Superior Protocerebrum Research Articles

Multiple Antigenic Peptides Designed to Structurally Related Drosophila Peptides

Nichols, R., J. McCormick and I. Lim. Multiple antigenic peptides designed to structurally related Drosophila peptides.Peptides 18(1) 41–45, 1997.—We have isolated TDVDHVFLRFamide (DMS), FDDYGHMRFamide (DSK), and DPKQDFMRFamide from Drosophila melanogaster. These peptides, structurally related by a common C-terminus -XRFamide, where X = L or M, are encoded by three different genes. To determine cellular expression, we have generated antisera to multiple antigenic peptides and performed double-label immunofluorescence using antisera raised in the same species host animal. Our results indicate that DMS and DSK immunoreactive materials have unique, non-overlapping expression patterns, while DMS and DPKQDFMRFamide immunoreactive materials colocalize in two superior protocerebrum neurons, and DSK and DPKQDFMRFamide immunoreactive materials colocalize in one superior protocerebrum neuron, one subesophageal ganglion neuron, and three thoracic ganglia neurons.

Immunocytochemical mapping of serotonin and neuropeptides in the accessory medulla of the locust, Schistocerca gregaria

Accumulating evidence suggests that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla of the insect brain are involved in circadian pacemaking functions. We have used immunocytochemical techniques to investigate the neurochemical organization of the accessory medulla in the locust Schistocerca gregaria. Local neurons with arborizations largely restricted to the accessory medulla are immunoreactive with antisera against serotonin, Manduca sexta allatotropin, and Diploptera punctata allatostatin 7. Projection neurons with arborizations in the accessory medulla and fibers to the lamina and/or several areas in the midbrain including the posterior optic tubercles, the inferior and the superior protocerebrum show Phe-Met-Arg-Phe (FMRF)amide-, gastrin/cholecystokinin-, crustacean cardioactive peptide-, and substance P immunoreactivities. A unique neuron with tangential ramifications in the medulla and lamina and varicose terminals in the accessory medulla contains a peptide related to locustatachykinin I/II. Double-label experiments show colocalization of pigment-dispersing hormone-immunoreactivity with substances related to gastrin/cholecystokinin, FMRFamide, substance P, or crustacean cardioactive peptide in certain projection neurons of the accessory medulla. The results suggest that neuropeptides and biogenic amines play major neuroactive roles in the accessory medulla of the locust. The abundance and extensive colocalization of neuropeptides in the locust accessory medulla is discussed with respect to the possible involvement of this brain area in circadian pacemaking functions.

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.

Cellular expression of the Drosophila melanogaster FMRFamide neuropeptide gene product DPKQDFMRFamide. Evidence for differential processing of the FMRFamide polypeptide precursor.

DPKQDFMRFamide is one of five different FMRFamide-containing peptides encoded in the Drosophila FMRFamide gene. To study the cellular expression of DPKQDFMRFamide, we have generated antisera to DPKQD, the N-terminal sequence of the peptide, to avoid crossreactivity with other -FMRFamide-containing peptides. The antisera were purified and the specificity characterized. DPKQDFMRFamide immunoreactive material is first observed in the embryonic central nervous system (CNS) in one cell of the subesophageal ganglion and one cell in each of the three thoracic ganglia. This pattern of expression is observed in larval, pupal, and adult neural tissue, albeit with increased signal intensity. In larva, pupa, and adult, additional cells in the superior protocerebrum, a thoracic ganglion, and an abdominal ganglion express DPKQDFMRFamide immunoreactive material. Immunoreactivity is observed in a cell in the lateral protocerebrum of pupa and adult and cells in the optic lobe of adult. No immunoreactive material was observed in gut tissue. DPKQDFMRFamide antisera stain a subset of cells previously identified by in situ hybridization and immunocytochemistry to express the FMRFamide transcript and polypeptide precursor. These data suggest that the Drosophila FMRFamide polypeptide precursor undergoes differential processing to produce DPKQDFMRFamide immunoreactive material in a limited number of cells expressing the FMRFamide precursor.

Pigment‐dispersing hormone‐like peptide in the nervous system of the flies Phormia and Drosophila: Immunocytochemistry and partial characterization

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab (Rao et al., '85) is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects were limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons was performed for the entire nervous systems of Phormia and the fruitfly Drosophila with a beta-PDH antiserum. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neurophils of the brain of both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies were found (only six were found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. We also found that some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hind-gut.

Galanin immunoreactivity in the blowfly nervous system: Localization and chromatographic analysis

