed outward current isolation. A : response in ASW to which TTX and Co2+ have been added. Membrane potential is stepped from a resting level of - 65 mV to a depolarized level of + 5 mV for 2.5 s. B : after adding approximately 50 mM TEA, the delayed outward current is blocked and the fast transient outward current is uncovered. C : isolated delayed outward current. Response in B is subtracted from response in A to yield a delayed outward current uncontaminated by the fast transient outward current and leakage current. The delayed outward current has a rapid activation phase followed by a lower exponential inactivation phase.

640 J. H. BYRNE

-26 HV

-16 HV

-6 HV

. l

. 5 HV .

.

.L

.

.

. . -se - -30 -10 38 68 .

- -I 5r ,’ -J ;Ir HEHBRRNE POTENTIAL OIV)

.5 SEC

FIG. 9. Delayed outward current Z-V relationship. A: membrane currents in response to various step depolarizations. The membrane potential is stepped for 2.5 s from a holding potential of -65 mV to various depolarized levels ranging between -45 to +45 mV. Currents are isolated by pharmacologic means as in Fig. 8. B: Z-V relationship. The peak current extrapolated to time of clamp onset for data of A are plotted as X’S.

Additional data from three experiments where the peak delayed outward current was examined at small depolarizations are plotted as stars.

outward current. A series of double pulses clamp procedure is repeated after blocking are delivered to a cell where the fast inward the delayed outward current with TEA (Fig. current is blocked by TTX (Fig. 12A). The 12A3). The currents are subtracted and the

Fl B

0.2 . : -.

X

158 @-@-12a ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ NA -100 -80 -68 -40 -20 0 :c

tlEtl6RANE POTENTIAL ItlVl

1 l 0- V -- X X x

X

0.8- X

E - x x r a > 8.6- E z - z -0.4- X

E X

388 MSEC

FIG. 10. Delayed outward current inactivation. A : membrane potential is stepped to various holding levels ranging from -93 to - 13 mV. After 5 s the membrane potential is stepped to a new fixed value of -3 mV. Traces shown are currents produced by that second step. Currents isolated by pharmacologic means as illustrated in Fig. 8. B : inactivation plot. Pooled data from two experiments showing ratios between the current obtained at depolarized holding levels and the maximum current obtained at a hyperpolarized holding level. See text for best-fitting equation.

R 2 +TER

R 3

INK MOTOR CELL ANALYSIS

B

80 -

z5 : 60- a > ;I : z 40- W S

20 -

641

0; 0 10 20 30 48 50

I

TIME (SEC) 400 NA

100 MSEC

FIG. 11. Delayed outward current recovery from inactivation. A 1: membrane potential is stepped from resting level to a fixed +20 mV depolarization for 5 s to inactivate the delayed outward current while cell is perfused in a 5% Na+, 5% Ca2+ solution. The 5-s pulse is then followed by a second pulse (500 ms duration) also to +20 mV. Responses illustrated are currents produced by the second pulse at 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 s after the first pulse. The largest current corresponds to the response 100 s after the inactivating pulse. An interval of between 50 and 100 s is required for the peak current produced by the second pulse to return to the level produced by the first pulse. A,: clamp procedure was repeated after adding 30 mM TEA to the perfusing solution. The delayed outward current is blocked leaving leakage and the fast transient outward current. Note that the fast transient outward current also appears to have a slow recovery from inactivation. A,: isolated delayed outward current recovery from inactivation. Responses in A2 are subtracted from responses in A, at each time so as to obtain an index of the delayed outward current recovery from inactivation uncontaminated by other currents. B: plot of delayed outward recovery from inactivation. Complete recovery from inactivation appeared to occur by 100 s and complete inactivation was arbitrarily defined as the current obtained with the 0.05-s pulse. Intermediate currents were scaled accordingly. There is a rapid phase of recovery with a time constant of about 1 s, followed by a slower phase with a 30-s time constant. The smooth curve fitting the entire range of data points is the sum of two exponential functions (see text). Individual curves for the slow and fast phases are independently plotted.

tails extrapolated to the onset of the second clamp pulse. Figure 12B illustrates that a reversal occurs at about -75 mV. The instantaneous I-V relationship for the delayed outward currents, like the fast outward current, is nonlinear.

