Insect Control: Biological and Synthetic Agents - Index of

6: The Spinosyns: Chemistry, Biochemistry, Mode of Action, and Resistance 223
hypothesis was confirmed by showing that the maximal
force exerted by the metathoracic femur in response
to tetanic crural nerve stimulation faded more
rapidly as poisoning progressed, and eventually disappeared
concomitant with paralysis. The inhibitory
neuromuscular synapses, which use g-aminobutyric
acid (GABA) as the neurotransmitter and are blocked
after prostration with the GABA gated chloride channel
blocker dieldrin, were shown to function normally
in insects prostrated by spinosyn A (Salgado,
1998). In in vitro experiments on larval housefly
muscle, spinosyn A at concentrations up to 1 mM
had no effect on neuromuscular transmission, but
at 5 mM and higher, the cell was slightly depolarized
and the excitatory postsynaptic potential (EPSP)
was enhanced. These concentrations are too high
for these effects to be relevant during poisoning.
6.4.2.3. Spinosyns excite central neurons by
inducing an inward, depolarizing, current Because
spinosyns potently stimulate CNS activity (see Section
6.4.2.1), effects on neurons of the cockroach 6th
abdominal ganglion were investigated in situ with
intracellular microelectrodes. Dorsal unpaired median
(DUM) neurons were chosen because they are
spontaneously active and are the best-characterized
neurons in the insect CNS, and known to contain
many different receptors important for insecticide
action, including nicotinic acetylcholine receptors
(nAChRs) (Buckingham et al., 1997), GABA
(Le Corronc et al., 2002) and inhibitory glutamate
(Raymond et al., 2000) receptors, and a variety of
sodium, calcium, and potassium channels (Grolleau
and Lapied, 2000). As reported previously (Dubreil
et al., 1994), DUM neurons were spontaneously
activate and had random spontaneous unitary
inhibitory postsynaptic potentials (IPSPs). Furthermore,
stimulation of the connective evoked an IPSP
in the DUM cell (Pitman and Kerkut, 1970; Dubreil
et al., 1994) (Figure 9b). At 200 nM, but not at
50 nM, spinosyn A depolarized DUM cells in situ
(10 and 13 mV in two cells; compare the resting
levels of the traces in Figure 9b and c) and increased
the rate of spontaneous IPSPs considerably, but
did not block the spontaneous or evoked IPSPs,
even at concentrations as high as 1 mM (n ¼ 3)
(Figure 9c). It also increased the spontaneous firing
rate, as can be seen by the two action potentials in
this trace (the evoked IPSP abolished an action potential
that was just starting). Picrotoxinin at 10 mM
abolished the spontaneous and evoked IPSPs (Figure
9d), leading to further depolarization and excitation
(note the increased action potential frequency).
Thus, while spinosyn A did not appear to affect the
inhibitory postsynaptic receptors in DUM neurons,
or the ability to generate action potentials, it did
have clear effects on the cells. While the increased
spontaneous IPSP rates reflect changes occurring in
presynaptic cells, the depolarization and increased
firing rate appear to be due to a direct action of
spinosyn A on the DUM cells themselves. It can
be seen from the steady depolarization between
action potentials (Figure 9d) that this firing is
due to a continuous depolarizing stimulus flowing
into the cell. This suggests that a spinosyn-induced
inward current was responsible for the depolarization
and increased firing rate. The depolarization
was reversed by 10 mM methyllycaconitine (MLA),
a selective nicotinic antagonist (Figure 10), indicating
that it is mediated by nicotinic receptors. MLA
also blocked the IPSP, consistent with the proposed
multisynaptic pathway for this response, involving a
nicotinic synapse (Dubreil et al., 1994) (Figure 10).
6.4.2.4. Activation of nicotinic receptors by
spinosyns generates a depolarizing inward current
The intracellular recordings (Figures 9 and 10) indicated
that spinosyn-A-induced an inward current
in central neurons, probably mediated by nAChRs.
This current was then directly investigated with
voltage clamp experiments on isolated central neurons.
The cells studied were a mixed population of
neuronal somata, with diameters between 35 and
80 mm, isolated from the thoracic ganglia of the
cockroach P. americana and studied with the
single-electrode voltage clamp method (SEVC) as
detailed by Salgado and Saar (2004). In ten cells
tested, spinosyn A did indeed induce an inward current
at concentrations between 50 nM and 1 mM.
Furthermore, this current was completely blocked
by 100 nM a-bungarotoxin (a-BGTX), a highly specific
antagonist of nAChRs. Because the interaction
of both a-BGTX and spinosyn A with the receptors
was relatively slow, it was difficult to measure
the dose response for block quantitatively. In the
example in Figure 11, 3nMa-BGTX blocked the
spinosyn-A-induced current by approximately 75%
before washout was begun, while 10 nM a-BGTX
blocked the spinosyn-A-induced current by 85%,
and 1000 nM blocked it completely. This sensitivity
to a-BGTX is comparable to that of the nondesensitizing
nAChR, which is blocked 78 3% by
3 nM and 93 1% by 10 nM a-BGTX (Salgado
and Saar, 2004).
6.4.3. Nicotinic Receptors as the
Spinosyn Target Site
6.4.3.1. Two subtypes of nicotinic receptors
Two subtypes of a-BGTX-sensitive nicotinic receptors
have been identified in insect neurons (van den