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Mechanism of excitation block by the insecticide allethrin applied externally and internally to squid giant axons
TOXICOLOGY
AND
APPLIED
Mechanism Applied
PHARMACOLOGY
of Excitation Externally
and
529-547
10,
Block
(1967)
by the
Internally
Insecticide
to Squid
Allethrin
Giant
Axons’
TOSHIO NARAHASHI AND NELS C. ANDERSON Department
of Physiology,
Duke
University Received
Medical January
Center, 27,
Durham,
North
Carolina
27706
1967
The insecticide allethrin is a derivative of pyrethrins, which are the active ingredients of pyrethrum. Pyrethrins have long been known to stimulate insect nerves to discharge repetitive impulses and to block nerve conduction (Hayashi, 1939; Lowenstein, 1942; Yamasaki and Ishii, 1952). These actions were thought to be the major mechanisms responsible for insecticidal action. However, it was not until 1962 that the neurotoxic action of allethrin was subjected to electrophysiologic analyses at the cellular level (Narahashi, 1962a,b). Allethrin exerts at least three actions on cockroach giant axons: (a) at a low concentration ( 1 PM), it increases and prolongs the negative afterpotential (delayed repolarization) that follows the spike potential; (b) at that concentration, repetitive afterdischarges may also be elicited by a single shock; and (c) at higher concentrations, it blocks conduction. This report is concerned with the ionic mechanism whereby nerve conduction is blocked by allethrin. Previous intracellular microelectrode studies (Narahashi, 1965) h ave shown (a) that a slight depolarization by allethrin is not enough to cause blockage of action potential, (b) that delayed rectification manifested by cathodal depolarization is suppressed by allethrin, and (c) that the rising phase and falling phase of the action potential decreases at about the same rate during the course of allethrin blockage. These observations suggest that the increases of the nerve membrane conductance both to sodium and to potassium are inhibited by allethrin. Voltage-clamp experiments have been performed in order to obtain definite evidence for or against this hypothesis. The development of an intracellular perfusion technique with squid giant axons permitted an examination of the geometric or structural aspects of the site of action of blocking agents. This technique was also applied to the present study in an attempt to localize the site of action of allethrin in the nerve membrane. METHODS
The giant axons of the squid L&go pea& available at the Marine Biological Laboratory, Woods Hole, Massachusetts, were used as material. The axon diameter ranged from 400 to 500 + 1 Presented at the Annual Meeting of the Entomological Oregon, November 28 through December 1, 1966.
Society of America,
P&land,
530
TOSHIO
NARAHASHI
AND
NELS
‘2.
ANDERSON
Resting and action potentials from perfused axons. The method of internal perfusion was essentially the same as that described previously (Narahashi et al., 1967). The diagram of the nerve chamber and recording system is shown in Fig. 1. Briefly, the procedure of internal perfusion is as follows: a small glass cannula is inserted in one end of an isolated giant axon, and the axoplasm is squeezed out by a small roller. The crushed axon preparation is then perfused with internal solution. A small amount of axoplasm still remaining in the axon can be washed out by this procedure. Then, an internal electrode is inserted in the axon longitudinally.
a?-^&..--.-
In!et
Ag-!gCI
fi
3ururarea
_I
.........Ag II I/..-.-...o.~M KCI
KCI;agar I
!dnternal solution pool
xk@Current ’ / 3-+
1
stop-cock FIG.
ments
I /
i
Cart&da
Ax& Ag-kgCl
v
Voltage
Sea water pool
Hole
1. Diagram (not to scale) of the nerve chamber and recording system for measureof resting and action potentials from perfused squid axons. See text for explanation.
However, the measurements of membrane potential were made in a slightly different way (Fig. 1). A glass capillary of about 100 p in diameter, filled with 0.6 it4 KC1 solution, was used as the internal potential electrode. A bare silver wire of 25 p in diameter was inserted in the capillary to reduce the highfrequency impedance. The capillary electrode was connected to the input of a high-input impedance preamplifier via an 0.6 M KCl-agar and an Ag-AgCl wire. An enamel-coated silver wire 50 ,U in diameter, with the enamel insulation removed and silver chloride coated for a length of about 10 mm from the tip, was used as the current electrode. This was twisted around the potential capiflary electrode in such a way as to locate the tip of the latter at the middle of the lo-mm Ag-AgCl region. The external reference electrode was a glass capillary similar to that used as the internal potential electrode with no wire current electrode twisting around it, and was connected to the input of an-
ALLETHRIN
BLOCKAGE
OF
SQUID
AXONS
531
other high-input impedance preamplifier via an 0.6 M KCI-agar and an AgAgCl wire. The reference electrode was dipped into a side pool which contained the same internal solution as inside the axon preparation, and the side pool was connected with the bathing medium via a saturated KCI-agar bridge. The potential recording system can then be represented by the following scheme : input of amplifrer/Ag-AgCl/O.G M KCl/intemal solution/nerve membrane/external solution/saturated KCl-agar/intemal solution/O.6 M El/O.6 M KCl-agar/Ag-AgCl/input of amplifier. The internal electrode was first dipped into the side pool containing internal solution into which the external electrode had been dipped, and any existing potential difference was canceled by an additional DC source. This gave the zero potential level. The internal electrode was then inserted in the axon which was being perfused with the internal solution. The potential difference produced at this stage represented the membrane potential assuming that the saturated KCl-agar bridge connecting the external bathing medium to the internal solution pool abolished the junction potentials there. The axon preparation was continuously perfused both externally and interThe compositions of internal solutions are nally throughout the experiment. given in Table 1. Natural sea water (Woods Hole) was used as the external solution. The action potential and its derivative were simultaneously recorded. The peak derivative during the rising phase of the action potential or the maximum rate of rise is a measure of inward current at that moment. The experiments were done at room temperature ( 21°C ) . Voltage clamp experiments. The sucrose-gap voltage-clamp techniques for intact and perfused axons were essentially the same as those described (Moore et al., 1964) and modified (Moore et al., 196710; Narahashi et al., 1967) previously. Two streams of isotonic sucrose solution separated an axon into three portions; the central portion, about 150 JL in width, was continuously perfused with sea water or test solution, and the measurements were made on this “artificial node.” One of the two side pools was perfused with sea water, and the current was applied through this pool. The other side pool was perfused -with isotonic KC1 solution and gave the zero potential level. This sucrose-gap condition caused the artificial node to be hyperpolarized by 20-50 mv. The action potential was observed by applying constant-current stimuIation. For voltage-clamp experiments, the membrane of the node was kept hyperpolarized at -80 mv to -110 mv by a feedback circuit to remove sodium inactivation, and then was suddenly shifted to and kept at depolarized or hyperpolarized levels. The membrane currents associated with these depolarizations or hyperpolarizations were recorded and measured. The compositions of internal SO~Utions for this purpose are given in Table 1. The experiments were done at 7-9°C. Alkthrin. Allethrin2 was first dissolved in ethanol to make up a stock SO~Ution, and the latter in turn was diluted with external or internal medium to give ‘The alleth.rin samples pany, Osaka, Japan, and
used in this work were from ‘McLaughlin Gormley
obtained from Sumitomo Chemical ComKing Company, Minneapolis, Minnesota.
532
TOSHIO
NAFiAHASHI
AND
TABLE COMPOSITION
OF SOLUTIONS
A B C
bk
(zf)
400 400 350
Glutamate WI)
0 50 50
C.
ANDERSON
I
USED FOR INTERNAL
F Solution”
NELS
370 0 320
PERFUSION
OF SQUID
’ b’h 0 490 50
AXONS
HgP04* (mW
Sucrose Wf)
15 15 15
333 250 333
a Solution A was used for action potential experiments in 1966. Solution B was used for voltageclamp experiments in 1965. Solution C was used for voltage-clamp egperiments in 1966. All sob tions were at pH 7.3. * In the form of potassium dihydrogen phosphate.
the desired concentrations. The concentration of ethanol was less, and had no effect on the electrical properties studied.
0.3%
(v/v)
or
RESULTS
Resting and Action
Potentials
Allethrin at a concentration of 10-100 PM depressed and blocked the action potential from either side of the nerve membrane, Figure 2 illustrates a series of records of the action potential and its derivative before and after application of allethrin. Shortly after allethrin was applied internally, the negative afterpotential that followed the postspike undershoot developed and increased to the point that a repetitive afterdischarge could be produced by a single stimulus (Fig. 2B). The poisoned axon soon ceased firing repetitively, and the action potential decreased its amplitude together with progressive depolarization (Fig. 2C). Washing with normal internal perfusate caused partial recovery in resting and action potentials, but the negative afterpotential usually remained augmented (Fig. 2D). Introducing allethrin externally resulted in progressive depolarization and action potential blockage as in the case of internal application of allethrin, but repetitive afterdischarge was lacking (Fig. 2E). Washing with normal sea water could not restore excitability in this particular case (Fig. 2F). In other experiments when allethrin was first applied externally, the axons responded in the same way as shown in Fig. 2A-C, where allethrin was first applied internally. Therefore, it can be said that allethrin block appears to be the same from either side of the nerve membrane. The time course of changes in resting potential, action potential, and its maximum rate of rise after internal and external application of allethrin is illustrated in Fig. 3. It can be seen that the blockages by internal and external allethrin proceed at about the same rate, and that they are accompanied by a slight depolarization ( 510 mv ) . It should be noted that this amount of depolarization was not enough for conduction block, because cathodal depolarization of the same magnitude in intact axons did not cause the same amount of decrease in action potential (Narahashi, 1965 1.
