Effects of Insecticides on Excitable Tissues

Effects of Insecticides on Excitable Tissues TOSHIO NARAHASHI Department of Physiology and Pharmacology Duke University Medical Center Durham. North Cizrolina. U.S.A. Introduction . . . . . . . . . . . . . . . . . . Process of Insecticidal Action . . . . . . . . . . . . 111. Mechanism of Nerve Excitation . . . . . . . . . . . . A . Excitation and Conduction in Nerve Fibers . . . . . . B. Synaptic and NeuromuscularTransmission . . . . . . IV . Functional Changes Caused by Insecticides in Nerve and Muscle . A . DDT . . . . . . . . . . . . . . . . . . . B. Lindane . . . . . . . . . . . . . . . . . . C . Cyclodienes . . . . . . . . . . . . . . . . D. Pyrethroids . . . . . . . . . . . . . . . . E. Rotenone . . . . . . . . . . . . . . . . . F. Organophosphates . . . . . . . . . . . . . . V . Mechanisms of Functional Changes Caused by Insecticides in . . . . . . . . . . . . . . . . Nerve and Muscle A. DDT . . . . . . . . . . . . . . . . . . . B . Allethrin . . . . . . . . . . . . . . . . . VI . Temperature Coefficient ot lnsectlcldal Action . . . . . . . A . DDT . . . . . . . . . . . . . . . . . . . B . Pyrethroids . . . . . . . . . . . . . . . . VII . Insecticide Resistance . . . . . . . . . . . . . . . A. Nerve Sensitivity to Insecticides . . . . . . . . . . B. Genes Controlling the Nerve Sensitivity . . . . . . . VIII . Structure-activity Relation . . . . . . . . . . . . . A . DDT . . . . . . . . . . . . . . . . . . . B . Pyrethroids . . . . . . . . . . . . . . . . Rotenone . . . . . . . . . . . . . . . . . C. IX . Road to the Molecular Mechanisms . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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I . INTRODUCTION

Mode of action of insecticides has been studied extensively for the past two decades since the development of a variety of synthetic AIP-i

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insecticides. One of the most remarkable achievements in this field is the study of the metabolism of insecticides which includes activation and degradation. Another contribution worthy of note is the study of the inhibition of cholinesterases (ChE’s) by a number of insecticides, most of which are either organophosphates or carbamates. However, in view of the fact that most insecticides are potent nervous poisons, it is surprising to find that a less amount of effort has been devoted to the study of the effects of various insecticides on the nervous system, especially on its excitable mechanism. As described in this articl6, it was not until the mid-1 960s that the cellular mechanism of action of certain insecticides on the nerve was satisfactorily elucidated. The action of insecticides on the nervous system may be classed into three categories: (1) functional changes in the nervous system as a result of insecticide intoxication; (2) biochemical mechanisms which are responsible for the functional changes; (3) biophysical or physico-chemical mechanisms which are responsible for the functional changes. First of all, the symptoms of poisoning caused by an insecticide must be interpreted in terms of disorders of various tissues. In most cases, the target site is the nervous system. The site of action of the insecticide in the nervous system must be determined, and changes in the nervous function must be observed. Since electric potential change or action potential is the only signal easily observable while the nerve is in the excited state, electrophysiological techniques are the most straightforward way of studying this problem. The biochemical aspects of the mechanism of insecticidal action on the nerve require some comments, because this problem is often misunderstood. As will be described later (Section I11 AS), the excitation and impulse propagation of the nerve fiber are not directly dependent upon the metabolic energy. In other words, the enzyme system is not directly involved in excitation. The only region where the enzyme system plays an immediate role in excitation is synapse and neuromuscular junction. At such junctions, the transmitter substance must be produced in the nerve terminals by enzymatic reactions, and the transmitter substance released from the nerve terminals upon excitation must be destroyed rapidly by the action of enzymes t o regulate the transmitter action on the postsynaptic element. Cholinesterase is the enzyme hydrolyzing the released acetylcholine at the cholinergic junctions. The inhibition of ChE causes severe disturbances of impulse transmission across the

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synapses. This is the major mechanism of action of a number of organophosphorus and carbamate insecticides. The mechanisms of ChE inhibition by these insecticides are out of the scope of the present article. Only physiological aspects of synaptic disturbances will be discussed. It is obvious from the explanation given above that the biophysical or physico-chemical aspects are of utmost importance in understanding the mechanism of action of insecticides. Electrophysiological techniques prove to be powerful in this study also. In view of these considerations, the present article covers the following aspects. First of all, the mechanism of the nerve excitation will be briefly described to help readers t a fully understand the subsequent sections. In the second place, changes in the nervous function caused by insecticides are described and discussed. This is the first step of the study of the mode of action. In the third place, the action on the nerve membrane is discussed in detail. This is the mechanism of action at the cellular or membrane level. In the fourth place, the electrophysiological techniques are applied to various problems of the mode of action of insecticides. This includes the mechanism involved in the temperature effect on insecticidal activity, the resistance of insects to insecticides, and the structure-activity relationship. It must be emphasized that the present article is not intended to cover these areas evenly and comprehensively. Most of the insecticides covered here are those in which the author has directly been involved for the past 20 years. For more comprehensive aspects of the mode of action of various insecticides, readers are urged to consult review articles (Brown, 1951, 1960, 1964; Casida, 1963; Colhoun, 1960, 1963; Dahm, 1957; Dahm and Nakatsugawa, 1968; Wilkinson, 1968; Fukuto, 1961; Hayes, 1959; Kearns, 1956; Lipke and Kearns, 1960; March, 1958; Metcalf, 1955, 1967, 1968; Perry, 1960; Roan and Hopkins, 1961 ; Smith, 1962; Spencer and O'Brien, 1957; Terriere, 1968; Winteringham and Lewis, 1959; Gordon, 1961; Hoskins and Gordon, 1956; O'Brien, 1966, 1967; Yamamoto, 1970; Winteringham, 1969). 11. PROCESS OF INSECTICIDAL ACTION

Before discussing the major problems of the present article, it will be appropriate to describe the process of insecticidal action, especially that in insects, because there are some reactions which are

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not common for other drugs or other animals. This is also important to visualize the role of each reaction in the whole intoxication process. Figure 1 illustrates a schematic process of the intoxication of insect by an insecticide. The insecticide may enter the insect body through the integument, the mouth, or the stomata. It may be

I1 1 4 DETOXICATION

ACCUMULATION

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ACTIVATION

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DETOXICATION

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4 *

ACCUMULATION

Lr_'irl EXCITABLE

MEMBRANE

ENZYME

I AUTOTOXIN

DEATH

Fig. 1. Process of toxic action of contact insecticide (Narahashi, 1964a).

insecticidally active as its original form (e.g. DDT) or may have to be converted into an active form t o exert the toxic action. For example, parathion becomes effective in inhibiting ChE's after having been oxidized t o paraoxon. The insecticide may be detoxified (e.g. from DDT to DDE) and excreted, or may be stored in the adipose tissue without exerting any toxic effect. In any case, the insecticide or its activated form finally reaches the site of action, which is in many cases the nervous system. However, there are generally diffusion barriers surrounding the nerve such as the nerve sheath. After having

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penetrated the nerve sheath, the process of activation or detoxication may take place. The insecticide can now exert its toxic action at the real site of action, e.g. at the nerve membrane or at the synaptic junctions. There are at least two ways by which the insecticide works there: (1) the direct physico-chemical action on the nerve membrane; (2) the action through the inhibition of enzymes. Symptoms of poisoning develop, but these do not necessarily lead the poisoned insect to death. In some cases, the hyperactivity of the nerve caused by insecticides liberates a toxin or toxins which in turn stimulate and paralyze the nerve (Sternburg, 1960, 1963; Sternburg and Kearns, 1952; Shankland and Kearns, 1959; Blum and Kearns, 1956; Hawkins and Sternburg, 1964; Sternburg et al., 1959). Death in fact results from the complicated multiple actions of the insecticide, including the exhaustion of energy, the paralysis of nerve and muscle systems, etc. Unlike vertebrate animals, death cannot be caused by a sole disturbance of the vital organ such as the paralysis of the respiratory center or the stoppage of the heart beat, because many functions in insects are not centralized. 111. MECHANISM OF NERVE EXCITATION

It would be appropriate t o briefly describe here the mechanism of nerve excitation, because without having proper knowledge on this problem it will be impossible t o fully understand the mode of action of insecticides on the nerve. This is a very specialized field so that readers in other fields may not be familiar with it. For more detailed information, readers are urged to consult specialty articles or text books (Hodgkin, 1958, 1964; Ruch et al., 1965; Katz, 1962, 1966; Eccles, 1964; Nastuk, 1966; Davson, 1964; Narahashi, 1963a). A. EXCITATION AND CONDUCTION IN NERVE FIBERS

I . Structure of the Nervous System The unit of the nervous system is called “neuron”. A neuron is composed of a nerve cell from which a number of “dendrites” and a long “nerve fiber” or “axon” emerge. Such neurons are synaptically connected with each other, or make synaptic contact with effective organs such as the skeletal muscle or the smooth muscle. At the synapse, there is a gap of a few hundred Angstroms between the presynaptic and postsynaptic membranes. At some junctions,

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however, the membranes of both presynaptic and postsynaptic elements are in close contact with each other forming a tight junction. A nerve membrane, which surrounds the axoplasm, is only about 75 A in thickness. This is the site of excitation of the nerve. A number of studies based on electron microscopic observations, X-ray diffraction, etc. have now resulted in the general agreement in that the nerve membrane is composed of a double phospholipid layer sandwiched by two protein layers. However, there have been many arguments regarding the exact arrignment of these macromolecule components in the nerve membrane. Cholesterol is also contained in the membrane, and calcium ions are said to maintain the integrity of the membrane by means of their positive charges. The axoplasm usually contains a large amount of potassium and a small amount of sodium and chloride. In the external medium such as the blood serum, the concentrations of these ions are reversed. Therefore, there are concentration gradients with high potassium inside and high sodium and chloride outside. However, in some insects the concentration gradient is in the opposite direction (see review by Narahashi, 1963a). 2. Resting Membrane Potential

When a glass capillary microelectrode is inserted into a giant nerve fiber (Fig. 2), a steady potential difference is recorded with the Stimulator

Nerve

rnutw \ {Inward

A t D e p o b r i z a t i o n

V

_----_-_-

IHyperpdarizotion

Fig. 2. Diagram of two-microelectrode experiment. Lower part depicts membrane potential changes produced by square pulses of current of various magnitudes in either outward or inwatd direction across the nerve membrane.

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inside negative with respect to the outside. This is the resting membrane potential, and is usually of the order of -50 to -100 mV (Fig. 3). Since the concentration of potassium is higher inside than outside, and since the resting membrane is permeable to potassium but is scarcely permeable to sodium or chloride, the membrane behaves more or less like a potassium electrode and the resting mV

50r

- 501

Fig. 3. Action potential recorded by means of intracellular microelectrode from the giant axon of the cockroach. Two tracings are photographiqlly superimposed, one before and the other after inserting the electrade into the axon.

potential approaches the equilibrium potential for potassium (EK) which is given by the Nernst equation:

where R, T and F represent the gas constant, the absolute temperature, and the Faraday constant, respectively, and [ I, and [ I i are the concentrations in the outside and inside of the axon, respectively.

3. Action Potential (a) Initiation of Action Potential: When a brief electric shock is applied to a giant nerve fiber preparation via a pair of wire electrodes, an action potential can be recorded by means of a microelectrode inserted in the fiber (Fig. 3). The action potential recorded from the nerve is usually very brief in duration, lasting only about 1 ms. At the peak of the action potential, the membrane potential is reversed in polarity the inside of the axon becoming positive with respect to the outside. This overshoot amounts to 20-50 mV.

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The falling phase of the action potential may simply return t o the original resting potential level in some kinds of nerve fibers, whereas in others the initial quick falling phase is followed by a slow terminal phase which gradually returns to the resting level. This slow repolarization phase is sometimes called “negative after-potential”. It should be noted that during the negative after-potential the potential actually deflects in the positive direction, and that the term “negative” comes from the classical method of external recording of the monophasic action potential whereby the depolarizing direction is recorded as a negative deflection. In some other nerve fibers, the falling phase of the action potential is followed by an undershoot or positive phase which may return t o the resting level gradually or may be in turn followed by a small negative after-potential. Again in this case, the “positive phase” is in fact a negative deflection. Instead of stimulating the nerve fiber by means of a pair of wire electrodes which are in contact with the nerve, one can insert another microelectrode into the axon very close to the recording microelectrode (Fig. 2). When a square current pulse is applied in the inward direction across the nerve membrane, the membrane is slowly hyperpolarized and attains the steady state. Upon cessation of the current pulse, the membrane potential returns slowly to the resting level. The membrane hyperpolarization is increased with increasing the intensity of the inward current pulse. When outward current pulses are applied t o the membrane, the situation is somewhat different. With a weak outward current, the membrane is slowly depolarized and the potential change is a mirror image of that produced by an inward current of the same intensity. With increasing the outward current intensity, a hump may appear during the early phase of depolarization. Upon increasing the current intensity slightly, an action potential is produced from the hump. The latency between the onset of current and the foot of the action potential is shortened as the current intensity is increased, whereas the threshold membrane potential where the action potential is produced remains constant. If one of the microelectrodes shown in Fig. 2 is withdrawn and reinserted at varying distances from the other microelectrode, and similar measurements are repeated, it can be seen that the height of the action potential remains unchanged whereas the steady-state amplitude of the hyperpolarization (anelectrotonic potential) or of the depolarization (catelectrotonic potential) declines with the

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distance in an exponential manner. This demonstrates that, although the action potential is propagated along the axon without decrement, the electrotonic potential passively produced by current is not propagated but simply spread. (b) Conduction of Action Potential: The diagram of impulse conduction along a nerve fiber is illustrated in Fig. 4. Since the membrane potential is reversed in polarity at the peak of the action REF

ACT

REST

+_ +_ +_ +_ +-n + - - -n -+++++++ - ++++-------

-

u

w

_ _ - - - - ++++------+ + + + + + ----+++ ++++ IMPULSE

Fig. 4. Diagram of impulse conduction in an axon. REF, refractory; ACT, active; REST, resting state (Narahashi, 1965a).

potential, a potential gradient is established between the activated area and the adjacent areas of the axon membrane. Hence, a local circuit current will flow across the membrane in such a direction as to depolarize the adjacent areas. Under normal conditions, the local circuit current is 3-5 times stronger than the threshold current necessary to produce an action potential. Therefore, an action potential is initiated from the area of the axon ahead of the activated area. No action potential will be produced from the area behind the activated area because the membrane is in a refractory state. Thus, the action potential is propagated along the axon by means of the local circuit current. (c) Ionic Mechanism: The ionic mechanism of action potential production is schematically shown in Fig. 5. As described before, the permeability of the nerve membrane to sodium is very low at resting conditions. Upon depolarizing stimulus, however, the sodium permeability (or sodium conductance, g N a ) rapidly increases so that the membrane becomes almost exclusively permeable to sodium. Therefore, the membrane potential approaches the equilibrium potential for sodium (ENa)defined by the Nernst equation for sodium

Because of the concentration gradient for sodium, sodium ions now

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-60 -80

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t~rrssc)

-------------K T l M STATE

Fig. 5. Diagram of the mechanism of action potential production. RP, resting potential; AP, action potential; EN,, sodium equilibrium potential; E K , potassium equilibrium potential; ma,membrane sodium conductance; gK , membrane potassium conductance. See text for further explanation (Narahashi, 1965a).

flow across the nerve membrane in inward direction. The increased sodium permeability starts decreasing soon, and the potassium permeability (or potassium conductance, gK ) starts increasing beyond its resting level. These permeability changes make the membrane almost exclusively permeable t o potassium again, thereby bringing the membrane potential back to the resting level. Potassium ions flow outwardly across the membrane according t o the concentration gradient. When the falling phase of the action potential approaches the resting potential level, the potassium permeability may still be maintained at a higher level. This enables the membrane potential to approach the E K closer than at resting conditions producing a positive phase or undershoot of the action potential. Experimental analyses have shown that the negative after-potential

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in squid and cockroach giant axons is produced by the transient depolarization caused by an accumulation of the released potassium in the immediate vicinity of the nerve membrane (Frankenhaeuser and Hodgkin, 1956; Narahashi and Yamasaki, 1960b).

