Insect Control: Biological and Synthetic Agents - Index of

84 3: Neonicotinoid Insecticides
their potential to control target pest species. In contrast
antagonistic compounds of mammalian
nAChR were shown to be far less active as insecticides
(Nauen et al., 1999a). All neonicotinoid insecticides
act with nanomolar affinity against housefly
and other insect nAChRs, except thiamethoxam,
which exhibits a comparatively low affinity for the
[ 3 H]imidacloprid binding site. This low affinites
was attributed to its proneonicotinoid nature (see
Sections 3.3.5 and 3.3.5.1), as it was shown to be
activated to clothianidin in insects and plants
(Nauen et al., 2003).
The mode of action of neonicotinoid insecticides
has been described in several excellent reviews
which should serve as a primary source for further
informations (Kagabu, 1997a; Matsuda et al., 2001;
Nauen et al., 2001; Tomizawa and Casida, 2003).
These reviews describe our current knowledge of the
structure and function of insect nAChR, characterized
by receptor binding studies, phylogenetic
considerations regarding receptor homologies
between orthologs from different animal species,
and electrophysiological investigations, as well as
the description of a vast range of a structurally
diverse receptor ligands, of course with an emphasis
on neonicotinoid insecticides.
3.6. Interactions of Neonicotinoids
with the Nicotinic Acetylcholine
Receptor
3.6.1. Selectivity for Insect over
Vertebrate nAChRs
Electrophysiological measurements reported in
numerous studies have revealed that nAChRs are
widely expressed in the insect nervous system on
both postsynaptic and presynaptic nerve terminals,
on the cell bodies of interneurons, motor neurons,
and sensory neurons (Goodman and Spitzer, 1980;
Harrow and Sattelle, 1983; Sattelle et al., 1983;
Breer, 1988; Restifo and White, 1990). Schröder
and Flattum (1984) using extracellular electrophysiological
recordings were the first to identify that the
site of action of the nitromethylene nithiazine was
on the cholinergic synapse. A number of subsequent
electrophysiological and biochemical binding studies
revealed that the primary target of the neonicotinoids
were the nAChRs (Benson, 1989; Sattelle
et al., 1989; Bai et al., 1991; Leech et al., 1991;
Cheung et al., 1992; Tomizawa and Yamamoto,
1992, 1993; Zwart et al., 1992; Liu and Casida,
1993a; Tomizawa et al., 1996). Recent electrophysiological
studies indicate that imidacloprid acts as
an agonist on two distinct nAChR subtypes on
cultured cockroach dorsal unpaired motoneuron
(DUM) neurons (Buckingham et al., 1997), an
a-bungarotoxin (a-BGTx) sensitive nAChR with
‘‘mixed’’ nicotinic/muscarinic pharmacology and an
a-BGTx insensitive nAChR. Such electrophysiological
observations were supported by binding studies
with [ 3 H]imidacloprid in membrane preparations
from M. persicae. These ligand competition studies
revealed the presence of high and low-affinity nAChR
binding sites for imidacloprid in M. persicae (Lind
et al., 1998).
The identification of multiple putative nAChR
subunits by molecular cloning is consistent with a
substantial diversity of insect nAChRs (Gundelfinger,
1992). At present, at least five different subunits
have been cloned from Drosophila melanogaster
(Schulz et al., 1998), from the locust Locusta migratoria
(Hermsen et al., 1998), and from the aphid
M. persicae (Huang et al., 1999). Despite the considerable
number of subunits identified, only a few
functional receptors were obtained after expression of
different subunit combinations in Xenopus oocytes or
cell lines. Initial work suggested that some subunits
can form homooligomeric functional receptors when
expressed in Xenopus oocytes. This was shown for
La1 from the locust Schistocerca gregaria (Marshall
et al., 1990; Amar et al., 1995), and for the Mpa1and
Mpa2 fromM. persicae (Sgard et al., 1998). However,
the expression of these subunits was not very
effective and generated only small inward currents
(5–50 nA) following application of nicotine or
acetylcholine. Alternatively, all three Drosophila
a subunits (ALS, SAD, and Da2) can form functional
receptors in Xenopus oocytes when coexpressed with
a chicken neuronal b2 subunit (Bertrand et al., 1994;
Matsuda et al., 1998; Schulz et al., 1998), suggesting
that additional insect nAChR subunits remain to be
cloned. Radioligand binding studies using several
M. persicae a subunits coexpressed with a rat b2
subunit in the Drosophila S2 cell line also indicate
pharmacological diversity in M. persicae (Huang
et al., 1999). In these binding studies it was shown
that imidacloprid selective targets were formed by
Mpa2 andMpa3, but not Mpa1 subunits.These
examples indicate that our understanding of the
complexity of insect nAChR is still limited, and that
electrophysiology will play an essential role in determining
the significance of certain subunit combinations
in the mode of action of neonicotinoid and
other insecticidally active ligands.
3.6.2. Whole Cell Voltage Clamp of Native Neuron
Preparations
The use of isolated neurons from insect CNS
for electrophysiological studies is a suitable tool to