ALLETHBIN
BLOCKAGE
OF
SQUID
533
AXONS
I
IO00 vkec
FIG. 2. The of a perfused represents the represents the internally; (C) normal internal washing with
I msec
effect of internal and external application of allethrin on the action potential squid giant axon (Preparation C, 7/H/66). The lower tracing in each record action potential, and the upper tracing its derivative. The upper baseline also zero membrane potential level. (A) Control; (B) 2 minutes in 30 @U allethrin 11 minutes in 30 PM allethrin internally; (D) 29 minutes after washing with solution; (E) 11 minutes in 30 p&4 allethrin externally; (F) 19 minutes after normal sea water.
The action potential observed under the sucrose-gap conditions underwent an additional change by internal application of allethrin. Under the sucrose-gap conditions, the membrane was normally hyperpolarized by 20-50 mv. After application of allethrin internally,’ the action potential was found to be remarkably prolonged in its falling phase. A prolonged plateau phase was soon established, resembling the cardiac action potential (Fig. 4). The rising phase of the action potential was slightly slowed down. The action potential was eventually suppressed in magnitude. However, the degree of suppression was less than that observed by the internal electrode, and this can be ascribed to the hyperpolarization associated with the sucrose-gap conditions. Such a prolonged action potential was never observed in the internal electrode perfusion experiments where no hyperpolarization occurred, nor in the sucrose-gap experiments when allethrin was applied externally. Voltage
Clump:
Current-Voltage
Relations
ExternaZ application of albthrin. Records of membrane currents associated with step depolarizations before and during application of 10 PM allethrin to an intact axon are illustrated in Fig. 5. It can be seen that both the
534
TOSH.IO
309M 40
Allethrin J-
NARAHASHI
AND
Internally
.J/ 2..
,L..
l”
20 E
&
C.
ANDEltSON
30~M Allethrin Externally
;. -am*-
Normal ‘* Internal Sol. . .. 4
NELS
l
l
:
l
l
-
‘K.
l:.
l
Normal Externpl Sol.
..
+ 600
Ac;on
600
Potential
;; t 2
400
:: iz z
-60
200
-80
0
IO
20
30 40 Timetmin)
g [L
0
50
60
FIG. 3. Changes in the resting potential, the action potential, rise of the action potential by internal and external application fused squid giant axon (Preparation C, 7/18/f%).
and of 30
70 the
pM
80 maximum allethrin
rate of in a per-
early transient current which is carried mostly by sodium ions and the late steady-state current which is carried mostly by potassium ions are partially inhibited by allethrin. The inhibitions are more clearly shown in Fig. 6, in which the peak value of the early transient current and the steady-state value of the late current are plotted as a function of membrane potential after correction for Ieakage currents.
fi*.
.................. ...................
30;z2ii;&In,
min
Co”+ro,
I 8-18-66
I
8-17-66
2 msec
IO set
I
loom”
... .. . ... . . .. .. . ... 3OpM Allethrin Internally 23 min
FIG. 4. Prolongation of the action potential of squid giant axons by internal perfusion of 30 & allethnin under the sucrose-gap conditions. Because of sucrose hyperpolarization, the action potentia1 can still be produced despite extended application of allethrin. The upper and lower tracings were obtained from two different preparations.
ALLETHIUN
BLOCKAGE
OF
Control
/------’
SQUID
535
AXONS
IOJJMAllethrin Externally 2.5 min
-100mv . .. ... 80
---.
5
ma/cm2
2 msec
FIG. of -80 allethrin
5. Membrane currents mv in a voltage-clamped externally.
associated squid
with giant
step depolarizations axon before and
..lOOmv
$:e3 o il-40 :-5 0
from the holding potential during treatment with 10 PM
Internal application of akthrin. Internal perfusion of allethrin at a concentration of 10-100 PM also suppressed both the early transient current and the late steady-state current (Fig. 7). An example of the current-voltage relation is illustrated in Fig. 8. The late steady-state current, which was normally outward in direction, was converted into inward at membrane potentials ranging between -50 mv and -5 mv after perfusion with allethrin (Fig. 7). Since the potassium concentration was kept unchanged in allethrin, the inward steady-state current cannot be regarded as a potassium current. The time for the late steady-state current to attain a half-maximum was measured at the early transient equilib-
-
Control
---IO&M
Allethrin
8-4-65-C
FIG. 6. Current-voltage steady-state current (I.,) pM allethrin externally.
relations for the peak early in a voltage-clamped squid Holding potential, -90 mv.
tmnsient current axon before and
(I,,) during
and for the late exposure to 10
536
TOSHIO
NARAHASHI
AND
NELS
Control
C.
30 Nf
ANDERSON
Allethrin
Internally
12 mln
2 mssc FIG. 7. Membrane currents associated of -100 mv in a perfused voltage-clamped 30 yM allethrin internally. The dashed membrane current.
with
step depolarizations from the holding potential squid giant axon before and during exposure to line on the right of the record represents the zero
rium potential where no early transient current flowed (Table 2). The internal application of allethrin had no effect on the time to half-maximum. Therefore, the late steady-state current does not contribute to the observed change in the early transient current. This implies the presence of persistent sodium current or the inhibition of the sodium-inactivation mechanism. An attempt was made to correct for this residual sodium current. First of all, it is assumed that the early transient current is exclusively carried by sodium ions. Secondly, if the progressive increase in the apparent inward steady-state current with increas- 15 8-17-66
I
---
30~M
------
Corrected
Allethrin I,,
Internally I2 min
FIG. 8. Current-voltage relations for the peak early transient current (I,) and for the late steady-state current (I,.) in a perfused voltage-clamped squid giant axon before and during exposure to 30 PM allethrin internally. The dotted line represents the potassium current corrected for the delayed sodium cument as described in the text. Holding potential, -40 mv.
ALLETHBIN
BLOCKAGE
OF
SQUID
537
AXONS
ing depolarization is mostly ascribed to an increase in the residual sodium current (from -50 mv to -25 mv in Fig. S), then the true outward potassium current starts flowing at the membrane potentia1 at which the apparent inward steady-state current reaches a maximum (-25 mv in Fig. 8). Thirdly, since no sodium current flows at the sodium equilibrium potential, no correction for residual sodium current is necessary at this membrane potential. On these assumptions, the steady-state potassium current is given by a straight line connecting between the zero current at the membrane potential where the apparent inward steady-state current reaches a maximum and the late steady-state current at the sodium equilibrium potential in allethrin. This corrected steady-state potassium current is shown by a dotted line in Fig. 8. It should be noted that the corrected current-voltage curve is less steep than the apparent curve. TABLE
&
EFFECT OF INTERNAL APPLICATION OF ALLETHRIN ON THE TIME FOR THE LATE STEADY-STATE CURRENT TO ATTAIN HALF-MAXIMUM (TBB) AT TRE EQUILIBRIIJM POTENTIAL FOR THE EARLY TRANSIENT CURRENT Allethrin
Date
Preparation
Tss (-4
Cont. bm
Time (min)
Before
During
After
1.95 2.1 2.0
2.0 2.0 -
2.01
2.0
8-12-66
D
10
6
8-16-66 8-17-66
B A
SO SO
6
12
2.1 12.15 1.9
-
-
2.05
Mean:
Voltage
Clamp:
Membrane
Conductance
The membrane conductance at the peak of the early transient was calculated by the equation
current
(g,)
where I, is the peak early transient current, E is the membrane potential, and E, is the membrane potential where I, reverses its polarity. If I, is assumed to be exclusively carried by sodium ions, then g, represents the membrane sodium conductance and E, the sodium equilibrium potential. Because of the problems in identifying ion contributions and of the difhculty in estimating the equilibrium potential, the slope conductance ( gSs) was calculated for the late steady-state current ( ZSs): dI.8 Be.8 = dE
External application of alkthrin. The early transient conductances before and during application of allethrin externally are plotted as a function of membrane potential in Fig. 9. By the application of allethrin the whole conductance
538
TOSHIO
NARAHASHI
AND
NELS
C.
ANDWSON
curve was brought down along the ordinate (the conductance axis). It is not certain whether the small shift along the abscissa (the potential axis) in the normalized curves is genuine because of low conductances at the foot of the curve. The maximum values for the early transient conductance before and during application of allethrin are presented in Table 3, in which 10 PM and 30 PM of allethrin decrease the conductance to 49.7% of control on an average. Also given in Table 3 is the equilibrium potential ( E,), which is shifted toward the inside, less positive, membrane potential, i.e., toward left along the potential axis in Fig. 6. 8-4-65-C
gP
300
I
I
I
-60
-40
-20
0
(mmhokm2)
I
I
I
I
20
40
60
80
I
100
E (mv) FIG. 9. The membrane conductance for the peak early transient current as a function of membrane potential in a voltage-clamped squid giant axon before and during exposure to 10 pM allethrin externally. The membrane conductance was calculated by Eq. ( 1).