4. Voltage Clamp Experiment (a) Rationale and Membrane Currents: In order to interpret changes in action potential or excitability caused by experimental procedures such as applications of drugs, it is necessary to measure changes in ionic permeabilities of the membrane. This can be achieved most efficiently by means of voltage clamp techniques. Since membrane ionic permeabilities are directly related to membrane conductances to the ions in question, and since conductance is obtained by dividing current by potential, measurements ought to be made on both membrane potential and membrane current. Figure 6 depicts electrical equivalent circuits of an axon. Under normal conditions, the internal resistance (ri) or the axoplasm resistance and the external resistance (r,) or the resistance of the external fluid such as the blood serum or the physiological saline solution are much smaller than the membrane resistance.(r,). There is the membrane capacity (c,) in parallel with the membrane resistance. When a square pulse of electric current is passed across the membrane through a pair of electrodes, one inside the axon and the other outside, the current is spread along the nerve fiber as is shown by arrows in Fig. 6. The longitudinal current inside the axon decreases in intensity with distance, because part of the longitudinal current crosses the membrane at any particular point of the axon. Thus the membrane current is not uniform but declines in intensity along the axon. Moreover, there are two components of the membrane current, one through the membrane resistance (ionic current, i i ) and the other across the membrane capacity (capacitative current, i, ). Under these experimental conditions, it is very difficult to measure the membrane current and potential in any reasonable manner. If, however, a long wire electrode is inserted into the axon longitudinally, and another wire electrode is placed immediately outside of the axon, the longitudinal current in the axon and the membrane current become uniform throughout the entire length of the axon where the wire electrodes are applied (Fig. 6, middle diagram). The situation therefore becomes much simpler, but there are still two components of the membrane current, i.e. i, and ii. This

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L

0

C

a i m

Fig. 6. Diagram of current flow in an axon preparation. Top, current is applied to the axon through internal and external microelectrodes. The membrane current and longitudinal current are not uniform along the axon. Middle, current is applied through internal and external wire electrodes. The currents are uniform along the axon (space clamp). Bottom, the ionic current (ii) across the membrane can be measured under voltage clamp conditions. ic, capacitative current; i,, total membrane current; c,, membrane capacity; ro, external resistance; ri, internal resistance; r,, membrane resistance. See text for further explanation.

condition is called “space clamp” and is prerequisite to voltage clamping. In order to eliminate the capacitative current across the membrane, one can make use of the fact that the membrane capacity is generally very small (1 pF/cm2 for squid giant axons) thereby making the duration of the capacitative current short. Instead of applying a square pulse of current and observing the resultant potential change, the membrane potential is changed in a square manner with the aid of an electronic feed-back circuit, and the membrane current necessary t o change the membrane potential is observed. This method is called “voltage clamp”. Because of the small value for the membrane capacity, the capacitative current under this condition ends in a brief period of time. In fact no

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important change in membrane ionic current occurs during this period of time. Therefore, if one ignores the very beginning and the very end of the membrane current under voltage clamp conditions, membrane ionic currents can be observed as a function of time and membrane potential. A family of membrane currents associated with step membrane potential changes under voltage clamp conditions is shown in Fig. 7. When the membrane is hyperpolarized from - 1 15 mV t o - 155 mV, -155mv

-,

L

Fig. 7. Family of membrane currents recorded under voltage clamp conditions from the lobster giant axon. The membrane potential is suddenly changed from -115 mV to the values indicated. The top record represents the membrane current associated with a step hyperpolarization, and the other records the membrane currents associated with various magnitudes of step depolarizations (Narahashi e? al., 1964).

an ionic current flows inwardly across the membrane (top record). This is easy to interpret from the Ohm’s law. If, however, a depolarizing pulse is applied, the membrane ionic current flows in quite a different manner. A large inward ionic current is followed by a steady-state outward ionic current. The current pattern changes with a change in membrane potential; although the steady-state current simply increases in magnitude with increasing the depolarization (for example, compare the record at -45 mV and that at +15 mV in Fig. 7), the transient current, with increasing depolarization, first increases in magnitude (record at -25 mV), decreases again (records at -5 mV and +15 mV), and finally is converted into a transient outward current. (b) Current- Voltage Relations: When the peak value of the transient current and the final value of the steady-state current are plotted as a function of the membrane potential, current-voltage relations are obtained (Fig. 8). Extensive analyses of voltage clamp data have demonstrated that the peak transient current is carried mostly by sodium, whereas the late steady-state current is carried mostly by

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potassium (Hodgkin and Huxley, 1952a, b, c, d; Hodgkin et al., 1 952). Therefore, the membrane potential where the transient current reverses its polarity is the sodium equilibrium potential. The potassium equilibrium potential cannot directly be measuted from the current-voltage curves such as those shown in Fig. 8, but separate measurements show that it is of the order of -80 mV.

-10

t

Fig. 8. Current-voltage relations for peak transient sodium current ( 1 ~ and ~ ) for steady-state potassium current ( I K ) in the voltage clamped lobster giant axon. I,, membrane current; Em, membrane potential; Eh, holding membrane potential from which the membrane is depolarized to various membrane potential levels (Narahashi, 1964b).

(c) Membrane Conductances: The membrane conductances t o sodium (gNa) and potassium (gK) are given by the following equations:

where ZN, and ZK are sodium and potassium currents, respectively, and E is the membrane potential. When the logarithms of the membrane conductances are plotted against the membrane potential, curves such as shown in Fig. 35 are obtained. The conductances thus calculated are the “chord conductances”, and are different from the “slope conductances” defined by aZ/aE.

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(d) Sodium Inactivation: As can be seen in membrane current records, the sodium current is transient even when the membrane is kept depolarized. Therefore, there are two mechanisms associated with the sodium conductance change, one being the mechanism whereby the sodium conductance is increased upon depolarization and the other the mechanism whereby the increased sodium conductance is decreased during sustained depolarization. The latter is often called the “sodium inactivation”. The time course of the sodium inactivation can be measured by applying two potential pulses. A conditioning pulse with a constant amplitude and varying durations is immediately ’followed by a test pulse of a constant amplitude and duration. The amplitude of the sodium current associated with the test pulse is then plotted as a function of the duration of the conditioning pulse. An exponential curve is obtained showing the time course of the sodium inactivation. The time constant depends on the membrane potential during the conditioning step, and on the temperature. An alternative way of obtaining the sodium inactivation curve is to plot the falling phase of the sodium current. However, the membrane current must be corrected for the potassium current. This may be achieved by the use of a specific inhibitor. For example, tetrodotoxin (TTX) is known to block the sodium current without any effect on the potassium current (Narahashi et al., 1964). If the membrane current recorded in TTX solution is subtracted from that recorded before application of TTX, the sodium current can be obtained. Another example for specific inhibitors is tetraethylammonium (TEA) which blocks the potassium current only when applied inside of the squid giant axon (Armstrong and Binstock, 1965). Therefore, the membrane current recorded from the TEA-treated axon represents the sodium current. Sodium inactivation is also a function of membrane potential. This relationship can be measured by the following two-pulse voltage clamp method. A conditioning pulse with a constant duration (30 ms or longer) and varying amplitudes is immediately followed by a constant test depolarizing pulse, and the amplitude of the transient sodium current associated with the test pulse is measured. The sodium current is plotted against the membrane potential of the conditioning pulse, and a sigmoid curve is obtained (Fig. 9). This is the steady-state sodium inactivation, and represents the availability of the sodium current at each membrane potential. This is one of the very important parameters in connection with drug actions, because

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1

I

-100

a, -50

1

0

Membrane potential (mV)

Fig. 9. Steady-state sodium inactivation curve from a squid giant axon. h-, the peak amplitude of sodium current associated with test step depolarization in a value relative to its maximum value. The abscissa represents the membrane potential of the conditioning pulse preceding the test depolarization (Moore et nl., 1964a).

changes in excitability are often explained in terms of a shift of the steady-state sodium inactivation curve along the potential axis. 5. Role of Metabolism Owing to the concentration gradients for sodium and potassium across the nerve membrane, the axon could gradually gain sodium and lose potassium. However, such a change does not in fact occur in the living tissue in situ, because there is in the axon the metabolic energy that constantly pumps out sodium and retains potassium. As long as the proper concentration gradient is maintained across the nerve membrane, the axon is capable of producing action potentials upon stimulation. This metabolic mechanism is called “sodium pump”. Immediately after excitation, the axon gains a small amount of sodium and loses a small amount of potassium. These changes in the ionic concentrations inside of the axon stimulate the sodium pump, and the concentration gradient is restored to the original value. The changes in internal sodium and potassium concentrations caused by one impulse are indeed very small. In the case of the squid giant axon which is about 5 0 0 p in diameter, the net influx of sodium and the net efflux of potassium are about 4 x lo-’*

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mole/cmZ of the membrane per impulse (Keynes, 195 1 ; Keynes and Lewis, 1951). The sodium and potassium concentrations in the axoplasm are about 50 mM and 400 mM, respectively. Hence, the increase in axoplasm sodium concentration caused by one impulse is of the order of In fact, the squid axon is capable of producing a number of action potentials after its sodium pump has been completely inhibited by treatment with metabolic inhibitors such as iodoacetate and cyanide (Hodgkin and Keynes, 1955). Therefore, it can be said that the excitation is a metabolism independent process and not directly supported by the metabolic energy. B. SYNAPTIC AND NEUROMUSCULAR TRANSMISSION

I . Classification of Junctions At synapses or neuromuscular junctions, impulse propagation is in most cases mediated by a chemical substance called “transmitter”. In other cases, however, the membrane of the presynaptic and postsynaptic elements form a tight junction, and impulses are transmitted by means of a local circuit current in much the same way as in the axon. Examples of the electrical synapse are found in the synapse between the giant axon in the nerve cord and the motoneuron in the crayfish (Furshpan and Potter, 1959), and in the Mauthner cell of the goldfish (Furshpan, 1964; Furukawa and Furshpan, 1963; Furukawa, 1966). Presynaptic impulses could exert either excitatory or inhibitory effects on the postsynaptic element. This is true for both chemical and electrical synapses. In the excitatory synapse or neuromuscular junction, the presynaptic impulses stimulate the postsynaptic cell to cause excitation such as action potential or contracture. In the inhibitory junctions, the presynaptic impulses prevent the postsynaptic cell from being excited by the excitatory presynaptic impulses. These two different kinds of synapses and junctions form the basis of the complicated nerve network. Thus synapses and neuromuscular junctions are classed as follows: Mode of impulse transmission Electrical Chemical Role of junction Excitatory Inhibitory

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2. Mechanism of Impulse Transmission (a) Excitatory Junctions: The preparation in which the mechanism of excitatory impulse transmission has been most extensively studied is the neuromuscular junction of the frog or mammal. When an impulse arrives a t the nerve terminal, a large amount of the transmitter substance acetylcholine (ACh) is released, and depolarizes the end-plate membrane of the muscle fiber. Unlike the membrane of the nerve or muscle fiber, the end-plate membrane is highly sensitive to ACh. The depolarization of the end-plate causes a local circuit current to flow across the muscle membrane surrounding the end-plate, so that an action potential is initiated from the muscle membrane. Cholinesterases present in the junction area hydrolyze the released ACh quickly, so that the stimulating action of ACh does not last an unnecessarily long period of time. When the nerve-muscle preparation is treated with d-tubocurarine, the neuromuscular transmission is blocked although the conduction of nerve or muscle is not impaired. Under these conditions, a microelectrode inserted in the end-plate region will record a small and slow depolarizing response upon nerve stimulation. This response is called the “end-plate potential (e.p.p.)”, and represents the depolarization of the end-plate membrane (Fig. 10). Since

Fig. 10. Action potenitial recorded from a sartorius muscle fiber of the frog and end-plate potential recorded from ancither fiber after treatment with d-tubc)CUrarine (Urakawa et QZ., 1960).

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d-tubocurarine suppresses the sensitivity of the end-plate membrane to ACh, the e.p.p. does not reach the threshold membrane potential beyond which an action potential of the muscle fiber is produced. In normal muscle preparations, the e.p.p. reaches the threshold, and the action potential almost masks the e.p.p. The e.p.p. is seen to be augmented and prolonged by treatment with anticholinesterases, because the transmitter action lasts longer under these conditions. In non-curarized preparations, anticholinesterases may initiate repetitive afterdischarges of the muscle fiber, and the transmission may eventually be blocked by high concentrations of accumulated ACh. In normal muscle preparations, small depolarizing responses can be observed from the end-plate without any presynaptic stimulation. The amplitude is of the order of 0.5-1 mV, and the duration is the same as that of the e.p.p. The responses occur spontaneously at a frequency of about 1Is, but the frequency is quite variable. They are called the “miniature end-plate potentials (m.e.p.p.s)”, and are produced by spontaneous release of ACh from the nerve terminal. The ACh is in fact released in quanta, and one m.e.p.p. is indicative of the depolarization caused by one ACh quantum which contains several thousand ACh molecules. The frequency and amplitude of the m.e.p.p.s may be differently affected by application of drugs. The change in frequency is a measure of the ability of the nerve terminal to release ACh, whereas the change in amplitude is due either to a change in quanta1 size (the number of ACh molecules in one quantum) or to a change in the sensitivity of the end-plate membrane to ACh, or both. The sensitivity of the end-plate membrane to ACh can directly be measured by recording the depolarization produced by application of ACh. A glass capillary microelectrode filled with ACh is brought close to the end-plate area and a brief positive electric pulse is applied to the electrode. Since ACh is positively charged, a small amount of ACh is ejected from the electrode tip, the amount being calculated from the intensity and duration of the pulse. The depolarization of the end-plate caused by the ACh is recorded by another microelectrode inserted in the end-plate region. At the excitatory synapses, a similar sequence of events occurs during the impulse transmission. The postsynaptic response caused by the transmitter is called the “excitatory postsynaptic potential (e.p.s.p.)”. It should be noted that the transmitter substance at the excitatory synapses or excitatory neuromuscular junctions is not

20

T. NARAHASHI

necessarily ACh. The transmitter is most probably 1-glutamate in crayfish and insect neuromuscular junctions (Faeder, 1968; Takeuchi and Takeuchi, 1964; Kerkut et aZ., 1965; Usherwood and Machili, 1966; Usherwood et al., 1968), whereas it is noradrenaline in adrenergic synapses of mammals. (b) Inhibitory Junctions: There are two types of inhibition, i.e. presynaptic inhibition and postsynaptic inhibition. In the presynaptic inhibition, the inhibitory nerve terminates near the excitatory presynaptic nerve terminals. The excitation of the inhibitory nerve causes a depolarization of the excitatory presynaptic nerve thereby decreasing the magnitude of the action potential there. This in turn causes a decrease in the amount of the transmitter released from the excitatory nerve terminals and results in an inhibition. In the postsynaptic inhibition, the excitation of the inhibitory nerve usually causes a transient hyperpolarization producing an “inhibitory postsynaptic potential (i.p.s.p.)”. The inhibitory impulse arriving at the nerve terminals at about the same time as the excitatory presynaptic impulse causes an inhibition of synaptic transmission through a suppression of the e.p.s.p. Gamma aminobutyric acid is a possible inhibitory transmitter substance in some inhibitory synapses and neuromuscular junctions, but the evidence is not very confirmative (e.g. Takeuchi and Takeuchi, 1965, 1966, 1967, 1969). (c) Ionic Mechanism: The ionic mechanism responsible for the potential change of the postsynaptic membrane is entirely different from that of the axonal membrane. Detailed voltage clamp analyses have been performed with the end-plate membrane of the frog (Takeuchi and Takeuchi, 1959, 1960). These studies are based on the observation of the end-plate currents (e.p.c.s) produced by nerve impulses when the end-plate membrane potential is clamped at various levels. Since the end-plate area is much smaller than the space constant of the muscle fiber, space clamp conditions can be established by a microelectrode inserted in the end-plate area. Another microelectrode is also inserted closely and serves as the potential electrode. ACh causes both sodium and potassium conductances t o increase almost simultaneously. The equilibrium potential for the overall conductance is about - 15 mV. In other excitatory or inhibitory synapses, different conductance changes may be involved. For example, at the inhibitory postsynaptic membrane of the cat motoneurons and snail neurons,

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

21

an increase in chloride conductance is the major factor (Ito et al., 1962; Kerkut and Thomas, 1964). Because the equilibrium potential for chloride is more negative than the normal resting potential, the inhibitory transmitter action causes a transient hyperpolarizing i.p.s.p. The excitatory postsynaptic membrane of the cat mo toneurons becomes permeable to all ions upon transmitter action (Eccles, 1964). The equilibrium potential is therefore near zero membmne potential and the response is a depolarizing e.p.s.p.