Slow inward current In addition to the fast TTX-sensitive

(presumably Na+) current, the L14 cells also possess a slower inward current, which is blocked by low concentrations of Co2+ (10). The slow inward current is presumably

mediated by Ca2+ ions. The techniques utilized to isolate this current are illustrated in Fig. 13. Initially a clamp pulse is delivered with the fast inward current blocked by TTX, the fast outward blocked by 4-AP, and the delayed outward blocked by TEA. In the example of Fig. 13 the cell is clamped from a holding level of -78 mV to a depolarized level of +2 mV. Co2+ is added, the clamp procedure is repeated (Fig. 13B), and the two responses are subtracted (Fig. 13C). There is a rapid activation phase followed by a slower exponential inactivation phase. At

642 J. H. BYRNE

A

I

2TTX

3TTX tTEA

1 r

I 20 mV

-I 50nA IOOmSec

60

50 z c -

40 & E

30 z

20

IO

MEMBRANE POTENTIAL (mV)

FIG. 12 Delayed outward current equilibrium potential estimation. A: clamp protocol and pharmacologic separation. A 1 : membrane potential is stepped from resting level (-75 mV) to a fixed depolarization of - 15 mV. After 300 ms the membrane potential is stepped back to a series of less-depolarized levels. A,: typical response obtained by stepping the membrane potential back to -35 mV. Ganglion is perfused with ASW and TTX. A,: after adding 20 mM TEA to the perfusing solution the clamp series is repeated. The TEA blocked the delayed outward current leaving the fast transient outward current. B: tail current magnitude versus membrane potential. The tail currents extrapolated to time of second clamp pulse in the presence of TEA are subtracted from the tail currents in TTX for each clamp level. The smooth curve was drawn by eye.

the depolarized level of Fig. 13C the inactivation is incomplete. Figure 14A illustrates a series of such currents obtained when the cell is clamped from resting level of -78 mV to various depolarized levels ranging from - 18 to + 12 mV. The extrapolated peak currents versus the clamp level are plotted in Fig. 14B. The data are best fit by the equation:

I= 1.8

(1 + q(-16.1-V)/10.8)2 (V - 52) (10)

The estimated reversal potential is +52 mV. There was considerable variability in this estimate (see DISCUSSION). In two other experiments the estimated reversal potentials were +90 and + 120 mV. The time to peak of the slow inward current shows a slight voltage dependency ranging from 30 ms at -18 mV to 22 ms at +22 mV.

The slow inward current is also dependent on the holding potential. Figure 15A illustrates

the results of an experiment where a cell is clamped from holding levels of -74 to -4 mV to a fixed test level of +6 mV. The ratio of the peak current when the cell is clamped from various holding levels to the maximum current obtained with a hyperpolarized holding level is plotted in Fig. 15B. The data are best fit by the equation:

I 0.76 -= + 0.24 I (V+16.3)/7.9 2

max (1 + e ) W)

The half-inactivation point occurs at about -20 mV.

Synaptic input In addition to the fast outward current,

the features of the synaptic input to the L14 cells also appear to play a critical role in mediating inking behavior. During a several- second train of synaptic input, the EPSPs appear to build up and may thus contribute to the accelerating burst activity of action


potentials in the ink gland motor neurons. each of four different levels and 4.5-s trains The synaptic input appears to be due to the of electrical stimuli were delivered to the combined action of increased and decreased connectives to mimic the synaptic input conductance PSPs (8, 10, 13, 33). produced by a noxious stimulus to the head

To analyze the synaptic conductance of an intact animal (33). A typical experi- mechanisms further, cells were clamped at ment is illustrated in Fig. 16 where each

A ASW + TTX + TEA + 40AP

+ZmV : - J

. . .

C

150 msec

FIG. 13. Slow inward current isolation. A: response to 800-ms step depolarization from resting level (-78 mV) to +2 mV with ganglion perfused in ASW, TTX, TEA (40 mM), and 4-AP (5 mM). B: clamp is repeated after adding approximately 30 mM Co*+, which blocked the slow inward current. C: isolated slow inward current. Response in B is subtracted from response in A to yield a slow inward current uncontaminated by leakage currents. The slow inward current has a rapid activation phase followed by a slower exponential inactivation phase.