The maximum values of g,, before and during exposure to allethrin externally are given in Tabls 3. The average steady-state conductance is reduced to 67.3% by allethrin as against 49.7% for the early transient conductance. The difference between these two values is statistically significant at P = 0.05, i.e., the early transient conductance is more strongly inhibited by allethrin than the late steady-state conductance. Internal application of allethrin. The early transient conductance was computed by Eq. (1) and plotted as a function of membrane potential in Fig. 10. The conductance curve was shifted downward along the conductance axis by the internal treatment with allethrin, but little or no shift along the potential axis was observed. The small shift along the potential axis, which could be seen after normalization, was lacking in some other experiments. Numerical data on the maximum early transient conductance and the corrected maximum steady-state conductance before and during internal application of allethrin are given in Table 4. The late steady-state conductance is slightly more depressed than the early transient conductance, but the difference is not statis-
values
in parentheses
B C D F
S-5-65
Mean:
A B C D E
Preparation
8-4-65
0 The
TABLE
3
were
10 10 10 10
30 30 10 10 10
Cont. GJW
excluded
Allethrin
36 44 84 4O.Q
49
(44) (4Q) 43 40 36
Before
for the calculation
4 3 5 3 5.5
Time (min)
40 35 41 84 33.1
QQ 37 Q3
(‘3)
(75)
During
of mean
ED (mvY
because
of possible
76 110 93 156
117 15Q 195 19Q 160
Before
shifts
of membrane
36 75 53 88
40 60 6Q 141 73
During
gP (mmho/cm2)
potentiql
0.47 0.68 0.55 0.56 0.497
by artifacts
106 114 94 180
76 166 147 197 146
0.34 0.39 0.31 0.73 0.45
D/B
in test
70 77 71 103
50 120 7Q 174 68
Durine
gss (mmho/cma)
solution.
0.66 0.67 0.75 0.79 0.673
0.65 0.73 0.48 0.88 0.46
D/B
CURRENT (E,), THE MAXIMUM SLOPE CONDUCTANCE (gas)
Before
OF EXTERNAL APPLICATION OF ALLETHRIN ON THE EQUILIBRIUX POTENTIAL FOR THE PEAK EARLY TRANSIENT VALUE FOR THE EARLY TRANSIENT CONDUCTANCE (gJ, AND THE MAXIMUM VALUE FOR THE LATE STEADY-STATE
Date
EFFECT
EARLY
B A D
B A
8-10-65 8-12-65 8-12.-66
8-16-66 8-17-66 Meatb:
30 30 -
30 100 10
Cont. (PM)
ON THE
Allethrin
-
14
6
11 8 6
Time (min)
CONDUCTANCE
OF ALLETRRIN
TRANSIENT
PERFUSION
Preparation
FOR THE
OF INTERNAL
VALUE
Date
EFFECT
28 39 31.2
37 36 16
4
32 42 31.4
26 46 11
During
113 216
40 47 159
Before
91 139
112
21 38
During
g,, (mmho/cm2)
0.80 0.64 0.692
0.52 0.80 0.70
D/B
POTENTIAL FOR THE PEAK EARLY TRANSIENT THE MAXI?MUM VALUE FOR THE LATE STEADY-STATE
TABLE
EP bv)
AND
Before
(gp),
EQUILIBRIUM
THE
During 8 30 96 61 88 -
39 47 130 98 140
ysa (mmho/cmz)
CONDUCTANCE
(Ep),
Before
SLOPE
CURRENT
(gsJ
0.62 0.63 0.566
0.21 0.64 0.73
D/B
MAXIMUM
5 ii Fi 2
n
3 c,
n
$
6 3
0
ALLETHRIN
BLOCKAGE
OF
SQUID
541
AXONS
1300 I c------------ 100 100
-----------
---
gP
(mmhokmz)
-
30
-
IO IO
1
-60
I
-40
Control
----
30 I.~M Allethrin
3
I
0
-20
-
Internally I2 min
I
I
I
I
20
40
60
80
I
100
E(mv) FIG. 10. The membrane conductance for the peak early transient current as a function of membrane potential in a perfused voltage-clamped squid giant axon before and during exposure to 30 pM allethrin internally. The membrane conductance was calculated by Eq. ( I ) .
tically sign&ant at P = 0.05. This is in contrast to the external application of allethrin by which the early transient conductance is more strongly depressed than the late steady-state conductance (Table 3). Time to Peak Early Transient Current External application of alkthrin. Allethrin was also found to prolong the time to peak early transient current. In Fig. 11, the time is plotted against
8-4-65-D
12.5
I
-o-
Control
E(mv) FIG. 11. The a voltage-clamped
time to peak early squid giant axon
transient current as a function of membrane before and during exposure to 10 pM allethrin
potential in externally.
542
TOSHIO
NARAHASHI
AND
NELS
C. ANDERSON
membrane potential, and the prolongation by allethrin can be seen at all membrane potentials. Numerical data are given in Table 5. The average prolongation amounts to 18.2% at zero membrane potential, and is statistically significant at P = 0.02. TABLE EFFECT OF EXTERNAL EARLY TRANSIENT
5
APPLICATION OF ALLETHRIN ON THE TIME TO PEAK CURRENT (T,) AT ZERO MEMBRANB POTENTIAL Allethrin
Date
Preparation
T,
(msec)
Cont. (PM)
Time (min)
Before
During
D/B
8-4-65
A B C D E
30 30 10 10 10
4 3 5 3 5.5
0.52 0.50 0.45 0.46 0.41
0.70 0.44 0.60 0.51 0.51
1.34 0.88 1.98 1.10 1.24
s-5-G5
B C D F
10 10 10 10
3 2 2 2
0.54 0.42 0.40 0.48
0.63 0.44 0.59 0.52
1.16 1.04 1.47 1.08
-
-
I.1852
-
Mean:
Internal application of allethrin. The time to peak early transient current was also prolonged slightly by internal perfusion of allethrin as in the case of external application. Figure 12 shows an example of the time-membrane potential curve. The numerical data are given in Table 6. The average prolongation amounts to 12.8% at zero membrane potenCa1 and the difference is statistically significant at P = 0.05. TABLE EFFECT OF INTERNAL EARLY TRANSIENT
6
PERFUBION OF ALLETHRIN ON THE TIME TO PEAK CURRENT (TJ AT ZERO MEMBRANE POTENTIAL Allethrin
Date 8-10-65 S-12-65 S-12-66 8-16-66 8-17-66 Mean: -
Preparation B A D B A
Cone. (PM) 30 100 10 30 SO -
T, (msec) Time (min)
Before
During
D/B
11 8 6 6 12
0.55 0.65 0.55 0.65 0.62
0.80 0.55 0.55 0.72 0.77
1.45 0.84 1.00 1.11 1.24
-
-
1.128
-
ALLETHFUN
-60
I
I
-40
I
,-20
BLOCKAGE
OF
SQUID
543
AXONS
I
I
I
I
0
20
40
60
I
80 E(mv)
J
100
FIG. 12. The time to peak early transient current as a function of membrane potential in a perfused voltage-clamped squid giant axon before and during exposure to 30 aA4 allethrin internally. DISCUSSION
The present experimental results have definitely demonstrated the previous hypothesis that allethrin reduces both the early transient conductance and the late steady-state conductance of the nerve membrane (Narahashi, 1965). It is worthwhile to compare this blocking action of allethrin with those by tetrodotoxin and procaine which have been extensively studied. Allethrin resembles procaine in at least four respects: (1) both drugs inhibit the late steady-state conductance change as well as the early transient conductance change (Taylor, 1959; Shanes & al., 1959). (2) Both drugs exert these inhibitory actions from either side of the nerve membrane (Narahashi et al., 1967). ( 3) Both prolong the time to peak early transient current (Taylor, 1959). (4) Blockage can partially be restored by anodal hyperpolarization in both cases (Narahashi, 1964, 1965). However, allethrin differs from procaine in the following three respects: (1) In terms of the effective concentration to block the conductances, allethrin is some 100 times more effective than procaine (Taylor, 1959; Shanes et al., 1959). (2) Recovery after washing is much slower with allethrin than with procaine (Narahashi et al., 1967). (3) Allethrin causes slight depolarization both from inside and from outside the nerve membrane, whereas procaine does not change the resting potential from outside (Taylor, 1959) and causes slight hyperpolarization from inside (Narahashi et al., 1967).
544
TOSI-XIO
NARAHASHI
AND
NELS
C.