IV. FUNCTIONAL CHANGES CAUSED BY INSECTICIDES IN NERVE AND MUSCLE

In this section changes in nerve and muscle functions caused by insecticide intoxication will be described. Observations and experiments in this category are naturally descriptive and superficial, yet they will give a clue t o further exploration of the mechanism of action at the cellular and molecular levels. Experimental materials covered here are mostly limited to lower animals, especially to insects and other crustaceans. The studies described here also give the basis on which some important aspects of insect toxicology can be accounted for as will be described later (Sections VI, VII, andd VIII). A. DDT

1. Symptoms of Poisoning

The observation of the symptoms of poisoning is the first step in

the study of the mechanism of action of an insecticide. For example,

if ataxia, hyperactivity or convulsion is observed in the insects poisoned with the insecticide, one can naturally suspect neuromuscular actions of the insecticide. On the other hand, if only paralysis occurs, the major action could either be neuromuscular blockage or metabolic inhibition. Intoxication with DDT results in ataxia and discoordination of insects. Convulsions of appendages and somatic muscle follow and last for a while, the period of which depends on the dosage and the kind of insects. The poisoned insect is eventually paralyzed (Yamasaki and Ishii," 1954a). *Former name of T. Narahashi.

22

T. NARAHASHI

2. Effects on Nervous Functions

One of the most sensitive nervous tissues to the action of DDT is the campaniform sensilla in the trochanter of the cockroach. When injected directly into the leg, DDT is effective in initiating trains of impulses at a concentration of 10-7-10-8 M (Fig. 11) (Becht, 1958; Lalonde and Brown, 1954; Roeder and Weiant, 1946, 1948, 1951; Yamasaki and Ishii, 1954a, b). However, not all sensory cells are

A

B

t .---- -- ----- -- ----- -- ----- -- ---- - - - 100 rnsec

0.2 mV

Fig. 1 1 . Trains of impulses from the sensory cells of the cockroach leg after injection of DDT into the leg. A, before injection of DDT; B and C, after injection (Narahashi, 1966).

equally sensitive to DDT. For example, the sensory cells on the cerci of the cockroach are less sensitive to DDT in producing trains of impulses (Roeder and Weiant, 1948; Eaton and Sternburg, 1967). The chemical sense organs on the tarsus and labellum of the housefly become more sensitive to adequate stimuli such as those by sucrose after intoxication with DDT (Smyth and Roys, 1955; Soliman and Cutkomp, 1963). Although the sense organs in the cockroach legs are highly sensitive t o DDT, repetitive discharges from them do not seem to be the sole factor responsible for the symptoms of poisoning. In the DDT-poisoned insect and other animals, the spontaneous discharge in the central nerve cord is increased in frequency, and synaptic transmission is facilitated (Dresden, 1949; Harlow, 1958; Heslop and

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

23

Ray, 1959; Tobias and Kollros, 1946; Yamasaki and Ishii, 1952b, 1954a, c). The role of these functional changes in producing the symptoms of poisoning can be studied by changing the temperature after intoxication with DDT (Yamasaki and Ishii, 1954a; Eaton and Sternburg, 1964). As will be described later (Section VI), DDT has a large negative temperature coefficient of action. When given an appropriate dose, the symptoms of poisoning appear at low temperature ( 15" -20" C) but reversibly disappear upon raising the temperature to 29"-35°C. At the low temperature, the poisoned cockroach produces ataxia and convulsion, and both the sensory nerve of the leg and the abdominal nerve cord discharge impulses at high frequencies. Upon raising the temperature, the symptoms of poisoning disappear and the impulse discharge in the abdominal nerve cord decreases in frequency, yet the sensory nerve in the leg is still discharging repetitively. Therefore, it is concluded that the sensory repetitive discharge alone cannot produce the symptoms of poisoning. DDT also stimulates nerve fibers or nerve terminals to produce repetitive discharges (Gordon and Welsh, 1948; Narahashi and Yamasaki, 1960c; Shanes, 1949a, by 195 1 ;Welsh and Gordon, 1947; Yamasaki and Ishii, 1952a; Roeder and Weiant, 1948; Harlow, 1958; Bodenstein, 1946; Van den Bercken, 1968). As a result of the hyperactivity of the nervous tissue, an unidentified toxic substance is believed t o be released from the nerve. This substance, sometimes called "autotoxin", is able t o stimulate and then paralyze the nerve (Hawkins and Sternburg, 1964; Shankland and Kearns, 1959; Sternburg, 1960, 1963; Sternburg and Kearns, 1952; Sternburg et al., 1959). In summary, DDT stimulates the nerve t o cause hyperactivity, and the resultant toxic substance eventually paralyzes the nerve. Another important feature of DDT action on the nerve is an increase in negative after-potential. This will be discussed in detail in a later section (V A). B. LINDANE

The symptoms of poisoning in lindane-intoxicated insects are characterized by ataxia, convulsions and eventual paralysis. The convulsions are more severe than those observed in DDT-poisoned insects (Yamasaki and Ishii, 1954d).

24

T. NARAHASHI

In lindane-poisoned insects, the effect on the central nervous system dominates over that on the peripheral nervous system. The frequency of spontaneous discharges in the central nerve cord is increased significantly by treatment with lindane, and the synaptic after-discharge is greatly prolonged (Fig. 12) (Dallemagne and Philippot, 1948; Fritsch, 1952; Fritsch and Krupp, 1952; Harlow,

Fig. 12. Effects of lindane on the synaptic after-discharges recorded from the abdominal nerve cord (postsynaptic) of the cockroach. Single stimuli were applied to the cercal nerve (presynaptic). A, control; B, C and D, after direct application of lindane lo-’ M to the nerve, 1 h 25 min (B), 4 h 10 min (C), and 6 h 10 min (D). Time marker 50 C.P.S. (Yamasaki and Ishii, 1954d).

1958; Vidal-Sivilla and Larralde, 1949; Yamasaki and Ishii, 1954d). No remarkable effect of lindane is observed on the sensory cells, nor on the nerve fibers (Becht, 1958; Lalonde and Brown, 1954; Yamasaki and Ishii, 1952a). There is no paralyzing action of lindane on the nerve. C. CYCLODIENES

Based on the observation of the symptoms of poisoning of dieldrin and aldrin (Fig. 13), it was suggested that the major site of action

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

25

was at the central nervous system (Gianotti et al., 1956). It was in fact found that the frequency of spontaneous discharges in the central nerve cord of the cockroach was increased and the synaptic after-discharge was prolonged by treatment with dieldrin, although the effect was less pronounced than that of lindane (Yamasaki and Ishii, 1958a). However, later experiments with highly purified

t Aldrin ( I )

Dieldrin

(II)

CI’

Aldrin-Transdiol (V)

Fig. 13. Conversion of aldrin and dieldrin to their analogs (Wang etal., 1971).

dieldrin samples showed that dieldrin, when applied directly to the nerve, exerted no effect on the spontaneous discharge and synaptic after-discharge (Narahashi, unpublished observation; J. W. Ray, personal communication). Apparently, the previous finding was due to impurity in the dieldrin sample used. Recent experiments with several derivatives and metabolites of dieldrin strongly point out that dieldrin is converted into active forms before exerting the neural effects (Wang et al., 1971). First of all, observations were made of synaptic transmissions across the last abdominal ganglion and the metathoracic ganglion in the dieldrin-poisoned cockroach. Although the last abdominal ganglion

26

T. NARAHASHI

was only negligibly affected, the synaptic transmission across the me tathoracic ganglion was found to be facilitated. When dieldrin was directly applied to the exposed metathoracic ganglion, it took 45 min or longer for the effect of dieldrin t o become apparent. Aldrin-transdiol (Fig. 13) stimulated the ganglion very quickly, and the synaptic transmission began to be prolonged only 5 min after treatment. When injected into the cockroach leg, dieldrin itself was able to stimulate the sensory cells to initiate repetitive trains of impulses only after a latency of 45 min. A longer latency was reported with topical application of dieldrin on the leg (Lalonde and Brown, 1 954). However, aldrin-transdiol produced repetitive discharges in only 2.5 min. Since aldrin-transdiol is one of the dieldrin metabolites (Matthews and Matsumura, 1969; Klein et al., 1968), it seems reasonable to assume that it is one of the active forms of dieldrin. D. PYRETHROIDS

It has long been known that pyrethrum is a fast acting insecticide, stimulating and paralyzing insects in a brief period of time. The active ingredients, pyrethrins, and the synthetic pyrethroid, allethrin, stimulate the nerve to cause repetitive discharges (Fig. 14) and then paralyze it (Lowenstein, 1942; Narahashi, 1962a; Welsh and Gordon, 1947; Yamasaki and Ishii, 1952a). However, no repetitive trains of

Ab

5 msec

I

P

rnsec Fig. 14. Compound action potentials recorded from the abdominal nerve cord of the cockroach by means of external electrodes. Aa and Ab, control; Ba and Bb, 1 rnin 30 s after M. Temperature 28OC (Narahashi, 1962a). treatment with allethrin 3.3 x 50

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

27

impulses can be observed in the cockroach sensory cells by treatment with pyrethrins (Lalonde and Brown, 1954). As in the case of DDT, pyrethrins were found to produce a toxin in the intoxicated insect (Blum and Kearns, 1956). The toxin is presumably responsible for the paralysis of the poisoned insect together with pyrethrins themselves. E. ROTENONE

Rotenone has been known to cause flaccid paralysis of insects, decrease in oxygen consumption, and decrease in the frequency of the heart beat (Harvey and Brown, 1951; Tischler, 1936; Hatai, 1941; Krijgsman et al., 1950; Orser and Brown, 195 1; Yamasaki and Ishii, 1951). The major mechanism of action is the inhibition of the electron transfer from DPNH to cytochrome b (Fukami, 1961; Fukami and Tomizawa, 1956, 1958a, b; Lindahl and Oberg, 1961; Oberg, 1961). Rotenone blocks the conduction of nerve (Fukami et aL, 1959), and depolarizes the nerve membrane (Yamasaki and Narahashi, 1957~).However, it remains to be seen whether the depolarization is due to the accumulation of potassium around the nerve membrane caused by the inhibition of the metabolic pump or the direct action on the nerve membrane. The muscle is also paralyzed by rotenone, but the effect is brought about later than the nerve paralysis (Fukami, 1954, 1956). It is noteworthy that three effects of rotenone, i.e. the nerve blockage, the metabolic disturbance, and the insecticidal action, go parallel with each other among a number of rotenone derivatives tested (Fukami et al., 1959) (see Section VIII C). F. ORGANOPHOSPHATES

It has well been established that organophosphorus insecticides inhibit ChE’s as their original forms or after having been converted into active forms. The transmitter substances in synapses and other junctions in insects still largely remain to be explored, but there is some evidence in support of the idea that 1-glutamate is the transmitter substance at the neuromuscular junctions of insects (Faeder, 1968; Kerkut et al., 1965; Usherwood and Machili, 1966; Usherwood et al., 1968). It seems also likely that ACh is the transmitter substance at the synapses in the last (sixth) abdominal ganglion of the cockroach (Yamasaki and Narahashi, 1960; Callec

28

T. NARAHASHI

and Boistel, 1967; Kerkut et al., 1969). These synapses connect the cercal sensory nerve fibers with the giant axons in the abdominal nerve cord. The effects of various organophosphorus insecticides on the insect nerve and muscle system are explicable in terms of their antiChE activity.

I . Synaptic After-discharges When the cercal nerve of the cockroach is stimulated by a single shock, the postsynaptic response can be recorded from the abdominal nerve cord. The postsynaptic response is composed of an initial few spikes of large amplitudes followed by an after-discharge of small amplitudes. The after-discharge lasts for about 100 ms (Fig. 15). After treatment with an organophosphorus or other antiChE compound, the synaptic after-discharge is prolonged in duration and increased in amplitude. The effect becomes more pronounced with time, and eventually the postsynaptic neurons produce a burst of high-frequency discharges which is followed by a sudden cessation and paralysis. The synaptic transmission is blocked at that time. However, spontaneous discharges begin to appear soon and the synaptic transmission is restored in the continuous presence of the ChE inhibitor. This block-and-recovery process is repeated many times in the presence of the ChE inhibitor (Narahashi and Yamasaki, 1960a; Roeder, 1948; Roeder and Kennedy, 1955; Roeder et al., 1947; Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958c, 1960). Similar effects are observed in the locust (Harlow, 1958).

2. Postsynaptic Potential and Membrane Potential The mechanism of action of anti-ChE’s on the synaptic transmission was studied in more detail by recording the excitatory postsynaptic potential and membrane potential (Yamasaki and Narahashi, 1958c, 1960). When one external electrode is in contact with the last abdominal ganglion and the other with the abdominal nerve cord (e.g. the connective between the second and third abdominal ganglia), changes in the membrane potential of the last abdominal ganglion produced by drugs or presynaptic stimulation can be recorded together with the action potentials. Examples of such records of the postsynaptic responses are shown in Fig. 15. In normal physiological saline solution, the initial large spikes are followed by a slow depolarization on which small after-discharges are superimposed (far left record in A). After

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

29

M on the synaptic transmission across the last Fig. 15. Effects of eserine 7.7 x abdominal ganghon of the cockroach. A, from left to right, before, 12,19and 29 min after treatment with eserine, respectively. B, as in A, but 56 min after eserine. All records are arranged on the same base line shown on the record far left. B1,from left to right, responses at the time of presynaptic stimulation, 15, 30 and 45 s after stimulation. B2, continuation of B1, from left to right, 60, 75, 90, and 180s after stimulation. C, spontaneous depolarization and repolarization, accompanied by discharges, 81 min after treatment with eserine. The time intervals between successive records are 15, 30, 60, 30, 15,45,and 15 s, respectively. D, postsynaptic responses showing their dependence on the membrane potential, 240 min after treatment with eserine. Voltage calibration in A, 0.5 mV, applied to A, B, and C; voltage calibration in D, 5 mV. Time marker in C2,50c.P.s., applied to A, B, and C; time marker in D, 100 C.P.S. (Yamasaki and Narahashi,1960).

30

T. NARAHASHI

treatment with eserine 7.7 x lo-’ M, the late slow depolarization is increased in magnitude and prolonged in duration with an increasing number of spikes superimposed on it (A). Finally, the late slow depolarization reaches a threshold level beyond which discharges are blocked (B 1 and B2). The depolarizing phase may last as long as 10 s. At this stage, therefore, the prolonged slow depolarization and after-discharges initiated by a single presynaptic stimulus are followed by a cessation of discharges which are in turn followed by a reappearance of discharges as the membrane is slowly repolarized toward the resting level. The repolarizing phase may last as long as 3 min. This process involving depolarization and repolarization appears even spontaneously, and discharges can be seen at a certain depolarized level (C1 and C2). The synaptic transmission is blocked when the membrane is spontaneously depolarized beyond the threshold, and is restored as the membrane is repolarized (D). The mechanism whereby such a spontaneous depolarization-repolarization is produced under the influence of antiChE’s remains to be seen. The slow depolarization observed in the preparation treated with antiChE has been found to be a prolonged large e.p.s.p. Phenobarbital or urethan, when applied at appropriate concentrations, is capable of blocking synaptic transmission without affecting the conduction of the presynaptic nerve fibers. Under these conditions, the e.p.s.p. can be observed without being disturbed by spike discharges superimposed upon it. Examples of records of the e.p.s.p.s are shown in Fig. 16. It is clearly seen that the e.p.s.p.

