Characterization of tyramine β-hydroxylase, an enzyme upregulated by stress in Periplaneta americana

Octopamine (OA) is an important neuroactive substance that modulates several physiological functions and behaviors of various invertebrate species. This biogenic monoamine, structurally related to noradrenaline, acts as a neurotransmitter, a neuromodulator, or a neurohormone in insects. The tyramine β-hydroxylase (TBH) catalyzes the last step in OA biosynthesis and thus plays a key role in the regulation of synthesis and secretion of OA in neurons. The aim of this study was to characterize TBH in the cockroach Periplaneta americana and to get a better understanding of its regulation under stress conditions in this insect. First of all, five full-length cDNAs encoding TBH isoforms were cloned from the nerve cord of the physiological model P. americana. PaTBH transcripts were found mainly expressed in nervous tissues and in octopaminergic dorsal unpaired median neurons. In addition, a new ELISA assay was developed so as to allow determination of both OA level and TBH activity in stressed cockroaches. Mechanical stressful stimulation led to a significant increase in TBH activity after 1 and 24 h, with a higher induction after 1 h than after 24 h. Thus, TBH could be considered as a promising biomarker of stress in insects rather than OA.

Nervous System ofPeriplaneta americanaCockroach as a Model in Toxinological Studies: A Short Historical and Actual View

Nervous system ofPeriplaneta americanacockroach is used in a wide range of pharmacological studies, including electrophysiological techniques. This paper presents its role as a preparation in the development of toxinological studies in the following electrophysiological methods: double-oil-gap technique on isolated giant axon, patch-clamp on DUM (dorsal unpaired median) neurons, microelectrode techniquein situconditions on axon in connective and DUM neurons in ganglion, and single-fiber oil-gap technique on last abdominal ganglion synapse. At the end the application of cockroach synaptosomal preparation is mentioned.

Cys-Loop Ligand-Gated Chloride Channels in Dorsal Unpaired Median Neurons of Locusta migratoria

In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABAA nor GABAC receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter.

Dorsal Unpaired Median Neurons of Locusta migratoria Express Ivermectin- and Fipronil-Sensitive Glutamate-Gated Chloride Channels

Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels (GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median (DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 μM fipronil or 100 μM picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 μM ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl α subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlClα subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluClα transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria.