Characterization of a metabotropic glutamate receptor in the honeybee (Apis mellifera): implications for memory formation.

G-protein-coupled metabotropic glutamate receptors (GPC mGluRs) are important constituents of glutamatergic synapses where they contribute to synaptic plasticity and development. Here we characterised a member of this family in the honeybee. We show that the honeybee genome encodes a genuine mGluR (AmGluRA) that is expressed at low to medium levels in both pupal and adult brains. Analysis of honeybee protein sequence places it within the type 3 GPCR family, which includes mGlu receptors, GABA-B receptors, calcium-sensing receptors, and pheromone receptors. Phylogenetic comparisons combined with pharmacological evaluation in HEK 293 cells transiently expressing AmGluRA show that the honeybee protein belongs to the group II mGluRs. With respect to learning and memory AmGluRA appears to be required for memory formation. Both agonists and antagonists selective against the group II mGluRs impair long-term (24 h) associative olfactory memory formation when applied 1 h before training, but have no effect when injected post-training or pre-testing. Our results strengthen the notion that glutamate is a key neurotransmitter in memory processes in the honeybee.

Distribution of metabotropic glutamate receptor DmGlu-A in Drosophila melanogaster central nervous system.

L-glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogaster CNS were investigated, with focus on the study of DmGluRA, a G-protein-coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult Drosophila CNS, using a polyclonal antibody raised against the C-terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA-containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate-like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the Drosophila CNS, are discussed.

Conservation of the ligand recognition site of metabotropic glutamate receptors during evolution.

Mammalian metabotropic glutamate receptors (mGluRs) are classified into 3 groups based on their sequence similarity and ligand recognition selectivity. Recently, we identified a Drosophila mGluR (DmGlu(A)R) which is about equidistant, phylogenetically, from the 3 mGluR groups. However, both the G-protein coupling selectivity and the pharmacological profile of DmGlu(A)R, as analysed with mutated G-proteins and a few compounds, look similar to those of mammalian group-II mGluRs. In the present study we carefully examined the pharmacological profile of DmGlu(A)R, and compared it to those of the rat mGlu(1a), mGlu(2) and mGlu(4a) receptors, representative of group-I, II and III respectively. The pharmacological profile of DmGlu(A)R was found to be similar to that of mGlu(2)R, and only very small differences could be identified at the level of their pharmacophore models. These data strongly suggest that the binding sites of these two receptors are similar. To further document this idea, a 3D model of the mGlu(2) binding domain was constructed based on the low sequence similarity with periplasmic amino acid binding proteins, and was used to identify the residues that possibly constitute the ligand recognition pocket. Interestingly, this putative binding pocket was found to be very well conserved between DmGlu(A)R and the mammalian group-II receptors. These data indicate that there has been a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs.

DmGluRA, a Drosophila metabotropic glutamate receptor, activates G-protein inwardly rectifying potassium channels in Xenopus oocytes.

Xenopus oocytes were coinjected with cDNAs encoding the Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) and two mammalian G-protein inwardly rectifying potassium channel subunits (GIRK1 and GIRK2). Glutamate and two vertebrate group II mGluR agonists (order of potency: LY 354740 > glutamate > DCG IV) elicited inwardly rectifying potassium currents. These inward currents were sensitive to cesium and barium. They were also blocked by two group II specific antagonists MCCG and APICA (IC50s 97.5 and 200 microM, respectively) and not affected by a group I antagonist (AIDA). Finally, the A-protomer of PTX reduced the glutamate-induced GIRK currents. This study is the first characterization of an invertebrate mGluR-mediated GIRK currents via a PTX-sensitive G protein.

The G protein-coupling profile of metabotropic glutamate receptors, as determined with exogenous G proteins, is independent of their ligand recognition domain.

Metabotropic glutamate (mGlu), Ca2+-sensing, gamma-aminobutyric acidB, and a large number of pheromone receptors constitute a peculiar family of G protein-coupled receptors. They possess a large extracellular domain that has been proposed to constitute their ligand binding domain. The aim of the current study was to examine whether this large ligand binding domain had any influence on the G protein-coupling selectivity of the receptor, and vice versa. We chose mGlu receptors, which are classified into three groups according to their sequence homology and pharmacology, as representatives of this receptor family. To define a G protein-coupling profile for these receptors, we used a set of exogenous phospholipase C-activating G proteins in the same way that synthetic ligands are used to define agonist and antagonist pharmacological profiles. This set includes Galpha15, Galpha16, Galphaq, and chimeric Galphaq proteins with the last few amino acids of either Galphai2 (Galphaqi), Galphao (Galphaqo), or Galphaz (Galphaqz). Cotransfection of mGlu receptors with these G proteins and examination of their coupling to phospholipase C revealed that group I, II, and III receptors have distinct G protein-coupling profiles. By swapping the extracellular domains of the most distantly related mGlu receptors (the rat group I mGlu1a and the Drosophila melanogaster group II DmGluA receptors), we show that the extracellular domain determines the agonist pharmacological profile and that this domain does not modify the G protein-coupling profile determined by the seven-transmembrane-domain region of mGlu receptors.

Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS.