Fig. 16. Effects of eserine 1.5 x lo-’ M on the excitatory postsynaptic potential recorded from the last abdominal ganglion of the cockroach. The preparation is under the influence of urethan 5.6 x lo-’ M to stop discharges. A, before application of eserine; B, 8 min after application of eserine; D, 12 min after application of eserine. Three short records on the right are 1, 2, and 3 s after the stimulation, respectively. Voltage calibration in C, 0.5 mV, applies to all records. Time marker in C, 50 c.P.s., applies to A and B. Time market in D, 50 C.P.S. (Narahashi, 1965b).

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

31

observed under the influence of urethan 0.56 M is greatly augmented in magnitude and prolonged in duration after treatment with eserine 1.5 x 10-5 M.

3. Cholinesterase Inhibition in Nerve The effect of antiChE’s described in the foregoing section has been found t o be related to the inhibition of ChE in the nerve. The ChE activity in the nerve preparations that have started showing prolonged synaptic after-discharges after treatment with parathion, thiol-demeton, thiol-methyldemeton, or thiono-methyldemeton is partially inhibited (Yamasaki and Narahashi, 1960; Narahashi and Yamasaki, 1960a). The exact percentage of inhibition is difiicult to estimate because the ChE activity may be partly restored while the nerve preparation is homogenized and diluted for assay. It is reasonable t o assume that the persistent presence of the transmitter substance in the synaptic area causes a prolongation of the e.p.s.p. thereby producing a prolonged after-discharge.

4. Effects on Other Nerve-Muscle Systems When treated with TEPP, the sensory cells in the cockroach leg produce trains of impulses after a long latency. However, parathion and schradan have no effect on them (Lalonde and Brown, 1954). Although parathion has no effect on the chemo-receptors on the labellum of the housefly, paraxon stimulates it to increase the frequency of discharges (Leski and Cutkomp, 1962; Soliman and Cutkomp, 1963). The neuromuscular transmission of insects is not affected by anti-ChE’s (Colhoun, 1960; Harlow, 1958; Narahashi, unpublished observation) in agreement with the observation that the transmitter substance is likely t o be 1-glutamate.

V. MECHANISMS OF FUNCTIONAL CHANGES CAUSED BY INSECTICIDES IN NERVE AND MUSCLE A. DDT

1. After-potential and Repetitive Discharges

Increase in negative after-potential by treatment with DDT was discovered by Shanes (1 949b) for the first time using crab nerve as material. Shortly after that time, Yamasaki and Ishii (1952b) found that the cockroach nerve fibers underwent a similar change by

32

T. NARAHASHI

intoxication with DDT. These two studies were performed by means of external recording techniques. Detailed analyses of the increased negative after-potential in the DDT-poisoned cockroach giant axon were since undertaken with the aid of both extracellular and intracellular electrodes (see Fig. 20). (a) After-potential and Supernormal Phase: In the cockroach nerve, the negative after-potential is greatly prolonged after treatment with DDT. The effect can be observed with the crural nerve or with the abdominal nerve cord by means of external recording electrodes (Yamasaki and Narahashi, 1957a). Since there was general agreement that the negative after-potential is accompanied by a supernormal phase or a decrease in threshold and the positive after-potential is accompanied by a subnormal phase or an increase in threshold (Gasser, 1941 ;Gasser and Grundfest, 1936; Graham, 1930; Graham and Gasser, 193 1 ; Lehmann, 1937), changes in excitability were examined during the course of the increased negative after-potential in the DDT-poisoned cockroach axon (Y amasaki and Narahashi, 1957a). A supramaximum conditioning stimulus is followed by a test stimulus of just above threshold intensity. The height of the action potential produced by the test stimulus is plotted as a function of the interval between the two stimuli. As is shown in Fig. 17, there is no significant supernormal phase in the control normal nerve. In the DDT-poisoned nerve, however, the absolute refractory period is followed by a marked supernormal phase, its time course running almost parallel with the negative after-potential (Fig. 18). (b) After-potential as Studied by Microelectrodes: There was no doubt that the prolonged falling phase of the externally recorded action potential from the DDT-poisoned cockroach nerve bundle was not due to a summation of repetitively firing small action potentials but due to an increase in negative after-potential in each fiber, because the same effect was observed in action potentials from single fibers in the nerve bundle (Yamasaki and Narahashi, 1957a). However, it is necessary t o confirm this notion by means of intracellular microelectrodes. Moreover, more detailed analyses on the mechanism of action of DDT can be undertaken by this technique. The first observation of the DDT-induced large negative after-potential with intracellular microelectrodes was made by Yamasaki and Narahashi (1957b). More detailed analyses were performed later as described below.

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

33

Fig. 17. Recovery process after an action potential i n the cockroach abdominal nerve cord bathed in normal solution. Ordinate, the amplitude of the externally recorded action potential (in percentage) relative to that produced by the supramaximum stimulation. Abscissa, the interval between a maximum conditioning stimulus and a weak submaximum test stimulus. The broken horizontal line represents the amplitude of the action potential produced by the test stimulus alone, and the dots represent the responses by the test stimuli when they are preceded by a conditioning stimulus. The action potential produced by the conditioning stimulus is also drawn with the peak as 100%. Note the absence of the supernormal phase after the conditioning action potential (Yamasaki and Narahashi, 1957a).

Fig. 18. Recovery process after an action potential in the abdominal nerve cord from the DDT-poisoned cockroach. See Fig. 17 for explanation. Note that the increased and prolonged negative after-potential is accompanied by a marked supernormal phase (Yamasaki and Narahashi, 1957a).

34

T. NARAHASHI

(i) After-potential in Normal Cockroach Giant Axons: In the normal cockroach giant axon, the action potential is followed by an undershoot or positive phase of about 5 mV amplitude which in turn is followed by a small negative after-potential of about 1.5 mV amplitude (Fig. 19). The positive phase can be explained in terms of a persistent increase in membrane potassium conductance (Yamasaki and Narahashi, 1959). The negative after-potential has been

Fig. 19. Action potentials recorded intracellularly from the giant axon of the cockroach. Note that the spike is followed by an undershoot or positive phase which is in turn followed by a slight depolarizing phase or negative after-potential (Narahashi, 1965a).

demonstrated to be due to an accumulation of potassium in the immediate vicinity of the nerve membrane (Narahashi and Yamasaki, 1960b). The conclusion is based primarily on the analyses of the time course of the negative after-potentials during repetitive stimuli. (ii) After-potential in DDT-Poisoned Cockroach Giant Axons: After introducing DDT into the nerve chamber at a concentration of M ythe negative after-potential is slowly increased in magnitude and prolonged in duration (Fig. 20). Repetitive after-discharges are usually produced by a single stimulus as the negative after-potential is increased. The repetitive responsiveness disappears as the negative after-potential grows further, and the latter finally reaches about 30 mV or more in magnitude (Fig. 20). The resting potential remains essentially unchanged, and the rising phase and the peak magnitude of the action potential are unaffected (Narahashi and Yamasaki, 1960~). In contrast to the negative after-potential in the normal unpoisoned cockroach giant axon, the DDT-induced large negative after-potentials are not built up upon repetitive stimuli. This behavior is shown in Fig. 21 in which the initial height of the negative after-potential remains almost constant during repetitive stimuli. If the effect of DDT on the negative after-potential were due

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

0.1

msec

35

10 msec

Fig. 20. Changes in intracellularly recorded action potential of the cockroach giant axon M. A, from top to bottom, before 38 min after, and 90 min after treatment with DDT after treatment with DDT. The horizontal lines indicate zero potential level. B, as in A, but with slower sweep (Narahashi and Yamasaki, 1 9 6 0 ~ ) .

to an increase in potassium accumulation around the nerve membrane, the negative after-potentials would be built up during repetitive stimuli as has been observed in the normal axon. Therefore, the effect is possibly due t o changes in conductance parameters responsible for the falling phase of the action potential, i.e. the mechanism whereby the sodium conductance is decreased or the sodium inactivation, or the mechanism whereby the potassium conductance is increased upon stimulation, or both. When a square pulse of current is applied to the nerve membrane, the resultant electrotonic potential rises exponentially and attains a steady-state level. A current-voltage relation for the steady state shows a rectification in the depolarizing direction, increasing the intensity of outward current producing a smaller magnitude of steady depolarization than the corresponding steady hyperpolarization. This is called “delayed rectification”, and can be ascribed to the increase in potassium conductance of the membrane. The delayed rectification has been found to be suppressed by application of DDT (Narahashi and Yamasaki, 1960c). It was therefore suggested that the

36

T. NARAHASHI

Fin. 21. After-uotentials during reuetitive stimuli of varvine freauencies in the no1.mal

(A) and DDT-poisoned (B) cockro&h-giant axons. The spikk pkentials are too large to be recorded. The frequencies of stimuli are, from top to bottom in A, 50, 100, 150, and 200 c.P.s., and in B, single stimulus, 50, 100, 200 and 300 C.P.S. (Narahashi and Yamasaki,

1960b, c).

suppression of the potassium conductance increase by DDT was at least partly responsible for the increase in negative after-potential. The large negative after-potential in the DDT-poisoned cockroach giant axon is further augmented in magnitude by removal of potassium from the bathing medium, producing a plateau resembling cardiac action potentials (Narahashi and Yamasaki, 1960d). The DDT-poisoned axon in K-free medium resembles cardiac tissues not only in the shape of the action potential but also in its electrical properties. For example, anodal break response is easily produced, the action potentials are very often produced spontaneously

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

37

(Fig. 22), the plateau phase is abolished by application of anodal current, and the refractory period for the duration of the plateau phase is extremely long. Based on the measurements of membrane conductance during the plateau, it was suggested that the sodium conductance, after having risen to the normal value, declined slowly and the potassium conductance underwent little or no change during

Fig. 22. Action potentials produced by a single stimulus (top record), and spontaneously (middle and bottom records) in the cockroach giant axon bathed in K-free DDT medium. Spike potentials are too large to be recorded (Narahashi and Yamasaki, 1960d).

the plateau phase in the DDT-poisoned cockroach giant axon (Narahashi and Yamasaki, 1960d). This notion has now been subjected t o voltage clamp analyses (Section V A 2 ) . (c) Repetitive Discharge: Welsh and Gordon ( 1947) and Gordon and Welsh (1 948) made interesting observations on the role of calcium in the DDT-induced repetitive discharge in crustacean nerves. An increase in calcium concentration in the bathing medium generally suppresses the repetitive responsiveness induced by DDT. This observation is taken as indicating that DDT somehow disturbs the binding of calcium with the nerve membrane components thereby causing an unstabilizing effect. The increase in negative after-potential during the course of DDT

38

T. NARAHASHI

poisoning is no doubt one of the factors responsible for repetitive responsiveness, because a sustained depolarization works as a stimulant. However, it should be noted that the sustained negative after-potential is not the sole factor responsible for repetitive firing. A prolonged outward current applied to the normal cockroach giant axon does not produce repetitive firing (Yamasaki and Narahashi, 1959). Therefore, the DDT-poisoned axon must undergo changes in such a way as the sustained negative after-potential can initiate repetitive firing. It is also of interest that the repetitive responsiveness caused by DDT has a very high negative temperature coefficient of action as will be described later (Section VI A). 2. Effects on Membrane Ionic Conductances The hypothesis decribed in the foregoing section can be demonstrated by voltage clamp experiments whereby each component of membrane ionic conductances is measured. Detailed analyses have been performed using lobster giant axons as material (Narahashi and Haas, 1967, 1968). (a) Methods: Squid giant axons are most convenient for voltage clamp experiments because of their large diameter (about 5 0 0 ~ ) . However, it was found that they were extremely insensitive to DDT, external or internal application of DDT causing only a small increase in negative after-potential even at a very high concentration of 10-4 M. Therefore, lobster giant axons, which were sensitive to DDT as cockroach giant axons, were used as material for voltage clamp experiments. The lobster giant axon is only about 80 p in diameter on an average, but large enough to do voltage clamp experiments if the sucrose-gap apparatus is used. As described earlier (Section I11 A 4), measurements of membrane currents by voltage clamp techniques require the space clamp conditions in which membrane current and membrane potential are distributed uniformly in a limited area of the nerve preparation. This can be achieved by inserting a wire electrode longitudinally into the giant axon. The squid giant axon is large enough for the wire insertion, but such a technique is almost impracticable in the lobster giant axon. However, if the sucrose-gap insulation originally developed by S t h p l f i (1954) is combined with voltage clamp, one can measure membrane currents. This method called “sucrose-gap voltage clamp” was developed by Julian et al. (1962a, b) for the lobster giant axon, and has since been used not only for the lobster

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

39

giant axon but also for the squid and crayfish giant axons (e.g. Moore et al., 1964a, by 1967; Takata et al., 1966a, b; Narahashi et al., 1967a, b y 1969a, byc; Narahashi and Anderson, 1967; Frazier et al., 1969). A narrow portion (about 100 p wide) of an isolated giant axon, which is in contact with the physiological saline solution, is insulated from both ends of the axon by means of two isotonic sucrose solutions. One end of the axon across this sucrose insulation is in contact with the physiological saline solution, while the other end is in contact with the isotonic KCl solution to depolarize the membrane completely. All of the solutions are flowing continuously. Because the sucrose insulation under these conditions is almost perfect, the absolute value for the membrane potential, without any significant attentuation, can be measured on the central narrow portion called “artificial node” using the KC1 pool as the zero reference potential. The artificial node can be stimulated by application of current pulses between the artificial node and the physiological saline pool. Since the width of the artificial node is much smaller than the length constant of the axon, the space clamp conditions are established in this node. Thus it is possible to make voltage clamp measurements of membrane currents on the artificial node. The sucrose-gap voltage clamp method has several advantages and disadvantages over the conventional axial wire voltage clamp method. Details of the technique will be discussed elsewhere, and will not be described here. The only point worthy of mentioning in connection with the study of DDT action is the fact that the survival time of an artificial node is relatively short (less than 20 min) in the lobster giant axon. Since the action of DDT progresses slowly, it is necessary to measure membrane currents on normal control axons and on DDT-treated axons separately. The survival time of the artificial node under sucrose-gap conditions is longer for larger axons. This is probably due to leakages of internal ions into the sucrose solution across the nerve membrane. (b) Membrane Currents: The top set of records in Fig. 23 represents a family of membrane currents associated with step depolarizations of various magnitudes recorded from a normal lobster giant axon under sucrose-gap voltage clamp conditions. The second set from the top shows a similar family of membrane currents recorded from another lobster giant axon poisoned with 5 x M DDT for a period of 40 min. Two changes brought about by DDT treatment are

40

T. NARAHASHI

ma/crn2 10

4

mv

Normal

-5

5 X 10-4M DDT t 3 X I O-7M T T X - 4 min 0

-

-20

-5

6min

-

8 ,0 -60

-40

20

-__l_m

C

.

.

0

1

2

'

*

k

3

4

5

'

I

-28

\-40 -50

6 7 msec Fig. 23. Families of membrane currents associated with step deuolarizations in a normal lobster giant axon, and another axon treated with DDT 5 x M and with DDT and tetrodotoxin ('ITX) 3 x lo-' M. The third set of records shows changes in membrane current during the course of TTX action. The dotted lines in each set refer to the zero base line (Narahashi and Haas, 1967).

easily recognized: (1) the transient sodium currents, though they rise almost normally, fall more slowly in the DDT-poisoned axon; (2) at certain membrane potentials (-20mV, -40mV, and -50mV in Fig. 23), the transient sodium current is followed by an inward steady-state current in the DDT-poisoned axon. This is never observed in the normal axon. There are two possible explanations for the inward steady-state current flow in the DDT-poisoned axon. One of them is to assume potassium as its carrier. However, this possibility can be excluded by the fact that the resting potential remained essentially unchanged by

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

41

treatment with DDT. The constant resting potential suggests that the internal potassium concentration is not changed drastically. Hence it is not possible for potassium current to flow inwardly at those membrane potentials mentioned above. The other explanation would be that the inward steady-state current represents a residual component of prolonged sodium current. In order t o demonstrate this possibility, tetrodotoxin (TTX) was applied on the DDT-poisoned axon. Tetrodotoxin is the active principle of the puffer fish poison, and has been demonstrated to block the sodium current selectively without any effect on the potassium current (Narahashi et al., 1964). Changes in membrane currents during the application of TTX 3 x lo-' M to the DDT-poisoned axon are shown in the third set of records in Fig. 23. It is seen that the transient sodium current is completely blocked and the inward steady-state current is now converted into a small outward steady-state current 4 min after introduction of TTX. The difference between the membrane current .at 0 min in TTX and that at 4 min in TTX should represent the sodium current flowing in the DDT-poisoned axon. The bottom set of records in Fig. 23 represents a family of membrane currents associated with various step depolarizing pulses in DDT plus TTX. The sodium currents are completely blocked, whereas the potassium currents are suppressed in magnitude compared with those from the normal axon (note that the ordinate scale is different). (c) Current- Voltage Relations: The current-voltage relations for the peak value of the transient current and for the steady-state current are illustrated in Fig. 24. The current-voltage curve for the peak transient current is not appreciably affected by exposure t o DDT (open circles). However, the steady-state current undergoes a considerable change (open triangles). In the normal axon, the steady-state current flows in outward direction in the entire range of membrane potential studied (Fig. 24(a)). In the DDT-poisoned axon, however, the steady-state current is seen to be flowing in inward direction at the membrane potentials ranging from -60mV to -15 mV (Fig. 24(b)). Moreover, the amplitude of the steady-state current is suppressed at more depolarized membrane potential levels. The membrane currents in DDT plus TTX are depicted by closed symbols in Fig. 24(b)). The transient sodium current is almost completely inhibited (closed circles). The steady-state current is now flowing in outward direction in the entire range of membrane

42

T. NARAHASHI

(a)

2-13-67-Al Normal

3-10-67-02

5XIO*M DDT 3XIO-'M TTX

+

Fig. 24. Current-voltage relations for peak transient (sodium) current (Ip) and steady-state (potassium) current (1,J in a normal lobster giant axon, and in another axon treated with DDT 5 x M or with DDT and tetrodotoxin (TI'X) 3 x lo-' M. The broken line shows the residual component of the transient current and was obtained by subtracting I,in DDT plus TTX from I,in DDT (Narahashi and Haas, 1967).

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

43

potentials studied (closed triangles), and should represent the potassium current. Therefore, the residual component of the sodium current can be obtained by subtracting the steady-state potassium current in DDT plus TTX (closed triangles) from the steady-state current in DDT (open triangles), and is drawn by a broken line. It is noteworthy that the residual sodium current reverses its polarity at the membrane potential where the peak transient sodium current also reverses its polarity (+40mV). Thus it is clear that in the DDT-poisoned axon the sodium current is greatly prolonged in its falling phase and the potassium current is suppressed. Since these two are the mechanisms that are directly responsible for the falling phase of the action potential, the inhibition of both of them naturally causes a prolongation of the action potential as has actually been observed. (d) Time Course of Na Inactivation: The time course of sodium inactivation can be plotted from the membrane current corrected for the potassium component. The procedure is shown in Fig. 25. The upper set of tracings shows the time course of the sodium current in a normal lobster giant axon. In this case saxitoxin (STX) is used

Fig. 25. Separation of membrane current into sodium current ( 1 ~ and ~ ) potassium current ( I K ) by use of saxitoxin or tetrodotoxin 3 x lo-' M in a normal and in a DDT-treated lobster giant axon. The membrane current in saxitoxin and that in DDT plus tetrodotoxin represents I K . IN^ is obtained by subtraction of IK from the total membrane current (Narahashi and Haas, 1968).

44

T. NARAHASHI

instead of TTX. Saxitoxin is the toxic principle of the poison from the toxic Alaska butter clam, Saxidomas giganteus. It is suggested that STX in the clam originally derives from the dinoflagellates, Gonyaulax catanella (Kao, 1966; Schantz et al., 1966). It has been shown that STX behaves in almost the same way as TTX in selectively blocking the sodium conductance increase (Narahashi et al., 1967b). The net sodium current (INa)is obtained by subtraction of the membrane current in STX from that before STX. The net sodium current in the DDT-poisoned axon is obtained in the same manner by using TTX, and is illustrated in the lower half of Fig. 25. The falling phase of the sodium current is then plotted in Fig. 26 on a semilogarithmic scale. It is clearly seen that the sodium current in the normal axon declines exponentially, whereas in the DDT-poisoned axon it declines slowly in two or more exponential functions. The time constant of the falling phase of the sodium current is estimated as 0.65 ms on an average for the normal axons. The average value for the DDT-poisoned axon is 2.93 ms for the first phase and 1 1.9 ms for the second phase.

0

2

4

6 8 Tinr ( m i r )

10

12

I1

Fig. 26. Semilogarithmic plot of the time course of the falling phase of the peak transient (sodium) current (Ip) at -20 mV in a normal and in a DDT-treated lobster giant axon after correction for the steady-state (potassium) current in the same way as in Fig. 25. The straight lines were drawn by eye (Narahashi and Haas, 1968).

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

45

(e) Effects on Other Kinetic Parameters: Other kinetic parameters of membrane currents are also slowed by treatment with DDT, but the effect is much less than that on the time course of sodium inactivation. The time for the transient sodium current to reach its peak is slowed from the normal average value of 0.77-0.99ms at -20 mV membrane potential after exposure to DDT. The time for the steady-state potassium current to reach its half maximum is also slowed from the normal average value of 2.33 ms to 3.51 ms at 0 mV membrane potential by DDT intoxication. Thus, it can be concluded that the kinetics for the on-process of sodium current, the sodium inactivation, and the on-process of potassium current are all slowed by the action of DDT. The slowing of the sodium inactivation is most remarkable. ( f ) Discussion: The results of voltage clamp experiments account for the prolongation of the action potential by treatment with DDT. DDT inhibits the mechanism whereby the sodium conductance is turned off and that whereby the potassium conductance is turned on, and these mechanisms are directly responsible for the falling phase of the action potential. Therefore, the prolongation of the falling phase of the action potential by DDT can be ascribed to these changes in membrane conductances. These effects of DDT on membrane conductances were confirmed with the giant axon of the cockroach (Pichon, 1969a, b). The same effect of DDT on the time course of the sodium inactivation was found with the node of Ranvier of the frog (Hille, 1968). However, the potassium current is not appreciably suppressed by application of DDT. The experiments performed with both lobster and frog nerves show that the so-called “sodium channels” remain open for an unusually long period of time after intoxication with DDT. The sodium channel here simply refers to a conceptual pathway through which sodium ions flow according to the electrochemical potential gradient. It does not necessarily mean any anatomical hole or pore, or any carrier mechanism. It is also suggested that DDT has no effect on the sodium channels that are not open (Hille, 1968). B. ALLETHRIN

1. Effects on Action Potential

Intracellular microelectrode recordings of resting and action potentials from the cockroach giant axons have revealed three effects

46

T. NARAHASHI

of allethrin (Narahashi, 1962a): (1 ) the negative after-potential is increased and prolonged; (2) repetitive afterdischarges are produced by a single stimulus; (3) at a higher concentration of allethrin, the nerve conduction is eventually blocked. (a) After-potential and Repetitive Discharge: Figure 27 shows an example of a series of records of action potentials from the

Ca

Fig. 27. Action potentials of the cockroach giant axon before (Aa-c), 6 min after (Ba-c), M (Narahashi, 24 min after (Ca-c), and 88 min after (Da-c) treatment with allethrin 1962a).

cockroach giant axon before and after application of allethrin at a concentration of M. The rising phase of the action potential is only slightly slowed after application of allethrin, whereas the falling phase is greatly slowed and is followed by a prolonged negative after-potential. The resting potential remains essentially unchanged during the course of the allethrin action (Fig. 28). Repetitive discharges are often superimposed on the large negative afterpotential in the allethrin-poisoned axon. This phenomenon is especially remarkable at high temperature beyond about 27"C, as will be discussed later (Section VI B).

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

47

ARP

W AP 12 120

80

20

Fig. 28. Changes in the amplitude of the action potential (AP), the initial amplitude of the negative after-potential (NAP), and the resting potential (RP) in the cockroach giant M (Narahashi, 1962a). axon after treatment with allethrin

A number of experiments have been performed by means of the microelectrode technique in an attempt to elucidate the mechanism of the increase in negative after-potential by allethrin (Narahashi, 1962b). Upon repetitive stimuli the negative after-potentials of the allethrin-poisoned axon are built up to some extent as in the case of the normal axon (Fig. 29). This is in sharp contrast with the situation of the DDT-poisoned axon in which no remarkable addition of the negative after-potentials is observed. This observation was taken as indicating that a depolarizing substance is accumulated outside or inside of the nerve membrane during the course of the repetitive stimuli.

A

B

C

Fig. 29. After-potentials produced by repetitive stimuli of varying frequencies in the cockroach giant axon. Only the positive phase and the negative after-potential are seen; the spike phase is too large to be recorded. A, in normal saline solution, 50 c.P.s.; B, 10 min M, 50 c.P.s.; C, 11 min, 10 C.P.S. (Narahashi, 1962b). after treatment with allethrin

48

T. NARAHASHI

However, the following experiment illustrated in Fig. 30 excludes the possibility that the depolarizing substance is potassium ion. When the potassium concentration is raised from the normal value of 3.1 mM to 30 mM, the after-potential of the allethrin-poisoned axon undergoes a considerable change in shape (Fig. 30, Ab). The after-potential associated with the second action potential elicited during the course of the after-potential of the first action potential

r-pF

Aa

Ba

J

111

Ab

J

5mv

50 msec

50 msec

I

Fig. 30. Effects of K-rich solution and conditioning action potentials on after-potentials of the allethrin-treated cockroach giant axon. Aa, in 3.3 x lo-' M allethrin; Ab, after treatment with 30 mM K; Ac, after washing with 3.3 x lo-' M allethrin. Bas, the test action potentials are produced at various moments during the course of the negative after-potential associated with the conditioning impulse (Narahashi, 1962b).

also undergoes a change, but in an entirely different way from the change brought about by high potassium (Fig. 30, Ba, Bb, Bc). Therefore, the large negative after-potential in the allethrin-poisoned axon cannot be ascribed to the accumulation of a large amount of potassium. Later experiments with voltage clamp techniques have demonstrated that this effect of allethrin is due to changes in sodium inactivation and potassium conductance increase mechanism (Section V B 2). (b) Conduction Block: At a high concentration of 3.3 x M, allethrin eventually blocks the action potential of the cockroach giant axon. The membrane is slightly and progressively depolarized during the course of this allethrin action, but the depolarization is not enough to account for the conduction block. An example of

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

49

such experiments is illustrated in Fig. 3 1, in which the maximum rate of rise of the action potential is plotted as a function of membrane potential. The maximum rate of rise of the action potential is proportional to the inward ionic current (sodium current) at that moment, and therefore can be used as a good measure of excitability. Before application of allethrin (open circles) the maximum rate of rise of the action potential is increased by anodal hyperpolarization

*

-

a

-

-

-

a

0

e

w

-

a

0 Control. Fblorizotion

A Allethrin I m l . Course of block 0 Allethrin ldptnl. Wrizotion I

I

Membrane potential (mV) Fig. 31. The maximum rate of rise of the action potential plotted as a function of membrane potential before and after application of allethrin (steady-state sodium inactivation curve). Cockroach giant axon. Open circles represent the measurements while the membrane potential is displaced from the resting potential (arrow) by polarizing currents. Filled triangles are the measurements during the course of allethrin action. Filled circles are similar measurements as the control after the membrane is depolarized by allethrin to the level shown by arrow (Narahashi, 1965a).

of the membrane and finally attains a steady value. It is decreased by cathodal depolarization and is finally blocked. When allethrin is applied, the maximum rate of rise of the action potential starts decreasing without any appreciable change in resting potential as shown by filled triangles. The resting potential then starts decreasing, and the excitability is completely blocked. However, the maximum rate of rise of the action potential is partially restored by anodal hyperpolarization (filled circles). This experiment strongly suggests that the mechanism by which the sodium conductance is increased AIP-3

50

T. NARAHASHI

upon stimulation is inhibited by allethrin. The notion was later demonstrated by the voltage clamp experiment (Section V B 2). Another point worthy of note in the experiment shown in Fig. 31 is that after treatment with allethrin the curve relating the maximum rate of rise of the action potential to the membrane potential (sodium inactivation curve) is shifted along the potential axis in the direction of hyperpolarization. This shift can be estimated from the membrane potentials where the respective curves attain 50% maximum value, or more directly if the two curves are normalized. Recent voltage clamp experiments with crayfish giant axons have demonstrated the shift of the sodium inactivation curve by poisoning with allethrin (unpublished observation). Thus this shift also contributes to the suppression of the action potential by allethrin. 2. Effects on Membrane Ionic Conductances Voltage clamp experiments on allethrin action have been performed with the giant axon of the squid (Narahashi and Anderson, 1967). Unlike DDT, allethrin exerts similar actions both on the cockroach giant axon and on the squid giant axon. It should be emphasized that the squid axon is much superior to the cockroach or lobster axon for voltage clamp analyses, because measurements on membrane currents can be made more accurately on the squid axon than on the other axons owing to its larger diameter and longer survival time under sucrose-gap conditions. (a) Methods: The sucrose-gap voltage clamp technique used for the squid giant axon is essentially the same as that for the lobster giant axon (Section V A 2a). Both external and internal applications of allethrin were attempted. It is somewhat surprising to find that allethrin exerts an additional effect when applied internally in view of its high lipophilic property. Two methods of internal perfusion of the squid giant axon were developed by different groups. Baker et al. (1961) developed a technique whereby the axoplasm was squeezed out by a small roller. When the crushed axon preparation was inflated by perfusion of internal media, the normal sized resting and action potentials were recorded and lasted for a few hours. Oikawa et al. ( 1961) developed a cannulation method. Two glass capillaries, one large (about 300 p in diameter) and the other small (about 150 p), were inserted longitudinally from both ends of the axon. After the capillaries met at the middle of the preparation, they were slowly withdrawn while internal media were introduced from the small capillary. The

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

51

axoplasm was gradually washed out from the large capillary and finally a continuous internal perfusion was established. In our experiments, a modified squeezing method was employed exclusively. The internal media generally contain potassium in the form of fluoride or glutamate salt. At the early stage of internal perfusion, potassium sulfate was often used (e.g. Narahashi, 1963b; Baker et al., 1962a, b), but it was later found that fluoride and glutamate were among the best anions for this purpose (Tasaki et al., 1965). In the present experiment on allethrin, the following two kinds of internal media were used: Solution I contained 400 mMK', 50 mM Na', 420 mM F-, 15 mM HzPO;, and 250 mM sucrose, and the pH was adjusted to 7.3. Solution I1 contained 350 mM K ' , 50 mM Na', 320 mM glutamate-, 50 mM F-, 15 mM H, PO,, and 333 mM sucrose and the pH was adjusted to 7.3. Both solutions gave essentially the same result. Before performing voltage clamp experiments, it was confirmed that the squid giant axon responds t o externally applied allethrin in the same manner as the cockroach giant axon, i.e. the negative after-potential is increased and prolonged, repetitive after-discharges

P-

............... -30pM ..................

