Insect Control: Biological and Synthetic Agents - Index of

42 2: Indoxacarb and the Sodium Channel Blocker Insecticides
Figure 7 RH-5529 raised the threshold in current-clamped crayfish stretch receptors, without affecting passive membrane
resistance. Records show voltage (upper) and current (lower) traces for current steps before, during, and after application of
10 mM RH-5529. Corresponding traces superimposed on the right show that the spike threshold was raised by RH-5529, with no
change in resting potential or in the response to hyperpolarizing pulses. (Reproduced with permission from Salgado, V.L., 1990.
Mode of action of insecticidal dihydropyrazoles: selective block of impulse generation in sensory nerves. Pestic. Sci. 28, 389–411;
ß Society of the Chemical Industry, permission is granted by John Wiley & Sons Ltd., on behalf of the SCI.)
(Salgado, 1990), but the slowly adapting stretch
receptor (SASR) of the crayfish Procambarus clarkii
(Wiersma et al., 1953) was selected for studies of the
cellular mechanism of action, because the greater
size of its neuron enables intracellular recordings
at the site of spike initiation.
Pyrazolines blocked the stretch receptor in crayfish
with a potency similar to that in insects. In mechanoreceptors,
membrane deformation resulting from
elongation activates stretch-sensitive ion channels
that induce a so-called generator current that depolarizes
the membrane of the spike initiation zone
to the threshold for action potential generation. In
other words, the generator current resulting from
mechanosensory transduction is encoded as a spike
train in the spike initiation zone. Insertion of a
microelectrode into the spike-initiation zone of an
SASR neuron allowed the function of this transduction
and spike-encoding process to be studied.
Figure 7 shows a SASR neuron studied under current
clamp, in which current pulses of varying amplitude
were injected to either hyperpolarize or depolarize
the membrane. Such currents bypass the sensory
transduction process and allow direct assessment of
the spike-encoding process. The voltage traces in response
to the injected currents are shown in the upper
row. For negative or hyperpolarizing pulses, represented
as downward deflections of both current and
voltage, RH-5529 did not affect the membrane
response, indicating that it did not affect passive
membrane properties (Figure 7). The singular effect
of RH-5529 was to raise the threshold for spike generation
in response to depolarizing pulses, making it
more difficult for injected currents and, by inference,
for generator currents, to elicit spikes. From this result,
it was immediately clear that voltage-dependent
Na þ channels, whose activation determines the
threshold and initiates the action potential, were
blocked by the pyrazoline. This was also likely the
mechanism of block in the spike initiation zones
of CNS pacemaker neurons, where spikes are also
generated by the activation of Na þ channels in
response to depolarization by summation of synaptic
inputs and pacemaker currents.
2.4.4. Mechanism of Na + Channel Block
In order to better understand the action of SCBIs on
Na þ channels, further studies were carried out on
crayfish giant axons treated with pyrazolines, with
techniques that allowed the study of Na þ channels
under highly controlled conditions. A first hypothesis
to explain the insensitivity of axonal Na þ channels
to pyrazolines was that the compounds block
Na þ channels in a voltage-dependent manner, and
are therefore selective for channels in the spike
initiation zone. Voltage-gated Na þ channels are
complex proteins, whose function is regulated by
membrane potential through voltage-dependent conformational
changes occurring on a timescale from
less than a millisecond to several seconds (Pichon and
Ashcroft, 1985). At the spike initiation zone, neurons
operate near the threshold for action potential generation,
in the range of 70 to 50 mV, where important
conformational changes in Na þ channels occur
prior to opening. In contrast, axons have a resting
potential near 90 mV, where Na þ channels are predominantly
in the resting state. However, when axons
are depolarized to the range where Na þ channels
begin to undergo conformational changes, they become
sensitive to pyrazolines (Salgado, 1990). Likewise,
the extracellularly recorded compound action
potential from motor nerves of M. sexta abdominal
ganglia, which was likewise highly insensitive to
DCJW, was rendered sensitive by depolarization of
the nerves with a high K þ saline (Wing et al., 1998).
Figure 8 shows membrane currents evoked by test
pulses to 0 mV before and after equilibration with
10 mM RH-3421 at various holding potentials. For
each trace, after initial rapid downward and upward
transients, the current became inward and peaked