The excitatory neurotransmitter glutamate plays important roles in the mammalian brain, ranging from synaptic plasticity to memory. To mediate these functions, glutamate activates two types of receptors: ligand-gated channels and metabotropic receptors coupled to G-proteins. Both families of glutamate receptors share no sequence homology and possess original structural features compared with other ligand-gated channels and G-protein-coupled receptors, respectively. Glutamate-gated receptor-channel subunits have already been characterized in invertebrates. Here we report the cloning and functional characterization of an invertebrate metabotropic glutamate receptor (DmGluRA) isolated from Drosophila melanogaster. This receptor displays 45 and 43% amino acid sequence identity with its mammalian homologs mGluR3 and mGluR2, respectively. Moreover, its pharmacology and transduction mechanisms are surprisingly similar to those of mGluR2 and mGluR3. DmGluRA is expressed in the CNS of the late embryo. These results indicate that the original structural features of both glutamate receptor types are conserved from insects to mammals and suggest that the functions of these receptors have been highly conserved during evolution.

atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system.

In the Drosophila peripheral nervous system, proneural genes of the achaete-scute complex (ASC) are required for formation of the precursors of external sense organs but not of chordotonal organs. We report the isolation of a gene, atonal (ato), with evidence that it is a proneural gene for the formation of chordotonal organs. This gene is expressed in the proneural clusters and sense organ precursors that give rise to the embryonic and adult chordotonal, but not external sense, organs. Chordotonal organs are eliminated in embryos carrying chromosomal deficiencies that remove ato. Like the ASC products, ato protein contains a basic-helix-loop-helix region and heterodimerizes with daughterless protein to bind to E boxes. Moreover, ectopic expression of ato promotes the formation of extra sense organs. Despite similar proneural properties, we find that ectopic expression of the ASC genes promotes external sense organ formation exclusively, whereas ato promotes chordotonal organ formation preferentially. Thus, proneural genes are major determinants of neuronal identity.

The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. VII. Characterization of the region around the snail and cactus loci.

The genetic interval 35C to 36A on chromosome arm 2L of Drosophila melanogaster has been saturated for mutations with visible or lethal phenotypes. 38 loci have been characterized, including several maternal-effect lethals (vasa, Bic-C, chiffon, cactus and cornichon) and several early embryonic lethals, including snail and fizzy. About 130 deletions have been used to order these loci. Complex interactions between mutant alleles have been uncovered in the immediate genetic environs of the snail gene, as has further evidence for an interaction between this region and that including the nearby genes no-ocelli and elbow.

An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily.

The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

The segment polarity gene costal-2 in Drosophila. II. The origin of imaginal pattern duplications.

Imaginal pattern duplications caused by hypomorphic expression of the segment polarity gene costal-2 are described. These affect the anteroposterior coordinate of the imaginal disc. A very small part of the pattern is deleted and a large number of additional pattern elements arise in a progressive order, anterior-most first followed by more and more posterior structures. Mosaic analyses show that the duplications arise nonautonomously in the larval stages but that the costal-2 gene is not required after early embryogenesis. Arguments that the duplications are the result of cell interactions and intercalary growth that themselves arise from an abnormal polarity of the embryonic segment are presented.

The segment polarity gene costal-2 in Drosophila. I. The organization of both primary and secondary embryonic fields may be affected.

A series of loss of function alleles at the costal-2 locus is described. Embryos mutant for lethal alleles that are derived from a mutant female germ line display polarity defects on the larval segments. A posterior part of the segmental denticle belt is missing and in its place is a mirror-image duplication of the anterior part including the segment boundary. Maternally rescued embryos are lethal but have normal morphology. Hypomorphic alleles escape to adults that display pattern duplications on the wings and halteres. Dominant gain of function alleles at the Costal-1 locus are also described and data are presented that argue that these are neomorphic and act in trans to impair functioning of costal-2. Some wild-type isoalleles of costal-2 are particularly sensitive to interference from Costal-1 mutations and different combinations of these alleles with Costal-1 can lead to embryos in which the primary embryonic field is disrupted (bicaudal phenotype) and adults with pattern duplications on the anterior compartment of most body segments.

Growth stimulation of the immature chick oviduct by androgens: the vagina as a new target tissue.

Testosterone when injected alone stimulates growth of the vagina but is inactive upon the other segments of the oviduct of the immature chicken. This action of testosterone can already be detected in embryos: it is expressed by the beginning of differentiation of the vaginal mesenchyme cells into smooth muscle cells. In the treated immature chicken, stimulation of growth is considerable and is specifically caused by androgens (testosterone and 5-alpha-dihydrotestosterone); the vaginal mesenchyme differentiates into two smooth muscular layers and vaginal epithelium cells differentiate into ciliated cells and goblets cells. [3H]testosterone binding has been found in the vagina of the immature chicken (data not shown). The characteristics of testosterone binding to cytoplasmic components of the chick vagina are consistent with its identity as a testosterone receptor.

Mutations and Chromosomal Rearrangements Affecting the Expression of Snail, a Gene Involved in Embryonic Patterning in DROSOPHILA MELANOGASTER.

Mutants at the snail locus are zygotically acting embryonic lethals that affect dorsoventral patterning. A comparison of seven mutant alleles shows considerable variation in expressivity and a graded effect along the dorsoventral axis: more extreme alleles result in the abnormal development of the dorsally derived ectoderm as well as the ventrally derived mesoderm, whereas weaker alleles affect only development of the mesoderm. Animals transheterozygous for different mutant alleles occasionally survive to adulthood; they frequently have missing halteres and more rarely are hemithorax. The mutant phenotype of snail is shown here to be enhanced zygotically by haploidy of two nearby regions on the second chromosome: the elbow to no-ocelli region and the interval defined by l(2)br36 and l(2)br37. It is concluded that the products of all of these genes function together in the process of specification of pattern in the embryo.