Allethrin Internally 10 min

Control

100mv

Fig. 32. Prolongation of the action potential of squid giant axons by internal perfusion of allethrin under sucrose+p conditions (Narahashi and Anderson, 1967).

are superimposed on the negative after-potential, and the action potential is eventually blocked. However, it should be noted that the effect on the negative after-potential is much more conspicuous when allethrin is applied internally than externally. The spike phase of the action potential is followed by a large and prolonged falling phase forming a plateau (Fig. 32). (b) Effects o n Membrane Currents b y External Application: Both peak transient sodium current and steady-state potassium current are

52

T. NARAHASHI

inhibited by application of allethrin at a concentration of lo-' M (Fig. 3 3 ) . When the peak current and the steady-state current are plotted as a function of the membrane potential, a current voltage relation can be obtained (Fig. 34). Control I------

-_

......IOOmv

......

10 JJMAllethrin Externally 2.5 min

..... ......

........

8-5-65

..100mv

......

.......... 0

:-s0 Fig. 33. Families of membrane currents associated with step depolarizations in a voltage clamped squid giant axon before and during treatment with allethrin externally (Narahashi and Anderson, 1967). '-5 0

2 mnc

-

Control

8-4-65-C

Fig. 34. Current-voltage relations for the peak transient (sodium) current (Ip) and for the steady-state (potassium) current (lsJ in a voltage clamped squid giant axon before and during exposure t o allethrin externally (Narahashi and Anderson, 1967).

The membrane chord conductance during the peak sodium current can be calculated from the equation:

where g , refers to the peak conductance, I p the peak current, E the membrane potential, and E , the equilibrium potential for I,. The membrane slope conductance during the steady-state potassium

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

53

current is calculated instead of the chord conductance because of difficulty in estimating the potassium equilibrium potential. The slope conductance (g), for the steady-state current (Iss) is given by 4

s

=dE-

The peak sodium conductance is suppressed t o an average of 53% normal control value by lo-’ M allethrin applied externally (seven experiments), whereas the steady potassium conductance is suppressed to an average of 67% (seven experiments). In Fig. 35, the logarithm of g , is plotted against the membrane 0-4-65-C

i

gP

300 (rnrnho/crnz)

I00

30

-

Control

10

I -60

1

-40

I

-20

1’ I

0

_ _ _IOpM - Allethrin

I

20

I

40

1

60

Externally 5 min

I

00 E (mv)

I

100

Fig. 35. The membrane conductance (g,) at the peak transient (sodium) current plotted

as a function of membrane potential in a voltage clamped squid giant axon before and during exposure to allethrin externally (Narahashi and Anderson, 1967).

potential before and during application of allethrin externally. The gp-E curve is shifted downward indicating the suppression of g , . (c) Effects on Membrane Currents b y Internal Application: Both the peak sodium current and the steady-state potassium current are suppressed by internal application of allethrin in a concentration of 10- -1 0-4 M. However, an additional effect has been found as might be expected from the extremely large negative after-potential when allethrin is applied internally (see Fig. 32). An example of membrane currents before and after application of allethrin internally is shown in Fig. 36. It is clearly seen that the peak sodium current, which is partially suppressed by allethrin, is followed by a steady-state inward

’

54

T. NARAHASHI

current at certain membrane potentials. This steady-state inward current cannot be due t o potassium ions, because the concentration gradient for potassium is maintained at a constant value by a continuous perfusion in both external and internal phases thereby eliminating the possibility of an inward flow of potassium at any membrane potentials. It is most likely that the peak sodium current 30 JIM Allethrin Internally 12 min

Control

........ 80mv ........ 60

...........8Omv

......... 40

........... 60

...........40

.........

..........20

.......

2 maec

Fig. 36. Families of membrane currents associated with step depolarizations in an internally perfused giant axon of the squid before and during exposme to allethrin internally. The dashed line on the right of each family represents the zero membrane current (Narahashi and Anderson, 1967).

is not terminated as quickly as normal but maintained for a while. This possibility is analyzed by drawing the current-voltage relatjon. Figure 37 shows that the peak sodium current is partially suppressed (open and filled circles). The steady-state current flows in inward direction at the membrane potentials ranging between -50 mV and -5 mV (filled triangles). Since the steady-state outward potassium current before treatment with allethrin starts flowing when the membrane is depolarized beyond -25 mV (filled triangles), and also since the steady-state inward current after treatment with allethrin reaches its maximum at the same membrane potential (filled triangles), it is reasonable to assume that the steady-state potassium current in the allethrin-poisoned axon also starts flowing at -25 mV membrane potential. The peak sodium current in the allethrinpoisoned axon reverses its polarity at +40 mV (filled circles) so that there should be no sodium component in the steady-state current at that membrane potential. Therefore, the potassium component in the steady-state current in the allethrin-poisoned axon can be approximated by a straight line connecting zero current at -25 mV and the steady-state current at +40 mV. This is shown by a dotted line in Fig. 37; the potassium current is now seen to be suppressed by

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

\

O\

55

30pM Allethrin Internally

....._ --- Ccfrected I,.

l2 rn'n

Fig. 37. Current-voltage relations for the peak transient current (Ip) and for the steady-state current (Id in an internally perfused giant axon of the squid before and during exposure to allethrin internally. The dotted line represents the potassium component in I,, corrected for the residual sodium current as described in the text (Narahashi and Anderson, 1967).

allethrin. Thus the sodium component in the steady-state current is the difference between the total steady-state current (filled triangles) and the potassium current (dotted line). Our recent voltage clamp experiments with crayfish giant axons have demonstrated the validity of this assumption. As in the case of DDT experiments (Section V A 2), TTX was used to eliminate the sodium component from the total membrane current recorded from the allethrin-poisoned axon. Detailed results will be reported elsewhere. (d) Effects on Kinetics of Conductance Change: In contrast to the marked prolongation of the kinetics of the sodium inactivation, the time for the sodium current to reach its peak is only slightly prolonged. The average prolongation amounts t o 18% and 12% for external and internal applications of allethrin, respectively. The time for the steady-state potassium current to reach 50% maximum is not affected by internal application of allethrin. (e) Discussion: The changes in conductance parameters by allethrin can adequately account for the changes in action potential. The suppression of the sodium conductance increase is directly responsible for the suppression or blockage of the action potential.

56

T. NARAHASHI

The suppression of the steady-state potassium current and the slowing of the sodium inactivation by internal application of allethrin are responsible for the slowing of the falling phase of the action potential. The fact that the slowing of the sodium inactivation is rather negligible when allethrin is applied externally reflects the small increase in negative after-potential. It is of interest t o see the differential effect of allethrin from both sides of the squid nerve membrane despite the fact that allethrin is highly lipid soluble. This might suggest that the mechanism whereby the sodium conductance is inactivated is located near the internal surface of the nerve membrane. VI. TEMPERATURE COEFFICIENT OF INSECTICIDAL ACTION A. DDT

It has long been known that the insecticidal action of DDT is stronger at low temperature than at high temperature (Barker, 1957; Dustan, 1947; Fullmer and Hoskins, 195 1; Guthrie, 1950; Efliger, 1948; Hoffman and Lindquist, 1949; Hoffman et al,, 1949; Kaeser, 1948; Lindquist et al., 1945, 1946; Menn et al., 1957; Nagasawa and Hoskins, 1962; Potter and Gillham, 1946; Pradhan, 1949; Rhoades and Brett, 1948; Richards and Cutkomp, 1946; Tahori and Hoskins, 1953; Tomaszewski and Gruner, 1951; Vinson and Kearns, 1952; Yamasaki and Ishii, 1953, 1954b; Yates, 1950). Because the rate of chemical reactions in general has a positive temperature coefficient, the negative temperature coefficient of the insecticidal action of DDT is of great interest from the viewpoint of the mode of action. At least four major factors must be taken into consideration to account for this phenomenon, i.e. (1) penetration of DDT through the integument, (2) detoxication of DDT, (3) accumulation of DDT in the adipose tissue, and (4) sensitivity of the target site or receptor to DDT. 1. Penetration of DDT Through the Integument When applied as a suspension, DDT was found to exert a stronger insecticidal action against the mosquito larvae at low temperature than at high temperature. However, when injected into the larvae, DDT was stronger at high temperature than at low temperature. Based on these observations, it was suggested that DDT was adsorbed more at low temperature than at high temperature (Fan et al., 1948).

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

57

However, the actual measurements o n the DDT penetration through the integument revealed that the rate of penetration was in fact faster at high temperature than at low temperature (Barker, 1957; Vinson and Kearns, 1952) (Table I). Therefore it can be concluded that the factor of penetration plays a role antagonistic to the negative temperature coefficient of the insecticidal action of DDT. Table I Qlo values of various actions of DDT in the cockroach (From Yamasaki and Ishii, 1954b)

Action of DDT

Temperature ("C)

-

Qio

Penetrability through the cuticle

15

Detoxication

15

1/LD50 by injection

15

1/LD50 by topical application

15

Potency in developing poisoning symptoms

15-35

<0.258

16-30

0.117-0.316

Nerve sensitivity

16

-

35 35 35 35

30

Source of data

1.414- 1.581 Vinson and Kearns (1952) 1.024 1.906 Vinson and Kearns (1952) 0.277 0.377 Vinson and Kearns (1952)

-

0.223

-

0.365 Vinson and Kearns (1952)

0.181

Vinson and Kearns (1952) YamasakiandIshii (1954b) Yamasaki and Ishii (1954b)

2. Detoxication of DDT DDT is detoxified into DDE, DDA or kelthane by the action of enzymes such as DDT dehydrochlorinase (e.g. Abedi et a/., 1963; Agosin et al., 1961 ;Bull and Adkisson, 1963; Miller and Perry, 1964; Perry, 1960; Perry et al., 1963; Tsukamoto, 1959, 1960, 1961). Because the action of enzymes has in general a positive temperature coefficient, it seems reasonable t o assume that the detoxication by enzymes plays a n important role in the negative temperature coefficient of the insecticidal action of DDT. This has been demonstrated to be the case (Barker, 1957; Menn et al., 1957; Vinson and Kearns, 1962) (Table I).

58

T. NARAHASHI

However, the detoxication factor is not sufficient t o account for the whole phenomenon in question. For example, when the amount of undetoxified DDT in the insect body is measured, a larger amount can be found in the survived individuals at high temperature than in the dead individuals at low temperature (Vinson and Kearns, 1952). In other words, the insect can tolerate a larger amount of DDT at high temperature than at low temperature. Another line of evidence in support of the idea that the detoxication is not the sole factor comes from the observation that the symptoms of DDT poisoning are reversible upon changing the temperature. When treated with an appropriate dose, the DDT-poisoned insect exhibits the symptoms of poisoning at a low temperature (1 5"C), but the symptoms disappear upon raising the temperature to 30°C. This process can be repeated several times by changing the temperature between the low and high values. If this reversibility is t o be explained in terms of detoxication, one must assume the reversible detoxication of DDT with respect to the temperature, but no DDT has been discovered in the insect injected with DDE o r DDA (Sternburg and Kearns, 1950). Therefore, it is not possible to account for the reversibility of the DDT symptoms in terms of the detoxication alone. 3. Accumulation of DDT in the Adipose Tissue Owing t o high lipid solubility, DDT is accumulated in the adipose tissue. The DDT molecules stored in the adipose tissue may not exert any toxic effect. Therefore, if DDT is discharged from the fat upon lowering the temperature, this might account for the stronger insecticidal action at the low temperature. In fact, the fat of the cockroach kept at low temperature for a while has a higher solubility and holding capacity of DDT than that kept at high temperature (Hurst, 1949; Munson, 1953; Munson et al., 1954). However, since no further quantitative data are available, this hypothesis remains speculative.

4. Sensitivity of Nerve to DDT From the foregoing discussion, it is naturally predicted that the sensitivity of the target site to DDT may change upon changing the temperature. This problem has been studied using the sensory neurons in the cockroach leg as material (Yamasaki and Ishii, 1953, 1954b). DDT was more effective in inducing trains of impulses in the

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

59

sensory cells at a low temperature (1 6°C) than at a high temperature (30°C). In Fig. 38, the percentage of the individuals that produce the trains after injection of DDT into the leg is plotted as a function of the concentration of DDT. The values for the effective dose fifty (ED50) are estimated as 0.95 x M M at 16°C and 10.41 x at 30°C. The value of Qlo is calculated t o be 0.181 (Table I). However, it should be noted that the frequency of appearance of

-

I00

80

60 40

20 0

lo

lo-

10-6

Mol.conc. of DDT

Fig. 38. Percentage of the individuals that produce trains of impulses in the sensory nerve of the cockroach leg after injection of various concentrations of DDT into the leg at 16°C (open circles) and at 30°C (filled circles) (Yamasaki and Ishii, 1954b).

trains is higher at 30" C than at 16°C when comparison is made at an equivalent concentration of DDT, for example at the ED50 for each temperature. This reflects the fact that the symptoms of poisoning are more intense and easier t o observe at 30°C than at 16°C. The effect of DDT on the trains of impulses was found t o be reversible upon changing the temperature. Figure 39 depicts changes in the frequency of appearance of trains when the temperature is altered. After injection of DDT at a concentration of 3 x M, the trains appear as the temperature is lowered from 30°C to 12"C, disappear as the temperature is raised to 30"C, and this process can be repeated. The .dose of DDT necessary to develop the initial symptoms of poisoning was compared at 16" C and 30" C by injection method. The concentration of DDT to produce the symptoms is lower at 16°C than at 30"C, the difference being 5-20 times. This reflects the difference in nerve sensitivity t o DDT, because under these conditions the effects of other factors such as detoxication can be

60

T. NARAHASHI

Time (midafter injection

Fig. 39. Reversibility of appearance of trains of impulses in the sensory nerve of the cockroach leg injected with DDT when temperature is altered. DDT is injected once at zero time in a concentration of 3 x lo-' M (Yamasaki and Ishii, 1954b).

-

effectively eliminated. The value of Qro is calculated as 0.3 16 0.1 17 (Table I). Table I summarizes the values of Qlo for various factors involved in the DDT action. It can be concluded that, although the detoxication of DDT contributes t o the negative temperature coefficient of the insecticidal action of DDT, the major factor appears to be the sensitivity of the nerve to DDT. The nerve sensitivity not only has a large negative temperature coefficient but also can adequately account for the reversible symptoms of DDT poisoning with respect t o the temperature. 5. Discussion Eaton and Sternburg (1964, 1967) also studied the effect of temperature on the trains of impulses induced by DDT. Part of their results seems to contradict t o our results described above. However, careful examinations of the experimental conditions have revealed that both data agree in essence. Eaton and Sternburg (1964) stated that the trains of impulses from the sensory cells of the DDT-poisoned cockroach leg show a positive temperature coefficient, increasing the temperature increasing the frequency of appearance of trains, whereas the trains from the abdominal nerve cord show a negative temperature coefficient decreasing the temperature increasing the frequency of appearance of trains (their Fig. 2 and Table I). However, the threshold concentration of DDT to induce the trains of impulses is much higher for the

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

61

abdominal nerve cord than for the sensory cells. Therefore, the dose of DDT used is well above the threshold for inducing the trains in the sensory nerve at both high and low temperatures. Thus, changes in temperature simply result in changes in the frequency of appearance of trains in the sensory nerve, increasing the temperature increasing the frequency. This agrees with the observation by Yamasaki and Ishii (1954b). For the abdominal nerve cord, however, the dose used is near the threshold to induce trains at high temperature. Therefore, the trains are not observed at the high temperature, and appear as the temperature is lowered. This also agrees with the observation by Yamasaki and Ishii ( 1954b) who applied the near-threshold concentration of DDT t o the sensory nerve. Eaton and Sternburg (1967) later performed similar experiments using the cercal nerve of the cockroach. The number of trains of impulses is markedly reduced as the temperature is lowered. Because of low threshold concentrations of DDT in the sensory nerve, this result is to be expected from the previous observations by Yamasaki and Ishii (1 954b) and by Eaton and Sternburg ( 1964). Thus, if the concentration of DDT is carefully chosen, the potency of DDT t o induce trains of impulses 'has a negative temperature coefficient both in the sensory nerve and in the abdominal nerve cord. In other words, the threshold concentration of DDT to produce trains becomes low as the temperature is lowered. Therefore, the DDT-poisoned insect shows reversible symptoms of poisoning upon changing the temperature if an appropriate dose is chosen. In the poisoned cockroach, the impulse trains in the central nervous system probably play an important role in exhibiting the symptoms of poisoning, because the trains disappear upon increasing the temperature in parallel with the symptoms of poisoning of insect, whereas the trains from the sensory nerve can still be observed at high temperature in the absence of the apparent symptoms of poisoning (Yamasaki and Ishii, 1954a; Eaton and Sternburg, 1964). B. PYRETHROIDS

The insecticidal activity of pyrethroids increases as the temperature is lowered (Blum and Kearns, 1956; Harries et al., 1945; Chamberlain, 1950; Guthrie, 1950; Hartzell and Wilcoxon, 1932). The effects of temperature on various aspects of allethrin action on the cockroach nerve have been studied. As described in an earlier section (IV D), allethrin causes the nerve to produce repetitive

62

T. NARAHASHI

discharges at relatively weak concentrations and blocks the conduction at higher concentrations.

