Cys-loop ligand-gated chloride channels in Dorsal Unpaired Median neurons of Locusta migratoria

Abstract

The economic and agricultural importance of locusts can easily be underestimated, given the widespread use of insecticides. However, areas in Africa and in Asia are frequently affected by locust swarms, causing devastating consequences to the local food security. Unfortunately, contrary to intuition, the wide use of broad-spectrum insecticides can increase the probability, duration and stability of locust outbreaks. Locusts and insects in general are highly adaptable, developing resistance to a wide range of insecticides and thus raising the need for the development of improved and novel antiparasitic drugs both more species-selective and more potent. Established target sites for insecticides are the members of the cys-loop ligand-gated chloride channels. They mediate important aspects of behaviour through fast inhibitory neurotransmission. In insects, the cys-loop ligand-gated chloride channels are composed of the glutamate-gated chloride channel (GluCI), the y-amino butyric-gated chloride channels (GABACI) and histamine-gated chloride channel (HisCI). They are excellent candidates for the development and design of new insecticidal compounds, and it is for this reason that our work has been concentrated on these receptors. In addition, the functional implication of cys-loop ligand-gated chloride channels in dorsal unpaired median (DUM) neurons deserved our attention. Efferent DUM neurons exert a variety of myo- and neuromodulatory effects and are involved in numerous behavioural aspects such as flying, jumping and walking. It is therefore plausible to assume that the cys-loop ligand-gated chloride channels could have modulatory effect on the excitability of DUM neurons, altering various aspects of the locust physiology and behaviour. Moreover, this work may provide new insight into the diversity of the cys-loop LGIC superfamily in different organisms and provide a better understanding of these channels in general The objectives

The main objective is to obtain a more detailed insight in the distribution, structure and functional properties of these cys-loop ligand-gated chloride channels on the cell surface of locust metathoracic DUM neurons. This was done using electrophysiological methods and molecular biological methods The functional expression of the glutamate-gated chloride channel, the GABA-gated chloride channel and the histamine-gate chloride channels will be studied and pharmacologically characterized, using the patch clamp techniques. The intention is to establish the DUM neuron as a viable in vitro model for the study of members of these cys-loop ligand­ gated chloride channel, in order to screen for compounds (i.e. toxins, drugs, insecticides,...) targeting these particular channels.

The identification of putative cys-loop ligand-gated chloride channel subunits will be attempted, using a degenerate RT-PCR and RACE strategy. The sequencing results will allow the comparison of these channels from different insect species and enhance our understanding of the cys-loop ligand-gated chloride channel functional genomics. Because the glutamate-gated chloride channels are exclusively present in invertebrates and thus form excellent targets for the development of more selective insecticides, our research will be mainly focused on this channel.

The identified cys-loop ligand-gated chloride channel subunits will be cloned and their functional expression in a heterologous system, such as the Xenopus laevis oocytes will be studied. The aim is to obtain a reliable in vitro model for the study of kinetics, biophysics and pharmacology of a single receptor type, without the interference of other receptor channels. These in vitro models can be exploited to design new pharmacological tools used in research or new drugs for specific diseases, and for screening of toxins acting on yet unknown targets.