Genome-wide analysis of ionotropic receptor gene repertoire in Lepidoptera with an emphasis on its functions of Helicoverpa armigera.

The functions of the Ionotropic Receptor (IR) family have been well studied in Drosophila melanogaster, but only limited information is available in Lepidoptera. Here, we conducted a large-scale genome-wide analysis of the IR gene repertoire in 13 moths and 16 butterflies. Combining a homology-based approach and manual efforts, totally 996 IR candidates are identified including 31 pseudogenes and 825 full-length sequences, representing the most current comprehensive annotation in lepidopteran species. The phylogeny, expression and sequence characteristics classify Lepidoptera IRs into three sub-families: antennal IRs (A-IRs), divergent IRs (D-IRs) and Lepidoptera-specific IRs (LS-IRs), which is distinct from the case of Drosophila IRs. In comparison to LS-IRs and D-IRs, A-IRs members share a higher degree of protein identity and are distinguished into 16 orthologous groups in the phylogeny, showing conservation of gene structure. Analysis of selective forces on 27 orthologous groups reveals that these lepidopteran IRs have evolved under strong purifying selection (dN/dS≪1). Most notably, lineage-specific gene duplications that contribute primarily to gene number variations across Lepidoptera not only exist in D-IRs, but are present in the two other sub-families including members of IR41a, 76b, 87a, 100a and 100b. Expression profiling analysis reveals that over 80% (21/26) of Helicoverpa armigera A-IRs are expressed more highly in antennae of adults or larvae than other tissues, consistent with its proposed function in olfaction. However, some are also detected in taste organs like proboscises and legs. These results suggest that some A-IRs in H. armigera likely bear a dual function with their involvement in olfaction and gustation. Results from mating experiments show that two HarmIRs (IR1.2 and IR75d) expression is significantly up-regulated in antennae of mated female moths. However, no expression difference is observed between unmated female and male adults, suggesting an association with female host-searching behaviors. Our current study has greatly extended the IR gene repertoire resource in Lepidoptera, and more importantly, identifies potential IR candidates for olfactory, gustatory and oviposition behaviors in the cotton bollworm.

Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction.

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-ß, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-ß or its intracellular domain were generated. When Neto-ß is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-ß mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-ß rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-ß restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.

Conserved cellular distribution of the glutamate receptors GluA2/3, mGlu1a and mGlu2/3 in isolated cultures of rat cerebellum.

To what extent the intrinsic glutamatergic system of the cerebellum is able to keep normal features in the absence of mossy and climbing fibres, is at present not known. To answer this question, immunocytochemistry for light and high resolution electron microscopy was used to reveal the cellular and subcellular distribution of glutamate receptors in isolated cerebellar cultures. The localization of the ionotropic glutamate receptor subunits GluA2/3 and the metabotropic glutamate (mGlu) 1a and mGlu2/3 receptor subtypes was carried out in 0 to 9-day-old rat parasagittal slices developed in vitro for 20-40 days. The typical localization of GluA2/3, mGlu1a and mGlu2/3 observed in Purkinje cells, granule cells, Golgi cells and unipolar brush cells was maintained in the organotypic cultures. Furthermore, the subcellular distribution of mGlu1a showed the characteristic in vivo perisynaptic position in Purkinje cell dendritic spines receiving parallel fibre synapses. We conclude that the cellular and subcellular localization of the studied ionotropic and metabotropic glutamate receptors is not affected by the removal of the two extrinsic cerebellar glutamatergic inputs, the mossy and climbing fibres.

Functional expression of ionotropic glutamate receptors in the rabbit retinal ganglion cells.

It has been known that retinal ganglion cells (RGCs) with distinct morphologies have different physiological properties. It was hypothesized that different functions of RGCs may in part result from various expressions of N-methyl-d-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-isoxazole-4-propinoic acid (AMPA), and kainic acid (KA) receptors on their dendrites. In the present study, we aimed to characterize the functional expression of AMPA and NMDA receptors of morphologically identified RGCs in the wholemount rabbit retina. The agmatine (AGB) activation assay was used to reveal functional expression of ionotropic glutamate receptors after the RGCs were targeted by injecting Neurobiotin. To examine the excitability of these glutamate receptors in an agonist specific manner, the lower concentrations of AMPA (2 µM) and NMDA (100 µM) were chosen to examine G7 (ON-OFF direction selective ganglion cells) and G11 (alpha ganglion cells) types of RGCs. We found that less than 40% of G7 type RGCs had salient AGB activation when incubated with 2 µM AMPA or 100 µM NMDA. The G11 type RGCs also showed similar activation frequencies, except that all of the OFF subtype examined had no AGB permeation under the same AMPA concentration. These results suggest that RGCs with large somata (G7 and G11 types) may express various heterogeneous functional ionotropic glutamate receptors, thus in part rendering their functional diversity.

Glutamate receptors in laryngeal cancer cells.

AIM: Despite recent improvements in treatment strategies, the results of chemotherapy in patients with advanced squamous cell carcinoma of the larynx are not satisfactory. Thus, the development of new approaches which influence specific metabolic pathways are needed. In the last decade, evidence has emerged implicating a role for glutamate as a signal mediator in tumors. MATERIALS AND METHODS: The presence of glutamate receptor subunits in two laryngeal cancer cell lines (RK33 and RK45) was evaluated by means of end-point PCR, real-time PCR, and immunocytochemistry. RESULTS: Glutamate receptor subunits are differentially expressed in laryngeal cancer cell lines. In addition, we show that selected ionotropic glutamate receptor antagonists and metabotropic glutamate receptor 5 antagonist inhibit proliferation of laryngeal cancer cells. Glutamate antagonists also affected activity of extracellular signal-regulated kinases 1/2 in tumor cells. CONCLUSION: Signaling through glutamate receptors may influence growth of laryngeal cancer cells and may constitute an adjunctive therapeutic target.

Glutamate regulation of GnRH neuron excitability.

The gonadotropin-releasing hormone (GnRH) neuronal network is the master controller of the reproductive axis. It is widely accepted that the amino acid transmitters GABA and glutamate play important roles in controlling GnRH neuron excitability. However, remarkably few studies have examined the functional role of direct glutamate regulation of GnRH neurons. Dual-labeling investigations have shown that GnRH neurons express receptor subunits required for AMPA, NMDA and kainate signaling in a heterogeneous manner. Electrophysiological and calcium imaging studies have confirmed this heterogeneity and shown that while the majority of adult GnRH neurons express AMPA/kainate receptors, only small sub-populations have functional NMDA or metabotropic glutamate receptors. Accumulating evidence suggests that one important role of direct glutamate signaling at GnRH neurons is for their activation at the time of puberty. Whereas in vivo studies have indicated the importance of NMDA signaling within the whole of the GnRH neuronal network, including afferent neurons and glia, investigations at the level of the GnRH neuron suggest that peripubertal changes in AMPA receptor expression may be dominant in the mouse. The sources of glutamatergic inputs to the GnRH neurons are only just beginning to be examined and include the anteroventral periventricular nucleus as well as the possibility that GnRH neurons may use glutamate as a neurotransmitter in recurrent collateral innervation. It is expected that a full understanding of the glutamatergic regulation of GnRH neurons will provide significant insight into the mechanisms underlying their control of reproductive function.