ANDERSON
In view of the lipid-soluble property of allethrin, it is naturally expected that the blocking action is exerted from either side of the nerve membrane. However, the percentage inhibitions of the early transient and the late steady-state conductance are not the same; the external application of allethrin inhibits the early transient conductance more effectively, while the internal application inhibits both conductances with no statistically significant difference (Tables 3 and 4). It was suggested that the gates for the early transient channel are located on the outer surface of the nerve membrane while those for the late steady-state channel are located on the inner surface of the nerve membrane (Narahashi et al., 1966a, 1967). This is based on the observations that tetrodotoxin, which is lipid-insoluble, blocks the early transient conductance selectively only from outside of the squid nerve membrane (Narahashi et al., 1966a, 1967) and that tetraethylammonium and cesium ions block the late steady-state conductance selectively only from inside of the squid nerve membrane (Chandler and Meves, 1965; Tasaki and Hagiwara, 1957; Armstrong and Binstock, 1965). The present results with allethrin are in keeping with this hypothesis. Allethrin is entirely different from tetmdotoxin in its mode of blocking action: ( 1) Unlike allethrin, tetrodotoxin blocks the early transient conductance exclusively (Narahashi et al., 1964; Moore et al., 196%). (2) Unlike allethrin, tetrodotoxin, is without effect on the time to peak early transient current (Takata et al., 1966). (3) Unlike allethrin, tetrodotoxin exerts its blocking action only from outside of the nerve membrane ( Narahashi et al., 1966a, 1967). (4) The effective concentration of tetrodotoxin is some 109 times lower than that of allethrin (Narahashi et al., 1964). (5) The recovery after washing is poorer in the allethrin poisoning than in the tetrodotoxin poisoning (Narahashi et al., 1966b). (6) Unlike allethrin, the tetrodotoxin blockage is not accompanied by a change in resting potential (Narahashi, 1964). (7) The allethrin blockage can partly be restored by anodal hyperpolarization whereas the tetrodotoxin blockage cannot be restored (Narahashi, 1964, 1965). There is no similarity between these two drugs with respect to the blocking mechanisms. In connection with the possible delay in the sodium inactivation as well as with the suppression of the late steady-state current by internal perfusion of allethrin, it is interesting to see the prolongation of action potential. This kind of prolonged plateau is to be expected from the Hodgkin-Huxley equations (Hodgkin and Huxley, 1952). However, the formation of a plateau by allethrin was observed only under the sucrose-gap, internal perfusion conditions. It was absent in the microelectrode experiments or in the sucrose-gap experiments with intact axons. Hence, it may be said that the internal application of allethrin and the hyperpolarization under the sucrose-gap conditions are necessary for the plateau formation and the delay in the sodium inactivation. In this connection, it should be recalled that internal perfusion with low ionic strength media prolongs the action potential forming a plateau (Narahashi, 1963) and that this is attributed to the slowing of the sodium-inactivation. The slowing of the sodium-inactivation and/or the inhibition of the steady-state potassium current were also observed by internal application of cesium which also caused a prolongation of action potential (Adelman and Senft, 1966; Chandler and
ALLETHBIN
BLOCKAGE
OF
SQUID
AXONS
545
Meves, 1965). Internal perfusion with potassium-free sodium fluoride solution also suppresses the sodium-inactivation (Chandler and Meves, 1966). It seems likely that changes in internal environment are in favor of the delay or suppression of the sodium inactivation. With respect to the mechanism whereby the early transient and late steadystate conductances are affected by allethrin, only two points can be mentioned at this stage. First of all, it is a remote possibility that these conductances are blocked by allethrin through inhibition of metabolism, It has been shown that the squid axons are capable of producing a number of action potentials after the metabolism has been inhibited by metabolic inhibitors such as dinitrophenol (Hodgkin and Keynes, 1955). Furthermore, allethrin blocks the conduction of cockroach nerve more effectively at lower temperatures than at higher temperatures, and this negative temperature coefficient of nerve blocking action is responsible for the negative temperature coefficient of insecticidal action of allethrin. These two facts appear to exclude the possibility of involvement of metabolic inhibition in the conductance blockage by allethrin. Secondly, there are at least two possible mechanisms by which drugs block membrane conductance change: ( 1) A drug may be plugged in the channels for early transient current and/or the channels for late steady-state current, thereby preventing sodium and/or potassium ions from flowing passively down the electrochemical gradient through these channels. Tetrodotoxin has been assumed to block the early transient channel in this way (Moore et al., 1967a). (2) A drug may b e sq ueezed into the phospholipid layers of the nerve membrane, thereby affecting the channels by lateral pressure, electrostatic force, or other means. This possibility has been suggested for procaine (Narahashi et al., 1967). The experimental data described in the present paper are rather in favor of the second mechanism for allethrin, i.e., the blockage of both early the higher effective concentration transient and late steady-state conductances, than that of tetrodotoxin, and the delay in the time course of early transient current. SUMMARY The mechanisms of excitability block by allethrin have been studied electrode, internal perfusion, and sucrose-gap voltage-clamp techniques the
by means of internal in the giant axons of
squid. Allethrin at a concentration of 10-100 pM blocked the action potential with slight depolarization either from inside or from outside the nerve membrane. The effect was partially reversible. The membrane currents associated with step depolarizations were inhibited by allethrin. With external application of allethrin, the early transient component of the membrane conductance was more strongly inhibited than the late steady-state component, whereas both components were inhibited to the same extent by internal application of allethrin. The time to peak early transient membrane current was slightly delayed either by external or by internal application of allethrin. There was an indication that with internal application of allethrin the sodium conductance increase was not as quickly inactivated as normal. The excitability block by allethrin can primarily be ascribed to the inhibition of the early transient membrane conductance. Further mechanisms of alletbrin action are discussed in comparison with other blocking agents.
546
TOSJXIO
NARAHASHI
AND
NELS
C.
ANDERSON
ACKNOWLEDGMENTS The ments. of the Mr. R. Mackey This NB06855)
authors wish to express Thanks are also due to electronic equipment, to N. Poston for technical for the analyses of the study was supported by and from the Duke
their gratitude to Dr. John W. Moore for his valuable comMr. E. M. Harris for construction and maintenance of much Mr. R. Solomon for construction of the nerve chamber, to assistance in the experiments in 1966, and to Miss B. L. data. grants from the National Institutes of Health (NB03437 and Endowment ( 84-0954). REFERENCES
ADELMAN, ternal
W. J., JR., and SENFT, J. P. (1966). cesium ion on sodium and potassium
Voltage currents
clamp studies in the squid
on the effect of ingiant axon. J. Gen.
Physiol. 50, 279-293. ADELMAN, W. J., JR., DYRO, F. M., and SENFT, J. (1965). Long-duration responses obtained fnom internaRy perfused axons. j. Gen. Physiol. 48 (No. 5, Part 2)) 1-9. ARMSTRONG, C. M., and BINSTOIZK, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. Gen. Physiol. 48, 859-872. CHANDLER, W. K., and MEVES, H. ( 1965). Voltage clamp experiments on internally perfused giant axons. J. Physiol. (London) 180, 788-820. CHANDLER, W. K., and ‘MEVEX, H. (1966). Incomplete sodium inactivation in internally perfused giant axons from Loligo forbesi. J. Physiol. (London) 186, 121-122P. HAYASHI, I. (1939). The action of pyrethrin I on insect nerve. Shokubutsu Oyobi Dobutsu 7, 2001-2008. HODGKIN, A. L., and HUXLEY, A. F. ( 1952). A quantitative desoription of membrane current and its application to conduction and excitation in nerve, J. Physiol. (London) 117, 500544. HODGKIN, A. L., and KEYNES, R. D. ( 1955). Active transport of cations in giant axons from Sepia and Loligo. J. Physiol. (London) 128, 28-60. LOWENSTEIN, 0. (1942). A method of physiological assay of pyrethrum extract. Nature 150, 760-762. MOORE, J. W., NARAHASHI, T., and ULBRICHT, W. (1964). Sodium conductance shift in an axon internally perfused with a sucrose and low-potassium solution. J. PhysioE. (London)
172, 163-173. MOORE, J. W., NARAHASHI, T., and SHAW, T. I. (1967a). An upper limit to the number of sodium channels in nerve membrane? J. Physiol. (London) 188, B-105. MOORE, J. W., BLAUSTEIN, M., ANDERSON, N., and NARAHASHI, T. (1967b). Basis of tetrodotoxin’s selectivity in blockage of squid axons. J. Gen. Physiol. 50, 1401-1411. NARAHASHI, T. ( 1962a). Effect of the insecticide allethrin on membrane potentials of cockroach giant axons. J. Cellular Camp. Physiol. 59, 61-65. NARAHASHI, T. (1962b). Nature of the negative after-potential increased by the insecticide allethrin in cockroach giant axons. J. Cellular Comp. Physiol. 59, 67-76. NARAHASHI, T. (1963). Dependence of resting and action potentials on internal potassium in perfused squid giant axons. J. Physiol. (London) 169, 91-115. NARAHASHI, T. ( 1964). Restoration of action potential by anodal polarization in lobster giant axons. J, Cellular Comp. Physiol. 64, 73-96. NWASHI, T. (1965). The physiology of insect axons. In: The Physiology of the Insect Central Nervous System (J. E. Treherne and J. W. L. Beament, eds.), pp. l-20. Academic Press, New York. NARAHASHI, T., MOORE, J. W., and Scorr, W. R. ( 1964). Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J. Gen. Physiol. 47, 965-974. NARAHASHI, T., ANDERSON, N. C., and MOORE, J. W. (1966a). Tetrodotoxin does not block excitation from inside the nerve membrane. Science 153, 765-767. NAFIAHASHI, T., MOORE, J. W., and POSTON, R. N. ( 1966b). Specific action of tetrodotoxin derivatives on nerve. Science 154, 425.
ALLETHBIN
NARAHASHI, T., ANDERSON,
BLOCKAGE
OF
SQUlD
AXONS
547
N. C., and MOORE, J. W. ( 1967). Comparison of tetrodotoxin and procaine in internally perfused squid giant axons. J. Gen. Physiol. 50, 1413-1428. SHANES, A. M., FREYGANG, W. M., GRUNDFEST, H., and AMATNIEK, E. (1959). Anesthetic and calcium action in the voltage-clamped squid giant axon. J. Gen. PhysioZ. 42, 793-802. TAKATA, M., MOORE, J. W., KAO, C. Y., and FUHRMAN, F. A. (1936). Blockage of sodium conductance increase in lobster giant axon by tarichatoxin (tetrodotoxin). J. Gen. Physiol. 49, 977-988. TASAKI, I., and HAGIWARA, S. (1957). D emonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride. J. Gen. Physiol. 40, 859-885. TAYLOR, R. E. ( 1959). Effect of procaine on electrical properties of squid axon membrane. Am. J. Physiol. 196, 1071-1078. YAMASAKI, T., and ISHII, T. ( 1952). Studies on the mechanism of action of insecticides (IV). The effects of insecticides on the nerve conduction of insect. Oyo-Kontyu (J. Nippon Sot. Appl. Entomol.) 7, 157-164.