644

f=l

J. H. BYRNE

B

-* “” 7; -10

z z Y c(

x -30

a o -50

-70 30 -70 -50 -30 -10 10 30 MR HEHBRFINE POTENTIFIL (HV)

208 tlSEC

FIG. 14. Slow inward current Z-V relationship. A : membrane currents in response to various step depolarizations. Membrane potential is stepped for 800 ms from resting level (-78 mV) to various depolarized levels ranging between - 18 and +22 mV (+22 mV response not illustrated). Currents were isolated by pharmacologic means as in Fig. 13. B: I-V relationship. Data are peak currents extrapolated to time of clamp onset for currents of A. Estimated reversal potential for this experiment occurs at +52 mV.

response is the average of four trials. A linear regression analysis was then performed on the four membrane potential levels and the four corresponding synaptic currents at

R B

I 30 NR

75 MSEC

each of 500 sample times (sample rate, 100 Hz). The synaptic current was taken as the difference between the holding current and the nerve-evoked current. With this tech-

1 l e-

@.8-

0.2-

d

0.0; 11, II,, , , ,l -90 -70 -58 -30 -16 10

MEMBRANE POTENTIAL (MVI

FIG. 15. Slow inward current inactivation. A: membrane potential is stepped to various holding levels ranging between -74 and -4 mV. After 1 s the membrane potential is stepped to a new fixed level of +6 mV. Traces shown are currents produced by that second step. Currents isolated by pharmacologic means as illustrated in Fig. 13. B: inactivation plot. Data points are ratios between currents obtained at depolarized holding levels and the maximum current obtained when clamping from a hyperpolarized holding level. See text for best-fitting equation.


nique, an instantaneous synaptic equilibrium about 0.95. The results of three such potential and conductance can be obtained. experiments are averaged and plotted in Fig. With the exception of the first several 17. The prestimulus synaptic conductance samples the correlation coefficients averaged and equilibrium potential were arbitrarily

A 1

- 54 mV

A 2

- 74 mV

A 3

- 94 mV

a 114 mV

10 nA

500 msec

FIG. 16. Synaptic input. Membrane potential is sequentially clamped at each of four levels (-54, -74, -94, and - 114 mV) while an identical train of electrical stimuli (6 Hz for 4.5 s) is delivered to the left and right connectives, which normally link the abdominal ganglion to the head. In order to avoid axon spikes invading the clamped soma, stimulus intensity was adjusted to be just subthreshold for action-potential initiation in the unclamped cell. While the synaptic input was subthreshold for initiating action potentials, EPSPs showed a char- acteristic slow buildup typical of the L14 cells (For examples see Ref. 9, Fig. 15A; Ref. 10, Fig. 2A; Ref. 12, Fig. 1). Current was sampled every 10 ms. Each trace represets the average of four responses. Stimulus artifacts recorded on the digitized current recordings were deleted from data file and were substituted by values midway between the pre- and postartifact level.

646 J. H. BYRNE

110 - 7 > -

m t- 0

70- Q-

- 58 I

E =0.24- > 3 -

W c-l

=0.18- a l- c-l 3 0 z ~0.12-

S.SS~j-

0 I I 1 I I I I 1 I I

1000 2000 3000 4000 5000

TIME (MSECI


set to zero. With the start of the train the synaptic conductance increases, but this is followed by gradual decline over a several- second period. The equilibrium potential remains fairly constant for about the first 2-2.5 s, but at about 2-2.5 s a gradual rise in the estimated equilibrium potential occurs.

DISCUSSION

The purpose of these experiments is to extend to a more quantitative level previous work on the ionic conductance mechanisms of the L14 ink gland motor neurons (10, 12). In addition, the experiments provide a data base for a quantitative model developed in the following paper (9). As discussed below, the voltage- and time-dependent properties of the identified ionic conductance mechanisms of the ink gland motor cells appear qualitatively similar to those observed in other molluscan cells. Despite this similarity, however, a number of possible sources of error are present in the analysis. These include problems with spatial uniformity of the clamped cell, possible lack of specificity of the pharmacological blocking agents, errors due to series resistance, and uncertainty in determining inward current reversal potentials. It will be shown in the following paper that, although errors exist, the analysis is sufficiently precise to lead to a quantitative model, which predicts the firing pattern of the ink motor cells to constant-current steps and trains of synaptic input.

Early inward current

As previously found (lo), the fast inward current is blocked by TTX (Fig. 2) and is presumably mediated by sodium. It thus appears similar to the early inward current in squid (21). Its rapid-activation phase occurs at about -25 mV, which is similar but occurs at a slightly more depolarized level than in squid, Dorid, or Helix (15, 21, 29). The estimated reversal potential of +67 mV is consistent with reversal potentials measured by others (15, 21, 29), and the Na+ equilibrium potentials between + 69 and + 74

mV for other Aplysia neurons calculated by direct means by Brown and Kunze (5).

