Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila.

The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein-coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT(1B) and 5-HT(7)), a dopamine receptor (DopR), the metabotropic GABA(B) receptor (GABA(B)R), the metabotropic glutamate receptor (DmGluR(A)) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABA(B)R, DmGluR(A,) and sNPFR1, whereas only GABA(B)R and DmGluR(A) were localized to the PB. Finally, strong expression of DopR and DmGluR(A) was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression.

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2),

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Differential distribution of isoforms of Leucophaea tachykinin-related peptides (LemTRPs) in endocrine cells and neuronal processes of the cockroach midgut.

Five isoforms of tachykinin-related peptides (TRPs), designated LemTRP-1-5, have been identified in the midgut of the cockroach Leucophaea maderae. These peptides have a conserved C-terminus hexapeptide (GFX1GX2Ramide; X1 and X2 are variable residues) and variable N-termini. Here, we address the question of whether these five isoforms are all colocalized in the two types of cells in the cockroach midgut, the endocrine cells and the neuronal processes. We also investigate whether the N-terminally extended isoforms LemTRP-2 and -3, which contain putative endoproteolytic cleavage sites, are expressed in intact form or are cleaved in the midgut cells. To this end, we used two approaches. (1) Extracts from portions of the midgut containing each of the cell types were subjected to reverse-phase high performance liquid chromatography (HPLC) and the fractions monitored in a radioimmunoassay (RIA) with an antiserum to the conserved C-terminus of insect TRPs. (2) Antisera were raised to the variable N-termini of the extended LemTRP-2 and -3 and used for immunocytochemistry. The HPLC-RIA and immunocytochemical findings indicate that LemTRP-1 and 4-5 are present in the neuronal processes and in endocrine cells of the midgut proper and of the gastric cecae. The two extended forms LemTRP-2 and -3 display a differential distribution: LemTRP-2 was found in endocrine cells of midgut and gastric cecae, but not in neuronal processes, whereas LemTRP-3 was seen in neuronal processes and endocrine cells of the midgut proper, but not in the gastric cecae. LemTRP-3 and -4 have not been identified in the brain, suggesting further cell- and tissue-specific expression of LemTRPs. The mechanisms behind the cell-specific expression of the LemTRPs are not yet understood, but the demonstration of differential distribution of the peptide isoforms provide a first indication that the isoforms may have different actions.

Characterization of actions of Leucophaea tachykinin-related peptides (LemTRPs) and proctolin on cockroach hindgut contractions.

The nine Leucophaea Tachykinin-Related Peptides (LemTRP 1-9) isolated from the midgut and brain of the cockroach, Leucophaea maderae, all induced increases in spontaneous contractions of the L. maderae hindgut. Synthetic LemTRP 1 and 3-9, were equally potent in inducing contractions of the hindgut. More than seven of the nine C-terminal residues of the closely related locust peptide locustatachykinin I (LomTK I) are required for full activity of the peptide on the L. maderae hindgut. Proctolin, a well characterized myostimulatory neuropeptide, was shown to be more potent than LemTRPs. LemTRP 1 and proctolin did not have synergistic actions in potentiating the amplitude and tonus of contractions of the L. maderae hindgut. Several differences could be seen in actions of LemTRP 1 and proctolin. In contrast to proctolin, LemTRP 1 could not override the inhibitory action of 10(-9) M of the myoinhibitory peptide leucomyosuppressin. Spantide I, an antagonist of the mammalian tachykinin receptors, at a concentration of 5 microM, blocked the response to LemTRP 1, but not to proctolin. The competitive proctolin receptor antagonist [alpha-methyl-L-tyrosine2]-proctolin blocked the action of both proctolin and LemTRP 1 when applied at 1 microM, whereas cycloproctolin had no antagonist action on either peptide. Verapamil, a blocker of voltage gated Ca2+-channels, and the less specific Ca2+-channel blocker Mn2+, abolished the action of LemTRP 1, but not of proctolin. The results obtained indicate that LemTRPs act on receptors distinct from those of proctolin. Double label immunocytochemistry revealed that all LomTK-like immunoreactive fibers impinge on the proctolinergic fibers in the hindgut. This finding and the inhibitory actions of Ca2+-channel blockers on TRP responses and of the proctolin receptor antagonist on both peptides, may suggest that the LemTRP receptors are not on the hindgut muscle fibers but on the terminals of the proctolinergic neurons. Thus, LemTRPs may induce release of proctolin on the hindgut. An alternative is that LemTRPs act by mechanisms clearly distinct from those of proctolin.

Multiple members of the leucokinin neuropeptide family are present in cerebral and abdominal neurohemal organs in the cockroach Leucophaea maderae.

Two neurohemal organs of the cockroach Leucophaea maderae, the corpora cardiaca and the lateral heart nerve are known to contain leucokinin immunoreactive material. We examined the corpora cardiaca and the lateral heart nerve to establish whether these neurohemal organs store all 8 known leucokinin isoforms or if the leucokinins have a differential distribution. Extracts of corpora cardiaca and abdominal hearts with attached lateral heart nerve were separated on reversed phase high performance liquid chromatography (rpHPLC), then tested for leucokinin immunoreactivity by a radioimmunoassay (RIA) able to detect all 8 leucokinin isoforms. Extracts from brain and optic lobes were also separated and assayed in the RIA. Synthetic leucokinin 1-8 were subjected to rpHPLC and their different retention times established by RIA for reference. Leucokinin immunoreactive material originating from the corpora cardiaca and lateral heart nerves eluted in fractions corresponding to those of the synthetic leucokinin 1-8. In this study we have thus demonstrated that probably all 8 leucokinin isoforms are stored in the corpora cardiaca and the lateral heart nerve. These observations suggest that all 8 leucokinins are likely to be released as neurohormones into the circulation.