I . Repetitive Discharge The repetitive discharge in the allethrin-poisoned axon shows a positive temperature coefficient. Figure 40 illustrates a series of records from the allethrin-treated cockroach giant axon when the

Fig. 40. Effects of temperature on the action potentials recorded from the allethrinpoisoned giant axon of the cockroach. Temperature 33°C (A), 28°C (B), 26.5"C (C), and 26°C (D) (Narahashi, 1962a).

temperature is gradually lowered from 33°C in record A (Narahashi, 1962a). As the temperature is lowered from 26.5"C in record C to 26OC in record D, the axon stops firing repetitively, leaving small oscillations of potential. Repetitive firing at high temperatures can be attributed, at least in part, t o the increase in negative after-potential upon raising the temperature (Narahashi, 1963a). The effectiveness of allethrin in producing repetitive discharges in the isolated abdominal nerve cord of the cockroach was studied at high and low temperatures (Y.Suzuki, personal communication). The allethrin concentrations to cause this effect in 50% of the individuals are estimated as 2.07 x M at 30°C and 1.1 x lod6 M at 15°C.

2. Nerve-blocking Action In contrast t o the positive temperature coefficient of the action of allethrin in producing repetitive discharges, the blocking action of

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

63

allethrin shows a negative temperature coefficient. Figure 4 1 depicts an example of experimental data in which the temperature and the amplitude of the action potential recorded externally from the cockroach nerve cord are plotted as a function of time (Narahashi, unpublished observation). Separate control experiments show that the action potential of the normal nerve cord increases in amplitude

0

5

10

15 20 Time after

25

30 35 - 4 0 g/ml Allelhrin (min.)

45

50

55

Fig. 41. Effects of temperature on the amplitude of the action potential recorded externally from the allethrin-poisoned abdominal nerve cord of the cockroach.

by only about 20% with lowering the temperature from 29°C to 15°C. In the nerve cord poisoned with allethrin 3.3 x M, however, the action potential is reduced in amplitude upon lowering the temperature from 29"C, and eventually blocked when the temperature reaches 12°C. This process can be repeated as is shown in Fig. 4 1. Y. Suzuki (personal communication) also found that M allethrin blocked the action potential of the cockroach nerve cord in a few minutes at 15" C, whereas it took more than 50 min to block at 30°C. To block the conduction at 30°C in a few minutes, the concentration of allethrin had to be raised to M. These observations clearly demonstrate that the nerve-blocking action of allethrin has a negative temperature coefficient. The mechanism involved in the negative temperature coefficient of the blocking action of allethrin has been studied by means of intracellular microelectrodes using the cockroach giant axon as material (Narahashi, unpublished observation). As described in an earlier section (V B l ) , the curve relating the maximum rate of rise of the action potential to the membrane potential (sodium inactivation curve) is shifted along the potential axis in the direction of inside

64

T. NARAHASHI

more negative membrane potential by application of allethrin (Fig. 3 1). In the allethrin-poisoned axon, the sodium inactivation curve is further shifted in the same direclion upon lowering the temperature from 26.5"C t o 14°C (Fig. 42). The absolute magnitude of the maximum rate of rise of the action potential is also decreased by lowering the temperature. Also plotted in Fig. 42 are the

:

:

: 0

I 1000

-10

AP T p -20 (mV)

-30

.........o.........

v.m.9.

-40-..& -50

-

-m

......A,......... .......... 0

->--*-

d...

% , . doto b

00

@ ."'

%

--ocfl

--o--ai-a-

:j

,('

..

,,*.-;*4 0%.

4..

-

,

p...,

p.&.4..

,

800

6oo dV/dt

4oo (V/reC) 200

'4?&..n

Fig. 42. The amplitude (AP) and the maximum rate of rise (dV/dt) of the action potential and the threshold membrane potential (TP) plotted as a function of membrane potential displaced from the resting potentials (arrows at the top) before and during exposure to allethrin 3.3 x M. AP, before (0) and during (4 allethrin. dV/dt, before (0) and during (A) allethrin. Arrows on the curves show the membrane potentials where dV/dt is half maximum. TP, before (0) and during (m)allethrin.

amplitude of the action potential and the threshold membrane potential where the action potential arises as a function of membrane potential. The threshold membrane potential becomes inside less negative upon lowering the temperature. The resting membrane potential is decreased by lowering the temperature as shown by arrows with symbols in Fig. 42. All of these changes tend to suppress the conduction of the action potential. The amplitude of the action potential is increased upon lowering the temperature, so that once the action potential is produced it can reach a higher level at low temperature. In summary, the resting membrane potential is decreased, the threshold membrane potential is also decreased (inside less negative), the sodium inactivation curve is shifted along the potential axis in the direction of hyperpolarization, and the

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

65

maximum rate of rise of the action potential is suppressed by a lowering of the temperature, and all of these changes are responsible for the stronger blockage of the conduction of the action potential at low temperature.

3. Discussion Although the study on the mechanism underlying the negative temperature coefficient of the insecticidal action of pyrethroids is less extensive than that for DDT, it is no doubt that the sensitivity of nerve to allethrin plays an important role in this phenomenon. The nerve-blocking action of allethrin is probably the major factor, because the cycle of blockage-recovery of the nerve conduction can be observed by changing the temperature in parallel with the reversible symptoms of poisoning in insects. From the positive temperature coefficient of the allethrin action in producing repetitive firing, it is predicted that the cockroach poisoned with a low dose of allethrin will become more hyperactive at high temperature than at low temperature. Careful observations of the symptoms of poisoning as a function of dose, time and temperature are necessary to evaluate the validity of this notion. Since pyrethroids are known t o be metabolized in insects as well as in mammals (see review by Yamamoto, 1970), it is reasonable to assume that the metabolic degradation of allethrin is accelerated by a rise in the temperature. If this is the case, then the metabolism of allethrin will play an additional role in the negative temperature coefficient of the insectidical action of allethrin. VII. INSECTICIDE RESISTANCE

A number of studies have been performed in an attempt to elucidate the mechanism of resistance to insecticides (see reviews by O’Brien, 1966, 1967; Brown, 1960, 1961, 1964). Although different mechanisms are in fact involved in different strains of resistant insects and in different insecticides, four major factors are easily recognized from Fig. 1, i.e. (1) penetration of insecticides through the integument, (2) detoxication and excretion of insecticides, (3) store of insecticides in non-target tissues, and (4) sensitivity of nerve to insecticides. In addition, there is so-called “behavioral resistance”, in which insects develop the ability to avoid contact with insecticides. This is outside the scope of the present article, and will not be described here. AIP-4

66

T. NARAHASHI

Importance of the nerve sensitivity to insecticides in insecticide resistance can easily be seen in the observation in which the amount of undertoxified insecticide is compared between susceptible and resistant strains of insects. The survived resistant insects in many cases contain a larger amount of undetoxified insecticides than the dead susceptible insects (Babers and Pratt, 1953; Perry and Hoskins, 1951; Sternburg et al., 1950; Tahori and Hoskins, 1963). This indicates that the resistant insects can tolerate a larger amount of insecticides than the susceptible insects without showing any sign of intoxication. For this reason, factors other than the cuticule penetration and detoxication are suspected to play an important role in insecticide resistance, although the detoxication factor has been demonstrated, in a number of resistant strains of insects, to be one of the key factors for the resistance. There should be some defense mechanisms whereby the target site in the resistant insects is protected from the toxic action of insecticides.

A. NERVE SENSITIVITY TO INSECTICIDES

Low sensitivity of the nerve to insecticides is one of the most probable mechanisms whereby the resistant insects can tolerate a large amount of the insecticide present in the body. Earlier studies indicate that the nerves from the resistant strains are less sensitive to insecticides than those from the susceptible strains (Pratt and Babers, 1953; Smyth and Roys, 1955; Weiant, 1955). Detailed studies were performed using a variety of insecticide-resistant strains of houseflies (Yamasaki and Narahashi, 1958b, 1962; Narahashi, 1964a; Tsukamoto et al., 1965). The test solution containing insecticide is applied to the exposed thoracic ganglia and the impulse discharge is recorded by means of external silver wire electrodes inserted in the femur of the metathoracic leg. An example of such records is shown in Fig. 43. The top record (A) shows a burst of discharges produced by stimulating the normal housefly with an air puff. Spontaneous discharges are low in both amplitude and frequency in the normal M to the housefly (record B). Direct application of DDT 2.8 x exposed thoracic ganglia induces bursts of discharges which increase in intensity with time (records C, D, and E). Therefore, in the experiments described in the following sections, the increase in the frequency of discharges is taken as a measure of response.

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

67

Fig. 43. Discharges of motoneurons originating from the thoracic ganglia and innervating the femur muscle of the housefly (DDT-susceptible NAIDM strain). Recordings are made externally from the femur muscle by means of silver wire electrodes. A, burst of impulses induced by an air puff applied to the housefly. B, spontaneous discharges in another normal M to the housefly. C, burst of discharges 7 min after direct application of DDT 2.8 x exposed thoracic ganglia. D, 13 min after DDT. E, 26 min after DDT (Yamasaki and Narahashi, 1962).

1. DDT

The value of the effective dose fifty (ED50) in stimulating the nerve to increase the discharge frequency in 50% individuals is estimated as 3.4 x lo-' M for the strain susceptible to DDT (NAIDM), 2.1 x 10-6 M for the strain moderately resistant to DDT (CSMA), and 2.6 x M for the strain highly resistant to DDT (DKM). Thus the ED50 ratio DKM/NAIDM is 7.6 as against the LD50 ratio of 217 (Yamasaki and Narahashi, 1962). Another DDT-resistant strain of houseflies exhibits much lower nerve sensitivity to DDT. The ED50 ratio of the resistant strain to the susceptible strain (Lab) is estimated to be 100 (Tsukamoto et al., 1965) (Fig. 44). Recently, the labellar taste receptors of DDT-resistant houseflies were found to be also less sensitive t o DDT than those of DDT-susceptible houseflies (Browne and Kerr, 1967).

68

T. NARAHASHI x

104

Io4

DDT cu-centraticn (MI

o3

I

Fig. 44. Dose-response relations for DDT in inducing high frequency discharges from the thoracic ganglia of the housefly. Ordinate represents the percentage of the individuals that respond to various concentrations of DDT. Lab, oDDT-susceptible strain. R (bwb : ocra : ar : ac), DDT-resistant strain. F, , F, hybrid (R + x Labs d) (Tsukamoto ef a/., 1965).

2. Lindane The lindaneresistant strains of houseflies also exhibit low nerve sensitivity to lindane. Lindane exerts a similar effect as DDT on the thoracic ganglia of the housefly. The ED50 ratio of the resistant HR(2356) strain to the susceptible Lab strain is estimated t o be 124-162 as against the LD50 ratio of 59,000 (Narahashi, 1964a). 3. Dieldrin In the dieldrin-resistant strain (Hikone) of the houseflies, the effect of directly applied dieldrin in increasing the discharge frequency of the thoracic ganglia can be observed after a longer latency than in the susceptible strain (Takatsuki), the difference being 1.5 times (Yamasaki and Narahashi, 1958b). However, since impurity in the dieldrin sample used is suspected (Section IV C), it% possible that this difference in the latency does not represent the difference in the nerve sensitivity to dieldrin. Alternatively, this difference may reflect the difference in the ability of the nerve to convert dieldrin into an active compound for which aldrin-transdiol is a possible candidate (Wang et al., 1971, also see Section IV C). No

EFFECTS O F INSECTICIDES ON EXCITABLE TISSUES

69

significant difference has been found between susceptible and resistant strains of insects in the detoxication of dieldrin and in the penetration of dieldrin through the integument (Khan and Brown, 1966; Perry et al., 1964; Ray, 1963; Winteringham and Harrison, 1959). In view of these considerations, it is urged t o study the sensitivity of the nerve t o the activated dieldrin metabolites such as aldrin-transdiol. Matsumura and Hayashi (1966a, 1969) studied the binding of dieldrin with various components of the German cockroach nerve. The nerve from the dieldrin-resistant strain binds a less amount of dieldrin than that from the susceptible strain. However, it remains to be seen whether this factor is causally related to the resistance of the cockroach to dieldrin. 4. Diazinoti The nerve sensitivity t o insecticides plays a minor role in the diazinon-resistant strain of houseflies (Narahashi, 1964a). Since diazinon is converted into an active form diazoxon in insects, the sensitivity of the nerve was studied both for diazinon and diazoxon. The results are shown in Fig. 45, in which the nerve sensitivity to diazinon and diazoxon is only slightly lower in the resistant strain

90

- 70 ?i

50

5z

0 W

30

ti 10 I

10-6

I

I

10'~ CONCENTRATION

I 0-4

(M)

Fig. 45. Dose-response relations for diazinon and diazoxon in inducing high frequency discharges from the thoracic ganglia of the housefly. Ordinate represents the percentage of the individuals that respond to various concentrations of the insecticides. NAIDM, diazinon-susceptible strain. L-S-5,diazinon-resistant strain.

70

T. NARAHASHI

than in the susceptible strain. It is clear that the nerves from both strains are more sensitive to diazoxon than to diazinon. This is to be expected because diazoxon is more potent than diazinon in inhibiting ChE’s in vitro and also because the inhibition of ChE’s is directly responsible for the multiple discharges produced by organophosphorus insecticides (Section IV F). The results of experiments described above are consistent with the observation that ChE’s from both resistant and susceptible strains of houseflies are equally inhibited by diazoxon (T. Shono, personal communication).