The steady-state inactivation characteristics (Fig. 3B) also roughly parallel those of other gastropod neurons. The half-inactivation point occurs at -27 mV, which corresponds to a value of about -30 mV in Helix (29) and -28 mV in Dorid (15). The kinetics of the fast inward current are highly voltage dependent and similar to the time course of the Dorid cells, but considerably slower than the similar current in squid.

Early transient outward current The fast transient outward current appears

similar to the fast transient outward current previously described in gastropod neurons (10, 16, 20, 26, 29, 35). Like the Helix and Dorid currents, it is activated with depolarizations less than necessary to activate the early inward and delayed outward currents. The half-inactivation voltage for the early outward current is -54 mV, which compares to about -68 mV for Dorid (16), -65 mV (29) and -80 mV (26) for Helix, and -70 mV for Tritonia (35). Thus the early outward current in the L14 cells requires higher levels of depolarization for complete inactivation than the corresponding currents in Helix, Dorid, and Tritonia. A corollary to this find- ing is that at the resting potential (-75 mV) the inactivation is removed. Thus the normally high resting potentials of the motor neurons and the characteristics of the steady- state inactivation ensures that at rest the inactivation of the early transient outward current is low. As a result, a depolarizing stimulus from the resting level can activate this current maximally. The relationship between this feature of the fast K+ current and the integrative action of the ink motor cells will be discussed in the second paper of this series (9).

The estimated equilibrium potential of -65 mV of the early outward current is similar to a value of -65 mV for Helix (29), -63 for Dorid (16), and - 60 mV for Tritonia (35). The deviation of the estimated equilibrium potential of the early outward current from that of the ‘delayed outward current

FIG. 17. Synaptic conductance and equilibrium potential. Sampled data of Fig. 16 provide 500 measurements of the synaptic current at each of four membrane potentials during the train of synaptic input. A linear regression is performed at each of these 500 points and an ongoing estimate of the synaptic conductance and equilibrium potential is obtained. Data are the averages of three experiments in three cells.

648 J. H. BYRNE

(-75 mV) and the directly estimated K+ equilibrium potential for other Aplysia neurons of -75 to -80 mV (5) may be partly due to the low temperature at which the reversal potential experiment was performed in the present study (31). Some of the deviation may also be due to an imperfect selec- tivity of the channels mediating the fast transient current. In addition, the tail currents examined to estimate the reversal potential may be contaminated with a tail reversing at the membrane potential of the poorly clamped axon. The nonlinear instantaneous Z-V plot is similar to that found by Neher (29) for the fast outward current in Helix.

Delayed outward current

The delayed outward current is similar to other delayed outward currents found in gastropod neurons. It begins to be activated at about -30 mV, it is selectively blocked by low concentrations of TEA, and has voltage-dependent time to peak activation. With small depolarizations from the resting potential the early outward current predomi- nates, but with large depolarizations the delayed outward current is predominant. The delayed outward current also inactivates like the Dorid, Helix, and squid delayed outward currents (15, 18, 26). The recovery from inactivation is slow compared to what might be expected from a system obeying Hodgkin and Huxley (24) kinetics. Similar slow recovery from inactivation has also been described for Helix neurons (26). It has been suggested that such a slow recovery from inactivation may be due to a depression or desensitization of a calcium-activated potassium conductance (17). This possibility seems unlikely for the case of the ink motor cells since the experiments were performed in low Ca2+, Na+ solutions, and currents were isolated by low concentrations of TEA.

Slow inward current Slow inward currents of the type analyzed

here have been previously described in a number of excitable membranes. (For review see Ref. 30.) In the ink motor cells the Ca2+ current begins to be significantly activated at about -30 mV. This compares to approximately - 15 mV for Ca2+ current in the Aplysia R2 cell (19), - 10 mV in the Aplysia R15 cell (36), -30 to -20 mV in Dorid (1)

neurons, and between -50 and -20 mV in Helix (2, 25, 34). The ink motor cell Ca2+ current also exhibits a potential-dependent inactivation. The half-inactivation point occurs at about -20 mV. This compares with approximately -30 mV for a +6-mV test pulse (2) and -24 mV for a + lo-mV test pulse (34) in Helix. A systematic investigation was not made of the effects of different values of test pulse on the ink motor cell Ca2+ steady- state inactivation (see Ref. 2). Most of the experiments indicated that the Ca2+ current did not completely inactivate (e.g., Figs. 14 and 15). Although a more detailed analysis is necessary, this appears similar to an incomplete inactivation of the slow inward current in Dorid neurons (14).