AND
APPLIED
Mechanism Applied
PHARMACOLOGY
of Excitation Externally
and
529-547
10,
Block
(1967)
by the
Internally
Insecticide
to Squid
Allethrin
Giant
Axons’
TOSHIO NARAHASHI AND NELS C. ANDERSON Department
of Physiology,
Duke
University Received
Medical January
Center, 27,
Durham,
North
Carolina
27706
1967
The insecticide allethrin is a derivative of pyrethrins, which are the active ingredients of pyrethrum. Pyrethrins have long been known to stimulate insect nerves to discharge repetitive impulses and to block nerve conduction (Hayashi, 1939; Lowenstein, 1942; Yamasaki and Ishii, 1952). These actions were thought to be the major mechanisms responsible for insecticidal action. However, it was not until 1962 that the neurotoxic action of allethrin was subjected to electrophysiologic analyses at the cellular level (Narahashi, 1962a,b). Allethrin exerts at least three actions on cockroach giant axons: (a) at a low concentration ( 1 PM), it increases and prolongs the negative afterpotential (delayed repolarization) that follows the spike potential; (b) at that concentration, repetitive afterdischarges may also be elicited by a single shock; and (c) at higher concentrations, it blocks conduction. This report is concerned with the ionic mechanism whereby nerve conduction is blocked by allethrin. Previous intracellular microelectrode studies (Narahashi, 1965) h ave shown (a) that a slight depolarization by allethrin is not enough to cause blockage of action potential, (b) that delayed rectification manifested by cathodal depolarization is suppressed by allethrin, and (c) that the rising phase and falling phase of the action potential decreases at about the same rate during the course of allethrin blockage. These observations suggest that the increases of the nerve membrane conductance both to sodium and to potassium are inhibited by allethrin. Voltage-clamp experiments have been performed in order to obtain definite evidence for or against this hypothesis. The development of an intracellular perfusion technique with squid giant axons permitted an examination of the geometric or structural aspects of the site of action of blocking agents. This technique was also applied to the present study in an attempt to localize the site of action of allethrin in the nerve membrane. METHODS
The giant axons of the squid L&go pea& available at the Marine Biological Laboratory, Woods Hole, Massachusetts, were used as material. The axon diameter ranged from 400 to 500 + 1 Presented at the Annual Meeting of the Entomological Oregon, November 28 through December 1, 1966.
Society of America,
P&land,
530
TOSHIO
NARAHASHI
AND
NELS
‘2.
ANDERSON
Resting and action potentials from perfused axons. The method of internal perfusion was essentially the same as that described previously (Narahashi et al., 1967). The diagram of the nerve chamber and recording system is shown in Fig. 1. Briefly, the procedure of internal perfusion is as follows: a small glass cannula is inserted in one end of an isolated giant axon, and the axoplasm is squeezed out by a small roller. The crushed axon preparation is then perfused with internal solution. A small amount of axoplasm still remaining in the axon can be washed out by this procedure. Then, an internal electrode is inserted in the axon longitudinally.
a?-^&..--.-
In!et
Ag-!gCI
fi
3ururarea
_I
.........Ag II I/..-.-...o.~M KCI
KCI;agar I
!dnternal solution pool
xk@Current ’ / 3-+
1
stop-cock FIG.
ments
I /
i
Cart&da
Ax& Ag-kgCl
v
Voltage
Sea water pool
Hole
1. Diagram (not to scale) of the nerve chamber and recording system for measureof resting and action potentials from perfused squid axons. See text for explanation.
However, the measurements of membrane potential were made in a slightly different way (Fig. 1). A glass capillary of about 100 p in diameter, filled with 0.6 it4 KC1 solution, was used as the internal potential electrode. A bare silver wire of 25 p in diameter was inserted in the capillary to reduce the highfrequency impedance. The capillary electrode was connected to the input of a high-input impedance preamplifier via an 0.6 M KCl-agar and an Ag-AgCl wire. An enamel-coated silver wire 50 ,U in diameter, with the enamel insulation removed and silver chloride coated for a length of about 10 mm from the tip, was used as the current electrode. This was twisted around the potential capiflary electrode in such a way as to locate the tip of the latter at the middle of the lo-mm Ag-AgCl region. The external reference electrode was a glass capillary similar to that used as the internal potential electrode with no wire current electrode twisting around it, and was connected to the input of an-
ALLETHRIN
BLOCKAGE
OF
SQUID
AXONS
531
other high-input impedance preamplifier via an 0.6 M KCI-agar and an AgAgCl wire. The reference electrode was dipped into a side pool which contained the same internal solution as inside the axon preparation, and the side pool was connected with the bathing medium via a saturated KCI-agar bridge. The potential recording system can then be represented by the following scheme : input of amplifrer/Ag-AgCl/O.G M KCl/intemal solution/nerve membrane/external solution/saturated KCl-agar/intemal solution/O.6 M El/O.6 M KCl-agar/Ag-AgCl/input of amplifier. The internal electrode was first dipped into the side pool containing internal solution into which the external electrode had been dipped, and any existing potential difference was canceled by an additional DC source. This gave the zero potential level. The internal electrode was then inserted in the axon which was being perfused with the internal solution. The potential difference produced at this stage represented the membrane potential assuming that the saturated KCl-agar bridge connecting the external bathing medium to the internal solution pool abolished the junction potentials there. The axon preparation was continuously perfused both externally and interThe compositions of internal solutions are nally throughout the experiment. given in Table 1. Natural sea water (Woods Hole) was used as the external solution. The action potential and its derivative were simultaneously recorded. The peak derivative during the rising phase of the action potential or the maximum rate of rise is a measure of inward current at that moment. The experiments were done at room temperature ( 21°C ) . Voltage clamp experiments. The sucrose-gap voltage-clamp techniques for intact and perfused axons were essentially the same as those described (Moore et al., 1964) and modified (Moore et al., 196710; Narahashi et al., 1967) previously. Two streams of isotonic sucrose solution separated an axon into three portions; the central portion, about 150 JL in width, was continuously perfused with sea water or test solution, and the measurements were made on this “artificial node.” One of the two side pools was perfused with sea water, and the current was applied through this pool. The other side pool was perfused -with isotonic KC1 solution and gave the zero potential level. This sucrose-gap condition caused the artificial node to be hyperpolarized by 20-50 mv. The action potential was observed by applying constant-current stimuIation. For voltage-clamp experiments, the membrane of the node was kept hyperpolarized at -80 mv to -110 mv by a feedback circuit to remove sodium inactivation, and then was suddenly shifted to and kept at depolarized or hyperpolarized levels. The membrane currents associated with these depolarizations or hyperpolarizations were recorded and measured. The compositions of internal SO~Utions for this purpose are given in Table 1. The experiments were done at 7-9°C. Alkthrin. Allethrin2 was first dissolved in ethanol to make up a stock SO~Ution, and the latter in turn was diluted with external or internal medium to give ‘The alleth.rin samples pany, Osaka, Japan, and
used in this work were from ‘McLaughlin Gormley
obtained from Sumitomo Chemical ComKing Company, Minneapolis, Minnesota.
532
TOSHIO
NAFiAHASHI
AND
TABLE COMPOSITION
OF SOLUTIONS
A B C
bk
(zf)
400 400 350
Glutamate WI)
0 50 50
C.
ANDERSON
I
USED FOR INTERNAL
F Solution”
NELS
370 0 320
PERFUSION
OF SQUID
’ b’h 0 490 50
AXONS
HgP04* (mW
Sucrose Wf)
15 15 15
333 250 333
a Solution A was used for action potential experiments in 1966. Solution B was used for voltageclamp experiments in 1965. Solution C was used for voltage-clamp egperiments in 1966. All sob tions were at pH 7.3. * In the form of potassium dihydrogen phosphate.
the desired concentrations. The concentration of ethanol was less, and had no effect on the electrical properties studied.
0.3%
(v/v)
or
RESULTS
Resting and Action
Potentials
Allethrin at a concentration of 10-100 PM depressed and blocked the action potential from either side of the nerve membrane, Figure 2 illustrates a series of records of the action potential and its derivative before and after application of allethrin. Shortly after allethrin was applied internally, the negative afterpotential that followed the postspike undershoot developed and increased to the point that a repetitive afterdischarge could be produced by a single stimulus (Fig. 2B). The poisoned axon soon ceased firing repetitively, and the action potential decreased its amplitude together with progressive depolarization (Fig. 2C). Washing with normal internal perfusate caused partial recovery in resting and action potentials, but the negative afterpotential usually remained augmented (Fig. 2D). Introducing allethrin externally resulted in progressive depolarization and action potential blockage as in the case of internal application of allethrin, but repetitive afterdischarge was lacking (Fig. 2E). Washing with normal sea water could not restore excitability in this particular case (Fig. 2F). In other experiments when allethrin was first applied externally, the axons responded in the same way as shown in Fig. 2A-C, where allethrin was first applied internally. Therefore, it can be said that allethrin block appears to be the same from either side of the nerve membrane. The time course of changes in resting potential, action potential, and its maximum rate of rise after internal and external application of allethrin is illustrated in Fig. 3. It can be seen that the blockages by internal and external allethrin proceed at about the same rate, and that they are accompanied by a slight depolarization ( 510 mv ) . It should be noted that this amount of depolarization was not enough for conduction block, because cathodal depolarization of the same magnitude in intact axons did not cause the same amount of decrease in action potential (Narahashi, 1965 1.