B. GENES CONTROLLING THE NERVE SENSITIVITY

The apparent low nerve sensitivity in DDT- and lindane-resistant strains of houseflies described in the preceding section does not exclude the possibility that hese insecticides are detoxified inside the nerve thereby making the nerve less sensitive. In fact, it has been shown that the activity of DDT dehydrochlorinase is higher in DDT-resistant houseflies than in susceptible houseflies (Miyake et al., 1957). There are at least two genes controlling DDT resistance in the housefly, one being located on the second chromosome and the other on the fifth chromosome. A single recessive gene pair on the second chromosome is known to control the inheritance of the so-called knockdown resistance to DDT (Harrison, 1951 ; Milani, 1954; Milani and Travaglino, 1957). A dominant resistance gene on the fifth chromosome controls dehydrochlorination of DDT (Tsukamoto and Suzuki, 1964). Since knockdown of DDT-poisoned houseflies is caused by the action of DDT on the nerve, it is possible that the knockdown resistance gene on the second chromosome controls the low nerve sensitivity to DDT. On the other hand, it is also possible for the dominant dehydrochlorination gene on the fifth chromosome plays a major role in the low nerve sensitivity to DDT, because the nerve can detoxify DDT (Miyake et al., 1957). The nerve sensitivity to insecticides was analyzed using multichromosomally marked resistant strain of houseflies, R (bwb : ocra : ar : ac), and a susceptible strain, Lab (Tsukamoto et al., 1965). As is shown in Fig. 44, the nerve from the resistant strain was much less sensitive to the directly applied DDT, the difference between the susceptible and resistant strains being about 100. The F1 hybrid between the females of the resistant strain and the males

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

71

of the susceptible strain showed an intermediate nerve sensitivity to DDT. In order t o analyze the genetic factor responsible for the nerve sensitivity to DDT, the males of the F1 hybrid were backcrossed to the females of the resistant strain. Since each autosome except for the fourth chromosome was labeled with a visible mutant marker, it was possible t o determine to which linkage group the recessive nerve sensitivity character belonged. Eight out of 16 phenotypes were examined for their nerve sensitivity to DDT, because the data with these eight phenotypes were sufficient to make the proposed analyses. Table I1

M Response of the nerve of the housefly t o 1.7 x DDT in different genetic make-ups of chromosomes Phenotype (2 : 3 : 5 : 6) +:

+:

+:+

+: +:ar:ac + : ocra: + : ac + : ocra : ar : +

bwb: +: +:ac bwb: +:ar:+ bwb : ocra : + : + bwb : ocra : ar : ac

Exp. 1

Exp.2

87.5 81.2 87.5 87.5 18.7 25.0 37.5 13.3

75 .O 58.3 79.1 45.8 12.5 8.3 16.6 12.5

Data are given in percentages of the houseflies that respond to DDT by an increase in discharge frequency from the motoneurons innervating the femur. The houseflies are obtained by backcross, R (bwb : ocra : ar : ac) x F1 [R(bwb : m a : ar : ac) ? x Lab dl d. (From Tsukamoto et al., 1965.)

Table I1 gives the results of experiments with these eight phenotypes. The data are expressed as the values in the percentage of the houseflies whose nerves are stimulated in response t o 1.7 x M DDT. These percentage values were transformed into the arc-sine unit, and the homozygous effect of each chromosomal factor o n inheritance of low nerve sensitivity was calculated by the partial factorial analysis. The result clearly shows that the recessive gene on the second chromosome is responsible for the low nerve sensitivity to DDT in the resistant strain, and the contribution of the fifth chromosomal factor to the nerve sensitivity is very small.

72

T. NARAHASHI

In view of the evidence that the nerve of the housefly can detoxify DDT (Miyake et aZ., 1957), it is tempting t o ascribe the fifth chromosomal factor described above to DDT detoxication in the nerve. However, it should be noted that we are here dealing with recessive genes. The gene on the fifth chromosome that controls DDT dehydrochlorination is a dominant one (Tsukamoto and Suzuki, 1964). Therefore, the recessive gene on the fifth chromosome is controlling the nerve sensitivity through some other mechanism or through the detoxication of DDT other than dehydrochlorination. The recessive gene on the second chromosome controls the nerve sensitivity to DDT. There are at least two possible mechanisms whereby the nerve exhibits low sensitivity to DDT, i.e. (1) low permeability of DDT through the nerve sheath, and (2) low sensitivity of the nerve excitable membrane to DDT. The present experiment does not distinguish these two possibilities, and this problem remains to be explored. Preliminary experiments with lindane-resistant houseflies show that the gene controlling low nerve sensitivity t o lindane is located neither on the second nor on the fifth chromosome (Tsukamoto et al., 1965). VIII. STRUCTURE-ACTIVITY RELATIONSHIP

A number of experiments have been carried out in an attempt to find out the structure-activity relationship of various insecticides. Most of the experiments were based on the observation of insecticidal activities using a wide variety of derivatives and analogs of any particular parent compound. Much progress has indeed been made in terms of creations of new compounds of potential use. Many insecticides currently developed emerged as a result of such broad searches of compounds. However, it should be emphasized that our knowledge on the structure-activity relationship remains poor despite an enormous amount of efforts so far made. This is at least in part due to the fact that the insecticidal activity is a v e complex ~ chain of various reactions. Figure 1 clearly shows the situation. Because we are dealing with the insecticide molecule on the one hand, it is almost impossible to relate the chemical structure to the complicated chain of reactions in the insect body. It is absolutely necessary to dissociate the whole reaction that leads to the death of the poisoned insect into each component such as the penetration through the

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

73

cuticule, the activation, the detoxication, the action on the nerve, etc. To elucidate the structure-activity relation for the primary toxic action for many of the insecticides, one would naturally be forced to compare the potency of action of various derivatives and analogs on the nervous function both qualitatively and quantitatively. In case the target site is known to be an enzyme system, the study will be relatively easy at least from a technical point of view, because one can perform in vitro experiments on that particular enzyme. This is true for ChE’s which ,are the target site of a number of organophosphorus and carbamate insecticides. For some other insecticides such as chlorinated hydrocarbons and pyrethroids, the study of structure-activity relationship involves a very time-consuming comparison of relative effectiveness of various derivatives on the nervous function. For this reason, not much progress has so far been made along this line of approaches. A. DDT

Preliminary electrophysiological experiments were performed using the sensory nerve of the cockroach leg and the giant axon of the crayfish. Since initiation of repetitive discharges and increase in negative after-potential are two major actions of DDT on the nervous function, the relative potency of a number of derivatives and metabolites of p,p’-DDT (I) in exerting these two effects was compared (Yamada and Narahashi, 1968).

(I)

Detailed results will be described elsewhere, and only a few points will be mentioned here. Amino substitute (11) is not effective on the nerve in agreement with the absence of insecticidal activity (Metcalf and Fukuto, 1968). However, nitro substitute (111) is effective in initiating trains of impulses in the sensory nerve of the cockroach leg and in increasing the negative after-potential in the crayfish giant axon, despite the lack of insecticidal activity (Metcalf and Fukuto, 1968; Holan,

74

T. NARAHASHI

Methyl substitute (IV) has an insecticidal activity, especially for mosquitoes (Metcalf and Fukuto, 1968). It is effective on the sensory nerve in producing trains of impulses, but has no effect on the negative after-potential of the crayfish axon.

(Is9

Substitution of chlorines at para positions by methoxy (-OCH3) (compound V) or ethoxy (-OC2 H, ) (VI) group still maintains the effectiveness on the sensory nerve.

However, although methoxy substitute is capable of increasing the negative after-potential of the crayfish axon, ethoxy substitute lacks this action. Both substitutes are insecticidally active (Metcalf and Fukuto, 1968). When the p,p’-substituents are increased in size (e.g. -OC4H,), the compound becomes inert on both types of nerve preparations. Metabolities of p,p’-DDT show an interesting spectrum of action on the nerve. Although o,p‘-DDT (VII) and p,p’-DDD (VIII) are effective in producing trains of impulses, they are ineffective in augmenting the negative after-potential. However, o,p’-DDD (IX) is somewhat effective in both respects.

(mI)

(nm)

(Ix)

The absence of the effect of p,p’-DDD on the negative after-potential was confirmed by Van den Bercken (1 969) using single nodes of Ranvier of Xenopus Zuevis. He also found that DDD suppressed the action potential. This action was never observed with p,p’-DDT. Dehydrochlorinated metabolite p,p’-DDE (X) and acid form metabolite p,p’-DDA (XI) have no effect on both nerve preparations.

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

75

This agrees with the general observation that they lack the insecticidal activity. CI O

f

O CCI,

C

J

CI

OF-@ COOH

(XI)

(XI

These observations on the effectiveness on the nervous function may be interpreted in terms of (1) the profile of formal charges on the chloroform group, (2) the steric factor, and (3) the hydrophobicity. Definite conclusion about the structure-activity relation awaits further experimental analyses. One important point emerging from these preliminary observations is that when the direct effect of insecticides on the nerve is examined as a measure of activity, there are qualitative as well as quantitative differences among derivatives or metabolites having very similar structures. These differences may not be elucidated if the insecticidal activity is simply compared. B. PYRETHROIDS

Several pyrethroid derivatives of a new type were compared for their insecticidal activity, mammalian toxicity, and ability to affect the nervous function (Berteau et al., 1968). Allethrin (XII) and four other pyrethroid-like compounds were used (Table 111). Compound XI11 is the keton analog of allethrin and lacks the ester function. In compound XIV, chrysanthemum-monocarboxylic acid of allethrin is replaced by tetramethylcyclopropanecarboxylicacid, and allethrolone by 5-benzyl-3-furylmethanol. In compound XV, tetrame thylcyclopropanecarboxylic acid of compound XIV is further replaced by a carbamate, tetramethylaziridine carboxylic acid. In compound XVI, the cyclopropane of compound XIV is substituted by N,N-diisopropylcarbamicacid. Some of the data on insecticidal activity and nerve activity are given in Table 111. It is clear that both activities run parallel with each other, and that all of the compounds tested do not loose the activities by changes in chemical structure described above. It is noteworthy that all of the compounds exert very similar actions on the crayfish giant axon, i.e. (1) slight and progressive depolarization, (2) increase in negative after-potential, and (3) repetitive afterdischarges by a single stimulus. Our recent voltage clamp

76

T. NARAHASHI

Table I11 Chemical structures and biological activities of allethrin and four structurally related compounds Compound

Structure

21

XI1 Allethrin

171

XI11 XIV

xv XVI

Toxicity to housefly LD5 0 (mg/kg)

0.9

Jm

Potency on nerve ED50 (PM 1

2.6 17 1.6

228

24

750

130

Nerve potency: micromolar level to decrease the maximum rate of rise of the action potential of crayfish giant axons to 50%normal. (Berteau etul., 1968.)

experiments with the crayfish giant axon show that compound XI11 exerts the same effects on membrane conductances as allethrin (compound XII), i.e. the sodium and potassium conductance increases are suppressed, and the sodium inactivation is greatly slowed. These results are in a way contradictory to the classical concept concerning the structure-activity relation of pyrethroids. It has been believed that the cyclopropane ring and the ester function are essential for the insecticidal activity. The results described here rather suggest that the configuration of the molecule, relative to appropriate size and shape to interact with the receptor of the nerve membrane, appears to be of critical importance in exerting the nerve action. The receptor for pyrethroids can be visualized as specific group(s) of macromolecules in the nerve membrane such as proteins and phospholipids which control the gate mechanism involved in conductance changes. Further experimental analyses, especially those by means of voltage clamp techniques, are necessary to explore the structure-activity relationship of pyrethroid-like compounds. C. ROTENONE

Rotenone (XVII) inhibits the electron transfer from DPNH to

EFFECTS OF INSECTICIDES ON EXCITABLE TISSUES

77

cytochrome b and blocks the nerve conduction as described earlier (Section IV E). cH2%-C cH3’

A% -CHI

OCH,

mm)

@33

Thirty-four derivatives of rotenone were examined for their insecticidal activity, and their potency in inhibiting the glutamic dehydrogenase activity. Some of them were also examined for their potency to block the nerve conduction (Fukami et al., 1959). In short, the three activities go parallel with each other for most of the derivatives, and those which have a strong insecticidal activity are, without exception, capable of inhibiting the enzyme activity and blocking the nerve conduction. Some of them, however, inhibit the enzyme activity and block the nerve conduction effectively, yet lack strong insecticidal activity. For example, rotenone hydrochloride (XVIII) is almost equipotent to rotenone for the enzyme inhibition and only slightly less active on the nerve, but it possesses only 20% insecticidal activity of rotenone. This may be due to rapid degradation of rotenone hydrochloride in the insect.

~xszllt)

See XVII for the rest of structure,

Detailed results will not be repeated here. The results confirm the earlier suggestion by Martin (1 946) that the asymmetric carbons at positions 7 and 8 are of critical importance in maintaining the activities. The presence of the chromanochromanone ring in the molecule is not essential, and the chromanochromanol ring can substitute for it. This is shown by experiments with rotenol (XIX), dihydro-rotenol (XX), and acetylrotenone (XXI), all of which are potent in exerting the three actions.

78

T. NARAHASHI

(XIXI

(xx)

(XXI)

See XVlI for the rest of structure.

IX. ROAD TO THE MOLECULAR MECHANISMS

Little has been known concerning the molecular mechanisms of action of insecticides. The fact that most insecticides interact with the nerve membrane makes the direct in vitro study of this problem very difficult. For example, it is first of all difficult to isolate the pure nerve membrane component without being contaminated by the components of other membranes such as those of Schwann cells and connective tissues. Even when this is accomplished satisfactorily, one will have to demonstrate that the interaction of insecticides with the isolated nerve membrane component is the same as that occumng in the nerve membrane in situ. It is also absolutely necessary to demonstrate that the interaction between the insecticides and the nerve membrane is directly related to the toxic action. In view of these considerations, there will be no single approach whereby one can obtain a clean-cut answer to this problem. The approach will have t o be multidisciplinary in nature. Classical electrophysiological techniques such as those by voltage clamp and microelectrodes will continue to be very useful and powerful in measuring the nerve activity in terms of membrane ionic conductances and membrane potential changes. These parameters, especially conductance changes, will provide us with the basis t o explore the molecular mechanisms involved. Attempts were made to isolate receptors for the insecticide action on the nerve membrane. This will give us the chemical basis of interpretation of the mode of action. DDT and dieldrin bind with various components of the nerve (Matsumura and Hayashi, 1966a, b, 1969; Hayashi and Matsumura, 1967; O’Brien and Matsumura, 1964; Matsumura and O’Brien, 1966a, b; Hatanaka et al., 1967; Brunnert and Matsumura, 1969). However, the role of such bindings in the toxic action of insecticides on the nerve remains to be explored.

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79

Artificial membrane systems, either the monomolecular film spread over the aqueous phase or the bimolecular membrane formed between two aqueous phases, will also be highly useful for elucidating the mechanism of action of insecticides at the molecular level. One of the advantages in this type' of experiment is that the artificial membrane is chemically defined. Experiments along this line have already been started. The potassium conductance of the lecithindecane membrane is increased by application of valinomycin, and DDT partially antagonizes the valinomycin action and decreases the potassium conductance (Hilton and O'Brien, 1970). The effect of DDT on the membrane potassium conductance is, on the surface, at least in the same direction as that found in the lobster axon membrane (Narahashi and Haas, 1967, 1968). However, the potassium conductance of the natural nerve membrane and that produced by valinomycin in the artificial membrane are not necessarily the same in every respect. Despite this, the experiments with artificial membranes will provide us with a clue to approach the molecular mechanism of action of insecticides. In addition to the mechanism of action of insecticides at the membrane or molecular level, electrophysiological techniques will be extremely useful for studies of other aspects as has been described in this article. Among many possible applications is the study of the structure-activity relationship of insecticides. It should be emphasized that the knowledge of the structure-activity relation for any particular type of insecticides is useful not only for creation of new insecticides but also for interpretation of insecticide-receptor interactions at the molecular level. In this connection, the hypotheses put forward by Mullins (1 954, 1955, 1956) and by Holan (1968) in an attempt to explain the structure-activity relationship of DDT and its analogs are worth while to note. Mullins (1954, 1955, 1956) proposed a model in which the molecules of DDT and its analogs must fit into an interspace formed by membrane macromolecules. For example, iodo-DDT in which two chlorine atoms on the phenyl rings are substituted by two iodine atoms does not fit because the p,p'-substituents are too large, and in fact it is ineffective as the insecticide. In DDE, the tetrahedral bond angle is changed and causes non-fit. Holan (1969) modified the Mullins' original hypothesis to explain the structure-activity relationship of new DDT analogs, 1,-l-di(p-chlorophenyl)-2,2-dichlorocyclopropane and its derivatives. Part of the insecticide molecule containing the phenyl rings locks itself into the overlaying protein

80

T. NARAHASHI

layer in the nerve membrane by forming a molecular complex with it. An attempt is made t o explain the prolongation of sodium current while the whole insecticide molecule is locked in the membrane. Projection of the van der Waals outline of the active insecticides can adequately explain the fit of all of the active compounds into the membrane. ACKNOWLEDGEMENTS

Part of the results described in the present article was supported by a grant from the National Institute of Health (NS 068SS), and by a contract with the National Institute of Environmental Health Sciences (PH-43-68-73). I wish t o thank Mrs. R. M. Crutchfield and Mrs. C. A. Munday for their secretarial assistance.

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