The calculated mean Ca2+ equilibrium potential of +87 mV is at best a rough estimate of the actual value due to the use of the estimation technique and the fact that in each case only several points on the ascend- ing limb of the Z-V curve were available. In addition, incomplete block of any of the other voltage-dependent currents would tend to reduce the estimated equilibrium potential. Despite the limitations of the techniques, the reversal potential estimates reported here are within the range of those reported by Ahmed and Connor (1) in Dorid, and Kostyuk et al. (25) and Standen (34) in Helix. Akaike et al. (2) report values near about +88 mV for some Helix cells, but the major- ity of their cells failed to show any clear reversal with test potentials up to + 175 mV.

The kinetics of the ink motor cell Ca2+ current appear similar to those of the Aplysia R15 Ca2+ current (36), but somewhat slower than the Helix Ca2+ current (2, 25). The time to peak of the slow inward current in the motor neurons is probably about an order of magnitude longer than that of their early inward current. The activation of the slow inward current occurs at levels more depolarized than for the early outward current, but at similar levels as the delayed outward and early inward currents. Synaptic current

The analysis of the synaptic input to the L14 ink motor cells confirms previous reports of the complex nature of the synaptic input onto these cells (8, 10, 13). During the initial portions of the train the synaptic conductance is high and equilibrium potential


near 0 mV, which is consistent with an increased conductance mechanism with a reversal potential between the Na+ and K+ equilibrium potentials. With time there is a fall in conductance and a concomitant increase in equilibrium potential, consistent with the late appearance of a dec reased conductance synaptic mechanism. A n indiv idual neuron that mediates a slow decreased conductance EPSP onto L14 has recently been identified (8 and unpublished observations). Firing this cell produces a slow EPSP that increases in amplitude over a several-second period, corresponding to the shift in reversal potential observed in Fig. 17. Nevertheless, care should be exercised in interpretating Fig. 17 (for discussion see Ref. 13). In addition, the present analysis does not permit a precise quantitative estimation of the relative contribution made by each conductance mechanism. The synaptic input used in the present analysis is obtained by electrical stimulation of the connectives that normally

REFERENCES

1. AHMED, Z. AND CONNOR, J. A. Measurement of calcium influx under voltage clamp in molluscan neurones using the metallochromic dye Arsenazo III. J. Physiol. London 286: 61-82, 1979.

2. AKAIKE,N.,LEE, K. S., AND BROWN, A.B.The calcium current of Helix neuron. J. Gen. Physiol. 71: 509-531, 1978.

3. ALVING, B. 0. Spontaneous activity in isolated somata of Aplysia pacemaker neurons. J. Gen. Physiol. 51: 29-45, 1968.

4. Box, M. J. A new method of constrained optimization and a comparison with other methods. Com- puter J. 8: 42-52, 1965.

5. BROWN, A. M. AND KUNZE, D. L. Ionic activities in identifiable Aplysia neurons. In: Ion Selective Microelectrodes, edited by H. J. Berman and N. C. Herbert, New York: Plenum, 1974, p. 57-73.

6. BYRNE, J. A quantitative description of the firing pattern of ink gland motoneurons in Aplysia. Biophys. J. 21: 163a, 1978.

7. BYRNE, J. H. Quantitative reconstruction of the firing pattern of motor neurons mediating a simple behavior in Aplysia. Proc. Joint Automatic Control Conf. 4: 53-58, 1978.

8. BYRNE, J. Premotor controls of inking behavior in Aplysia californica. Federation Proc. 38: 1394, 1979.

9. BYRNE, J. H. Quantitative aspects of contributing ionic conductance mechanisms contributing to firing pattern of motor cells mediating inking behavior in Aplysia californica, 43: 651-668, 1980.

10. BYRNE, J. H., SHAPIRO, E., DIERINGER,N., AND KOESTER, J. Biophysical mechanisms contributing to inking behavior in Aplysia. J. Neurophysiol. 42: 1233- 1250, 1979.

link the abdominal ganglion to the head. A recent analysis (8) of the individual neurons mediating this input indicates that there is a wide diversity of cell types. Interneurons activated by connective stimulation include cells producing increased conductance fast EPSPs, increased conductance fast IPSPs, increased conductance slow IPSPs, and decreased conductance slow EPSPs.