ALLETHBIN
BLOCKAGE
OF
SQUID
533
AXONS
I
IO00 vkec
FIG. 2. The of a perfused represents the represents the internally; (C) normal internal washing with
I msec
effect of internal and external application of allethrin on the action potential squid giant axon (Preparation C, 7/H/66). The lower tracing in each record action potential, and the upper tracing its derivative. The upper baseline also zero membrane potential level. (A) Control; (B) 2 minutes in 30 @U allethrin 11 minutes in 30 PM allethrin internally; (D) 29 minutes after washing with solution; (E) 11 minutes in 30 p&4 allethrin externally; (F) 19 minutes after normal sea water.
The action potential observed under the sucrose-gap conditions underwent an additional change by internal application of allethrin. Under the sucrose-gap conditions, the membrane was normally hyperpolarized by 20-50 mv. After application of allethrin internally,’ the action potential was found to be remarkably prolonged in its falling phase. A prolonged plateau phase was soon established, resembling the cardiac action potential (Fig. 4). The rising phase of the action potential was slightly slowed down. The action potential was eventually suppressed in magnitude. However, the degree of suppression was less than that observed by the internal electrode, and this can be ascribed to the hyperpolarization associated with the sucrose-gap conditions. Such a prolonged action potential was never observed in the internal electrode perfusion experiments where no hyperpolarization occurred, nor in the sucrose-gap experiments when allethrin was applied externally. Voltage
Clump:
Current-Voltage
Relations
ExternaZ application of albthrin. Records of membrane currents associated with step depolarizations before and during application of 10 PM allethrin to an intact axon are illustrated in Fig. 5. It can be seen that both the
534
TOSH.IO
309M 40
Allethrin J-
NARAHASHI
AND
Internally
.J/ 2..
,L..
l”
20 E
&
C.
ANDEltSON
30~M Allethrin Externally
;. -am*-
Normal ‘* Internal Sol. . .. 4
NELS
l
l
:
l
l
-
‘K.
l:.
l
Normal Externpl Sol.
..
+ 600
Ac;on
600
Potential
;; t 2
400
:: iz z
-60
200
-80
0
IO
20
30 40 Timetmin)
g [L
0
50
60
FIG. 3. Changes in the resting potential, the action potential, rise of the action potential by internal and external application fused squid giant axon (Preparation C, 7/18/f%).
and of 30
70 the
pM
80 maximum allethrin
rate of in a per-
early transient current which is carried mostly by sodium ions and the late steady-state current which is carried mostly by potassium ions are partially inhibited by allethrin. The inhibitions are more clearly shown in Fig. 6, in which the peak value of the early transient current and the steady-state value of the late current are plotted as a function of membrane potential after correction for Ieakage currents.
fi*.
.................. ...................
30;z2ii;&In,
min
Co”+ro,
I 8-18-66
I
8-17-66
2 msec
IO set
I
loom”
... .. . ... . . .. .. . ... 3OpM Allethrin Internally 23 min
FIG. 4. Prolongation of the action potential of squid giant axons by internal perfusion of 30 & allethnin under the sucrose-gap conditions. Because of sucrose hyperpolarization, the action potentia1 can still be produced despite extended application of allethrin. The upper and lower tracings were obtained from two different preparations.
ALLETHIUN
BLOCKAGE
OF
Control
/------’
SQUID
535
AXONS
IOJJMAllethrin Externally 2.5 min
-100mv . .. ... 80
---.
5
ma/cm2
2 msec
FIG. of -80 allethrin
5. Membrane currents mv in a voltage-clamped externally.
associated squid
with giant
step depolarizations axon before and
..lOOmv
$:e3 o il-40 :-5 0
from the holding potential during treatment with 10 PM
Internal application of akthrin. Internal perfusion of allethrin at a concentration of 10-100 PM also suppressed both the early transient current and the late steady-state current (Fig. 7). An example of the current-voltage relation is illustrated in Fig. 8. The late steady-state current, which was normally outward in direction, was converted into inward at membrane potentials ranging between -50 mv and -5 mv after perfusion with allethrin (Fig. 7). Since the potassium concentration was kept unchanged in allethrin, the inward steady-state current cannot be regarded as a potassium current. The time for the late steady-state current to attain a half-maximum was measured at the early transient equilib-
-
Control
---IO&M
Allethrin
8-4-65-C
FIG. 6. Current-voltage steady-state current (I.,) pM allethrin externally.
relations for the peak early in a voltage-clamped squid Holding potential, -90 mv.
tmnsient current axon before and
(I,,) during
and for the late exposure to 10
536
TOSHIO
NARAHASHI
AND
NELS
Control
C.
30 Nf
ANDERSON
Allethrin
Internally
12 mln
2 mssc FIG. 7. Membrane currents associated of -100 mv in a perfused voltage-clamped 30 yM allethrin internally. The dashed membrane current.
with
step depolarizations from the holding potential squid giant axon before and during exposure to line on the right of the record represents the zero
rium potential where no early transient current flowed (Table 2). The internal application of allethrin had no effect on the time to half-maximum. Therefore, the late steady-state current does not contribute to the observed change in the early transient current. This implies the presence of persistent sodium current or the inhibition of the sodium-inactivation mechanism. An attempt was made to correct for this residual sodium current. First of all, it is assumed that the early transient current is exclusively carried by sodium ions. Secondly, if the progressive increase in the apparent inward steady-state current with increas- 15 8-17-66
I
---
30~M
------
Corrected
Allethrin I,,
Internally I2 min
FIG. 8. Current-voltage relations for the peak early transient current (I,) and for the late steady-state current (I,.) in a perfused voltage-clamped squid giant axon before and during exposure to 30 PM allethrin internally. The dotted line represents the potassium current corrected for the delayed sodium cument as described in the text. Holding potential, -40 mv.
ALLETHBIN
BLOCKAGE
OF
SQUID
537
AXONS
ing depolarization is mostly ascribed to an increase in the residual sodium current (from -50 mv to -25 mv in Fig. S), then the true outward potassium current starts flowing at the membrane potentia1 at which the apparent inward steady-state current reaches a maximum (-25 mv in Fig. 8). Thirdly, since no sodium current flows at the sodium equilibrium potential, no correction for residual sodium current is necessary at this membrane potential. On these assumptions, the steady-state potassium current is given by a straight line connecting between the zero current at the membrane potential where the apparent inward steady-state current reaches a maximum and the late steady-state current at the sodium equilibrium potential in allethrin. This corrected steady-state potassium current is shown by a dotted line in Fig. 8. It should be noted that the corrected current-voltage curve is less steep than the apparent curve. TABLE
&
EFFECT OF INTERNAL APPLICATION OF ALLETHRIN ON THE TIME FOR THE LATE STEADY-STATE CURRENT TO ATTAIN HALF-MAXIMUM (TBB) AT TRE EQUILIBRIIJM POTENTIAL FOR THE EARLY TRANSIENT CURRENT Allethrin
Date
Preparation
Tss (-4
Cont. bm
Time (min)
Before
During
After
1.95 2.1 2.0
2.0 2.0 -
2.01
2.0
8-12-66
D
10
6
8-16-66 8-17-66
B A
SO SO
6
12
2.1 12.15 1.9
-
-
2.05
Mean:
Voltage
Clamp:
Membrane
Conductance
The membrane conductance at the peak of the early transient was calculated by the equation
current
(g,)
where I, is the peak early transient current, E is the membrane potential, and E, is the membrane potential where I, reverses its polarity. If I, is assumed to be exclusively carried by sodium ions, then g, represents the membrane sodium conductance and E, the sodium equilibrium potential. Because of the problems in identifying ion contributions and of the difhculty in estimating the equilibrium potential, the slope conductance ( gSs) was calculated for the late steady-state current ( ZSs): dI.8 Be.8 = dE
External application of alkthrin. The early transient conductances before and during application of allethrin externally are plotted as a function of membrane potential in Fig. 9. By the application of allethrin the whole conductance
538
TOSHIO
NARAHASHI
AND
NELS
C.
ANDWSON
curve was brought down along the ordinate (the conductance axis). It is not certain whether the small shift along the abscissa (the potential axis) in the normalized curves is genuine because of low conductances at the foot of the curve. The maximum values for the early transient conductance before and during application of allethrin are presented in Table 3, in which 10 PM and 30 PM of allethrin decrease the conductance to 49.7% of control on an average. Also given in Table 3 is the equilibrium potential ( E,), which is shifted toward the inside, less positive, membrane potential, i.e., toward left along the potential axis in Fig. 6. 8-4-65-C
gP
300
I
I
I
-60
-40
-20
0
(mmhokm2)
I
I
I
I
20
40
60
80
I
100
E (mv) FIG. 9. The membrane conductance for the peak early transient current as a function of membrane potential in a voltage-clamped squid giant axon before and during exposure to 10 pM allethrin externally. The membrane conductance was calculated by Eq. ( 1).
The maximum values of g,, before and during exposure to allethrin externally are given in Tabls 3. The average steady-state conductance is reduced to 67.3% by allethrin as against 49.7% for the early transient conductance. The difference between these two values is statistically significant at P = 0.05, i.e., the early transient conductance is more strongly inhibited by allethrin than the late steady-state conductance. Internal application of allethrin. The early transient conductance was computed by Eq. (1) and plotted as a function of membrane potential in Fig. 10. The conductance curve was shifted downward along the conductance axis by the internal treatment with allethrin, but little or no shift along the potential axis was observed. The small shift along the potential axis, which could be seen after normalization, was lacking in some other experiments. Numerical data on the maximum early transient conductance and the corrected maximum steady-state conductance before and during internal application of allethrin are given in Table 4. The late steady-state conductance is slightly more depressed than the early transient conductance, but the difference is not statis-
values
in parentheses
B C D F
S-5-65
Mean:
A B C D E
Preparation
8-4-65
0 The
TABLE
3
were
10 10 10 10
30 30 10 10 10
Cont. GJW
excluded
Allethrin
36 44 84 4O.Q
49
(44) (4Q) 43 40 36
Before
for the calculation
4 3 5 3 5.5
Time (min)
40 35 41 84 33.1
QQ 37 Q3
(‘3)
(75)
During
of mean
ED (mvY
because
of possible
76 110 93 156
117 15Q 195 19Q 160
Before
shifts
of membrane
36 75 53 88
40 60 6Q 141 73
During
gP (mmho/cm2)
potentiql
0.47 0.68 0.55 0.56 0.497
by artifacts
106 114 94 180
76 166 147 197 146
0.34 0.39 0.31 0.73 0.45
D/B
in test
70 77 71 103
50 120 7Q 174 68
Durine
gss (mmho/cma)
solution.
0.66 0.67 0.75 0.79 0.673
0.65 0.73 0.48 0.88 0.46
D/B
CURRENT (E,), THE MAXIMUM SLOPE CONDUCTANCE (gas)
Before
OF EXTERNAL APPLICATION OF ALLETHRIN ON THE EQUILIBRIUX POTENTIAL FOR THE PEAK EARLY TRANSIENT VALUE FOR THE EARLY TRANSIENT CONDUCTANCE (gJ, AND THE MAXIMUM VALUE FOR THE LATE STEADY-STATE
Date
EFFECT
EARLY
B A D
B A
8-10-65 8-12-65 8-12.-66
8-16-66 8-17-66 Meatb:
30 30 -
30 100 10
Cont. (PM)
ON THE
Allethrin
-
14
6
11 8 6
Time (min)
CONDUCTANCE
OF ALLETRRIN
TRANSIENT
PERFUSION
Preparation
FOR THE
OF INTERNAL
VALUE
Date
EFFECT
28 39 31.2
37 36 16
4
32 42 31.4
26 46 11
During
113 216
40 47 159
Before
91 139
112
21 38
During
g,, (mmho/cm2)
0.80 0.64 0.692
0.52 0.80 0.70
D/B
POTENTIAL FOR THE PEAK EARLY TRANSIENT THE MAXI?MUM VALUE FOR THE LATE STEADY-STATE
TABLE
EP bv)
AND
Before
(gp),
EQUILIBRIUM
THE
During 8 30 96 61 88 -
39 47 130 98 140
ysa (mmho/cmz)
CONDUCTANCE
(Ep),
Before
SLOPE
CURRENT
(gsJ
0.62 0.63 0.566
0.21 0.64 0.73
D/B
MAXIMUM
5 ii Fi 2
n
3 c,
n
$
6 3
0
ALLETHRIN
BLOCKAGE
OF
SQUID
541
AXONS
1300 I c------------ 100 100
-----------
---
gP
(mmhokmz)
-
30
-
IO IO
1
-60
I
-40
Control
----
30 I.~M Allethrin
3
I
0
-20
-
Internally I2 min
I
I
I
I
20
40
60
80
I
100
E(mv) FIG. 10. The membrane conductance for the peak early transient current as a function of membrane potential in a perfused voltage-clamped squid giant axon before and during exposure to 30 pM allethrin internally. The membrane conductance was calculated by Eq. ( I ) .
tically sign&ant at P = 0.05. This is in contrast to the external application of allethrin by which the early transient conductance is more strongly depressed than the late steady-state conductance (Table 3). Time to Peak Early Transient Current External application of alkthrin. Allethrin was also found to prolong the time to peak early transient current. In Fig. 11, the time is plotted against
8-4-65-D
12.5
I
-o-
Control
E(mv) FIG. 11. The a voltage-clamped
time to peak early squid giant axon
transient current as a function of membrane before and during exposure to 10 pM allethrin
potential in externally.
542
TOSHIO
NARAHASHI
AND
NELS
C. ANDERSON
membrane potential, and the prolongation by allethrin can be seen at all membrane potentials. Numerical data are given in Table 5. The average prolongation amounts to 18.2% at zero membrane potential, and is statistically significant at P = 0.02. TABLE EFFECT OF EXTERNAL EARLY TRANSIENT
5
APPLICATION OF ALLETHRIN ON THE TIME TO PEAK CURRENT (T,) AT ZERO MEMBRANB POTENTIAL Allethrin
Date
Preparation
T,
(msec)
Cont. (PM)
Time (min)
Before
During
D/B
8-4-65
A B C D E
30 30 10 10 10
4 3 5 3 5.5
0.52 0.50 0.45 0.46 0.41
0.70 0.44 0.60 0.51 0.51
1.34 0.88 1.98 1.10 1.24
s-5-G5
B C D F
10 10 10 10
3 2 2 2
0.54 0.42 0.40 0.48
0.63 0.44 0.59 0.52
1.16 1.04 1.47 1.08
-
-
I.1852
-
Mean:
Internal application of allethrin. The time to peak early transient current was also prolonged slightly by internal perfusion of allethrin as in the case of external application. Figure 12 shows an example of the time-membrane potential curve. The numerical data are given in Table 6. The average prolongation amounts to 12.8% at zero membrane potenCa1 and the difference is statistically significant at P = 0.05. TABLE EFFECT OF INTERNAL EARLY TRANSIENT
6
PERFUBION OF ALLETHRIN ON THE TIME TO PEAK CURRENT (TJ AT ZERO MEMBRANE POTENTIAL Allethrin
Date 8-10-65 S-12-65 S-12-66 8-16-66 8-17-66 Mean: -
Preparation B A D B A
Cone. (PM) 30 100 10 30 SO -
T, (msec) Time (min)
Before
During
D/B
11 8 6 6 12
0.55 0.65 0.55 0.65 0.62
0.80 0.55 0.55 0.72 0.77
1.45 0.84 1.00 1.11 1.24
-
-
1.128
-
ALLETHFUN
-60
I
I
-40
I
,-20
BLOCKAGE
OF
SQUID
543
AXONS
I
I
I
I
0
20
40
60
I
80 E(mv)
J
100
FIG. 12. The time to peak early transient current as a function of membrane potential in a perfused voltage-clamped squid giant axon before and during exposure to 30 aA4 allethrin internally. DISCUSSION
The present experimental results have definitely demonstrated the previous hypothesis that allethrin reduces both the early transient conductance and the late steady-state conductance of the nerve membrane (Narahashi, 1965). It is worthwhile to compare this blocking action of allethrin with those by tetrodotoxin and procaine which have been extensively studied. Allethrin resembles procaine in at least four respects: (1) both drugs inhibit the late steady-state conductance change as well as the early transient conductance change (Taylor, 1959; Shanes & al., 1959). (2) Both drugs exert these inhibitory actions from either side of the nerve membrane (Narahashi et al., 1967). ( 3) Both prolong the time to peak early transient current (Taylor, 1959). (4) Blockage can partially be restored by anodal hyperpolarization in both cases (Narahashi, 1964, 1965). However, allethrin differs from procaine in the following three respects: (1) In terms of the effective concentration to block the conductances, allethrin is some 100 times more effective than procaine (Taylor, 1959; Shanes et al., 1959). (2) Recovery after washing is much slower with allethrin than with procaine (Narahashi et al., 1967). (3) Allethrin causes slight depolarization both from inside and from outside the nerve membrane, whereas procaine does not change the resting potential from outside (Taylor, 1959) and causes slight hyperpolarization from inside (Narahashi et al., 1967).
544
TOSI-XIO
NARAHASHI
AND
NELS
C.