ACKNOWLEDGMENTS

I thank Dr. J. Koester for reviewing an earlier draft of the manuscript; C. Byrne, E. Oris, and H. Bloom- berg for programming assistance; G. Camp for typing the manuscript; and the University of Pittsburgh Com- puter Center for computational and graphics facilities.

This research was supported by National Institutes of Health Research Career Development Award NS 00200 and National Institutes of Health Research Grant NS 13511.

Received 1 May 1979; accepted in final form 10 September 1979.

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12. CAREW, T. J. AND KANDEL, E. R. Inking in Aplysia californica. II. Central program for inking. J. Neurophysiol. 40: 708-720, 1977.

13. CAREW, T. J. AND KANDEL, E. R. Inking in Aplysia californica. III. Two different synaptic conductance mechanisms for triggering the central program for inking. J. Neurophysiol. 40: 721-734, 1977.

14. CONNOR, J. A. Calcium current in molluscan neurones: measurement under conditions which maximize its visibility. J. Physiol. London 286: 41-60, 1979.

15. CONNOR, J. A. AND STEVENS, C. F. Inward and delayed outward membrane currents in isolated neural somata under voltage clamp. J. Physiol. London 24: l-19, 1971.

16. CONNOR, J. A. AND STEVENS, C. F. Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J. Physiol. London 213: 21-30, 1971.

17. ECKERT, R. AND Lux, H. D. Calcium-dependent depression of a late outward current in snail neurons. Science 197: 472-475, 1977.

18. FRANKENHAEUSEP, B. AND HODGKIN, A. L.The after effects of impulses in the giant nerve fibers ofLoligo. J. Physiol. London 131: 341-376, 1956.

19. GEDULDIG, D. AND GRUENER, R. Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents. J. Physiol. London 211: 217- 244, 1970.

20. HAGIWARA,S.,KUSANO, K., ANDSAITO,N. Mem-

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21. HODGKIN, A. L. AND HUXLEY, A. F. Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. London 116: 449-472, 1952.

22. HODGKIN, A. L. AND HUXLEY, A. F. The components of membrane conductance in the giant axon of Loligo. J. Physiol. London 116: 473-496, 1952.

23. HODGKIN, A. L. AND HUXLEY, A. F. The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J. Physiol. London 116: 497-506, 1952.

24. HODGKIN, A. L. AND HUXLEY, A. F. A quantitative description of membrane current and its appli- cation to conduction and excitation in nerve. J. Physiol. London 117: 500-544, 1952.

25. KOSTYUK, P. G., KRISHTAL, 0. A., AND DORO- SHENKO, P. A. Calcium current in snail neurones. I. Identification of calcium current. Pjkegers Arch. 348: 83-93, 1974.

26. KOSTYUK, P. G., KRISHTAL, 0. A., AND DORO- SHENKO, P. A. Outward currents in isolated snail neurons. I. Inactivation kinetics. Comp. Biochem. Physiol. C. 51: 259-263, 1975.

27. KUESTER, J. L. AND MIZE, J. H. Optimization Techniques with Fortran. New York: McGraw, 1973.

28. MEVES, H. AND PICHON, Y. The effect of internal

and external 4-aminopyridine on the potassium currents in intracellularly perfused squid giant axons. J. Physiol. London 268: 511-532, 1977.

29. NEHER, E. Two fast transient current components during voltage clamp on snail neurons. J. Gen. Physiol. 58: 36-53, 1971.

30. REUTER, H. Divalent cations as charge carries in excitable membranes. Prog. Biophys. Mol. Biol. 26: 3-43, 1973.

31. RUSSELL, J. M. AND BROWN, A. M. Active trans- port of potassium and chloride in an identifiable molluscan neuron. Science 175: 1475- 1477, 1972.

32. SHAPIRO, E. AND KOESTER, J. Ionic conductances in ink motor cells in Aplysia. Biophys. J. 17: 210a, 1977.

33. SHAPIRO, E., KOESTER, J., AND BYRNE, J.Aplysia ink release: central locus for selective sensitivity to long-duration stimuli. J. Neurophysiol. 42: 1223- 1232, 1979.

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