ANDERSON
In view of the lipid-soluble property of allethrin, it is naturally expected that the blocking action is exerted from either side of the nerve membrane. However, the percentage inhibitions of the early transient and the late steady-state conductance are not the same; the external application of allethrin inhibits the early transient conductance more effectively, while the internal application inhibits both conductances with no statistically significant difference (Tables 3 and 4). It was suggested that the gates for the early transient channel are located on the outer surface of the nerve membrane while those for the late steady-state channel are located on the inner surface of the nerve membrane (Narahashi et al., 1966a, 1967). This is based on the observations that tetrodotoxin, which is lipid-insoluble, blocks the early transient conductance selectively only from outside of the squid nerve membrane (Narahashi et al., 1966a, 1967) and that tetraethylammonium and cesium ions block the late steady-state conductance selectively only from inside of the squid nerve membrane (Chandler and Meves, 1965; Tasaki and Hagiwara, 1957; Armstrong and Binstock, 1965). The present results with allethrin are in keeping with this hypothesis. Allethrin is entirely different from tetmdotoxin in its mode of blocking action: ( 1) Unlike allethrin, tetrodotoxin blocks the early transient conductance exclusively (Narahashi et al., 1964; Moore et al., 196%). (2) Unlike allethrin, tetrodotoxin, is without effect on the time to peak early transient current (Takata et al., 1966). (3) Unlike allethrin, tetrodotoxin exerts its blocking action only from outside of the nerve membrane ( Narahashi et al., 1966a, 1967). (4) The effective concentration of tetrodotoxin is some 109 times lower than that of allethrin (Narahashi et al., 1964). (5) The recovery after washing is poorer in the allethrin poisoning than in the tetrodotoxin poisoning (Narahashi et al., 1966b). (6) Unlike allethrin, the tetrodotoxin blockage is not accompanied by a change in resting potential (Narahashi, 1964). (7) The allethrin blockage can partly be restored by anodal hyperpolarization whereas the tetrodotoxin blockage cannot be restored (Narahashi, 1964, 1965). There is no similarity between these two drugs with respect to the blocking mechanisms. In connection with the possible delay in the sodium inactivation as well as with the suppression of the late steady-state current by internal perfusion of allethrin, it is interesting to see the prolongation of action potential. This kind of prolonged plateau is to be expected from the Hodgkin-Huxley equations (Hodgkin and Huxley, 1952). However, the formation of a plateau by allethrin was observed only under the sucrose-gap, internal perfusion conditions. It was absent in the microelectrode experiments or in the sucrose-gap experiments with intact axons. Hence, it may be said that the internal application of allethrin and the hyperpolarization under the sucrose-gap conditions are necessary for the plateau formation and the delay in the sodium inactivation. In this connection, it should be recalled that internal perfusion with low ionic strength media prolongs the action potential forming a plateau (Narahashi, 1963) and that this is attributed to the slowing of the sodium-inactivation. The slowing of the sodium-inactivation and/or the inhibition of the steady-state potassium current were also observed by internal application of cesium which also caused a prolongation of action potential (Adelman and Senft, 1966; Chandler and
ALLETHBIN
BLOCKAGE
OF
SQUID
AXONS
545
Meves, 1965). Internal perfusion with potassium-free sodium fluoride solution also suppresses the sodium-inactivation (Chandler and Meves, 1966). It seems likely that changes in internal environment are in favor of the delay or suppression of the sodium inactivation. With respect to the mechanism whereby the early transient and late steadystate conductances are affected by allethrin, only two points can be mentioned at this stage. First of all, it is a remote possibility that these conductances are blocked by allethrin through inhibition of metabolism, It has been shown that the squid axons are capable of producing a number of action potentials after the metabolism has been inhibited by metabolic inhibitors such as dinitrophenol (Hodgkin and Keynes, 1955). Furthermore, allethrin blocks the conduction of cockroach nerve more effectively at lower temperatures than at higher temperatures, and this negative temperature coefficient of nerve blocking action is responsible for the negative temperature coefficient of insecticidal action of allethrin. These two facts appear to exclude the possibility of involvement of metabolic inhibition in the conductance blockage by allethrin. Secondly, there are at least two possible mechanisms by which drugs block membrane conductance change: ( 1) A drug may be plugged in the channels for early transient current and/or the channels for late steady-state current, thereby preventing sodium and/or potassium ions from flowing passively down the electrochemical gradient through these channels. Tetrodotoxin has been assumed to block the early transient channel in this way (Moore et al., 1967a). (2) A drug may b e sq ueezed into the phospholipid layers of the nerve membrane, thereby affecting the channels by lateral pressure, electrostatic force, or other means. This possibility has been suggested for procaine (Narahashi et al., 1967). The experimental data described in the present paper are rather in favor of the second mechanism for allethrin, i.e., the blockage of both early the higher effective concentration transient and late steady-state conductances, than that of tetrodotoxin, and the delay in the time course of early transient current. SUMMARY The mechanisms of excitability block by allethrin have been studied electrode, internal perfusion, and sucrose-gap voltage-clamp techniques the
by means of internal in the giant axons of
squid. Allethrin at a concentration of 10-100 pM blocked the action potential with slight depolarization either from inside or from outside the nerve membrane. The effect was partially reversible. The membrane currents associated with step depolarizations were inhibited by allethrin. With external application of allethrin, the early transient component of the membrane conductance was more strongly inhibited than the late steady-state component, whereas both components were inhibited to the same extent by internal application of allethrin. The time to peak early transient membrane current was slightly delayed either by external or by internal application of allethrin. There was an indication that with internal application of allethrin the sodium conductance increase was not as quickly inactivated as normal. The excitability block by allethrin can primarily be ascribed to the inhibition of the early transient membrane conductance. Further mechanisms of alletbrin action are discussed in comparison with other blocking agents.
546
TOSJXIO
NARAHASHI
AND
NELS
C.
ANDERSON
ACKNOWLEDGMENTS The ments. of the Mr. R. Mackey This NB06855)
authors wish to express Thanks are also due to electronic equipment, to N. Poston for technical for the analyses of the study was supported by and from the Duke
their gratitude to Dr. John W. Moore for his valuable comMr. E. M. Harris for construction and maintenance of much Mr. R. Solomon for construction of the nerve chamber, to assistance in the experiments in 1966, and to Miss B. L. data. grants from the National Institutes of Health (NB03437 and Endowment ( 84-0954). REFERENCES
ADELMAN, ternal
W. J., JR., and SENFT, J. P. (1966). cesium ion on sodium and potassium
Voltage currents
clamp studies in the squid
on the effect of ingiant axon. J. Gen.
Physiol. 50, 279-293. ADELMAN, W. J., JR., DYRO, F. M., and SENFT, J. (1965). Long-duration responses obtained fnom internaRy perfused axons. j. Gen. Physiol. 48 (No. 5, Part 2)) 1-9. ARMSTRONG, C. M., and BINSTOIZK, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. Gen. Physiol. 48, 859-872. CHANDLER, W. K., and MEVES, H. ( 1965). Voltage clamp experiments on internally perfused giant axons. J. Physiol. (London) 180, 788-820. CHANDLER, W. K., and ‘MEVEX, H. (1966). Incomplete sodium inactivation in internally perfused giant axons from Loligo forbesi. J. Physiol. (London) 186, 121-122P. HAYASHI, I. (1939). The action of pyrethrin I on insect nerve. Shokubutsu Oyobi Dobutsu 7, 2001-2008. HODGKIN, A. L., and HUXLEY, A. F. ( 1952). A quantitative desoription of membrane current and its application to conduction and excitation in nerve, J. Physiol. (London) 117, 500544. HODGKIN, A. L., and KEYNES, R. D. ( 1955). Active transport of cations in giant axons from Sepia and Loligo. J. Physiol. (London) 128, 28-60. LOWENSTEIN, 0. (1942). A method of physiological assay of pyrethrum extract. Nature 150, 760-762. MOORE, J. W., NARAHASHI, T., and ULBRICHT, W. (1964). Sodium conductance shift in an axon internally perfused with a sucrose and low-potassium solution. J. PhysioE. (London)
172, 163-173. MOORE, J. W., NARAHASHI, T., and SHAW, T. I. (1967a). An upper limit to the number of sodium channels in nerve membrane? J. Physiol. (London) 188, B-105. MOORE, J. W., BLAUSTEIN, M., ANDERSON, N., and NARAHASHI, T. (1967b). Basis of tetrodotoxin’s selectivity in blockage of squid axons. J. Gen. Physiol. 50, 1401-1411. NARAHASHI, T. ( 1962a). Effect of the insecticide allethrin on membrane potentials of cockroach giant axons. J. Cellular Camp. Physiol. 59, 61-65. NARAHASHI, T. (1962b). Nature of the negative after-potential increased by the insecticide allethrin in cockroach giant axons. J. Cellular Comp. Physiol. 59, 67-76. NARAHASHI, T. (1963). Dependence of resting and action potentials on internal potassium in perfused squid giant axons. J. Physiol. (London) 169, 91-115. NARAHASHI, T. ( 1964). Restoration of action potential by anodal polarization in lobster giant axons. J, Cellular Comp. Physiol. 64, 73-96. NWASHI, T. (1965). The physiology of insect axons. In: The Physiology of the Insect Central Nervous System (J. E. Treherne and J. W. L. Beament, eds.), pp. l-20. Academic Press, New York. NARAHASHI, T., MOORE, J. W., and Scorr, W. R. ( 1964). Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J. Gen. Physiol. 47, 965-974. NARAHASHI, T., ANDERSON, N. C., and MOORE, J. W. (1966a). Tetrodotoxin does not block excitation from inside the nerve membrane. Science 153, 765-767. NAFIAHASHI, T., MOORE, J. W., and POSTON, R. N. ( 1966b). Specific action of tetrodotoxin derivatives on nerve. Science 154, 425.
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NARAHASHI, T., ANDERSON,
BLOCKAGE
OF
SQUlD
AXONS
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N. C., and MOORE, J. W. ( 1967). Comparison of tetrodotoxin and procaine in internally perfused squid giant axons. J. Gen. Physiol. 50, 1413-1428. SHANES, A. M., FREYGANG, W. M., GRUNDFEST, H., and AMATNIEK, E. (1959). Anesthetic and calcium action in the voltage-clamped squid giant axon. J. Gen. PhysioZ. 42, 793-802. TAKATA, M., MOORE, J. W., KAO, C. Y., and FUHRMAN, F. A. (1936). Blockage of sodium conductance increase in lobster giant axon by tarichatoxin (tetrodotoxin). J. Gen. Physiol. 49, 977-988. TASAKI, I., and HAGIWARA, S. (1957). D emonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride. J. Gen. Physiol. 40, 859-885. TAYLOR, R. E. ( 1959). Effect of procaine on electrical properties of squid axon membrane. Am. J. Physiol. 196, 1071-1078. YAMASAKI, T., and ISHII, T. ( 1952). Studies on the mechanism of action of insecticides (IV). The effects of insecticides on the nerve conduction of insect. Oyo-Kontyu (J. Nippon Sot. Appl. Entomol.) 7, 157-164.