In this study chromatographic, immunochemical, and immunocytochemical methods provide evidence of a galanin-like peptide(s) in an invertebrate, the blowfly Phormia terraenovae. The major portion of the galanin-like immunoreactivity (GAL-LI) in fly heads was extractable in acetic acid but not in boiling water, which suggests that the peptide(s) may be highly basic in nature. GAL-LI was present both in the head and body portion of the blowfly in roughly the same amounts. Initial gel filtration data, using a G-50 Sephadex column and a weak phosphate-buffer (pH 6.5) as eluent, suggested that a fly GAL-LI peptide(s) from fly heads, eluting as an apparent single peak, was smaller than porcine GAL(1-29) and GAL(1-15). However, concomitant analysis using a G-25 Sephadex column and acetic acid (0.2 M) as eluent, spread the immunoreactive material over a great portion of the chromatogram, although the main portion of the material eluted in the same size range as porcine GAL(1-29). Taken together, the gel filtration data thus suggest that fly GAL-LI peptide(s) may be highly basic but presumably similar in size to vertebrate GAL(1-29). However, the hydrophobic properties of the fly GAL-LI peptide(s) differ from that of porcine GAL as demonstrated by the presence of several immunoreactive components eluting both early as well as late in the chromatogram when using reverse-phase high performance liquid chromatography (HPLC); early peaks may represent highly basic and/or possibly smaller GAL-immunoreactive peptide(s), whereas later peaks may represent less basic and possibly elongated forms. Immunocytochemistry indicated that GAL-LI was present in the nervous system of the blowfly. About 160 GAL-immunoreactive neurons were found in the brain and subesophageal ganglion, 26 in the fused thoracic ganglion and 30 in the fused abdominal ganglion. In the brain, GAL-immunoreactive fibers supply specific subdivisions of the central body, optic lobe, superior protocerebrum, and tritocerebrum as well as neuropil in the subesophageal ganglia. In the thoracico-abdominal ganglia, GAL-immunoreactive neuron processes are found inside synaptic neuropil as well as in the neural sheath of the ganglia and several of the dorsal nerve roots. Many of the GAL-immunoreactive neurons react also with an antiserum against porcine galanin message associated peptide, a peptide present in the preprogalanin protein. Immunocytochemical double-labeling indicated that some GAL-immunoreactive neurons also reacted with antisera against the molluscan peptides FMRFamide and SCPB, whereas no evidence could be found for colabeling with antisera against tyrosine hydroxylase, substance P and physalaemin.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin immunoreactivity in the optic lobes of the sphinx moth Manduca sexta and colocalization with FMRFamide and SCPB immunoreactivity.

In the optic lobes (OLs) of the sphinx moth Manduca sexta, 300-350 neurons per hemisphere are immunoreactive with an antiserotonin antiserum. Two groups of weakly serotonin-immunoreactive cells (OL1) appear to be amacrine cells of the medulla, whereas more intensely immunoreactive cells (OL2) are probably centrifugal neurons that innervate the lobula, medulla, and lamina, as well as the superior protocerebrum. At least one other OL2 cell is a local optic-lobe interneuron with arborizations in the dorsal medulla and lobula. The serotonin-immunoreactive cells are also immunoreactive with an antiserum against Drosophila melanogaster DOPA decarboxylase. All OL2 cells, but not the OL1 cells, are furthermore immunoreactive with an anti-FMRFamide antiserum and an anti-SCPB antiserum. This suggests that neuropeptides related or identical to FMRFamide and SCPB are localized and may serve as cotransmitters with serotonin in OL2 optic-lobe interneurons.

Physiology and morphology of projection neurons in the antennal lobe of the male moth Manduca sexta.

1. We have used intracellular recording and staining, followed by reconstruction from serial sections, to characterize the responses and structure of projection neurons (PNs) that link the antennal lobe (AL) to other regions of the brain of the male sphinx moth Manduca sexta. 2. Dendritic arborizations of the AL PNs were usually restricted either to ordinary glomeruli or to the male-specific macroglomerular complex (MGC) within the AL neuropil. Dendritic fields in the MGC appeared to belong to distinct partitions within the MGC. PNs innervating the ordinary glomeruli had arborizations in a single glomerulus (uniglomerular) or in more than one ordinary glomerulus of one AL (multiglomerular) or in one case, in single glomeruli in both ALs (bilateral-uniglomerular). One PN innervated the MGC and many or all ordinary glomeruli of the AL. 3. PNs with dendritic arborizations in the ordinary glomeruli and PNs associated with the MGC typically projected both to the calyces of the ipsilateral mushroom body and to the lateral protocerebrum, but some differences in the patterns of termination in those regions have been noted for the two classes of PNs. One PN conspicuously lacked branches in the calyces but did project to the lateral protocerebrum. The PN innervating the MGC and many ordinary glomeruli projected to the calyces of the ipsilateral mushroom body and the superior protocerebrum. 4. Crude sex-pheromone extracts excited all neurons with arborizations in the MGC, although some were inhibited by other odors. One P(MGC) was excited by crude sex-pheromone extract and by a mimic of one component of the pheromone blend but was inhibited by another component of the blend. 5. PNs with dendritic arborizations in ordinary glomeruli were excited or inhibited by certain non-pheromonal odors. Some of these PNs also responded to mechanosensory stimulation of the antennae. 6. The PN with dendritic arborizations in the MGC and many ordinary glomeruli was excited by crude sex-pheromone extracts and non-pheromonal odors and also responded to mechanosensory stimulation of the antenna.

Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system.

Mutations that eliminate acetylcholinesterase (AChE) activity were used to study the effects of disrupted acetylcholine metabolism on the form and function of the central nervous system in Drosophila melanogaster. Mutants in the Ace gene, which have no AChE activity, usually die in early development, but the postembryonic effects of this lesion can be studied in genetic mosaics, or with conditional mutants. Adult mosaics, which expressed Ace mutations in part of their CNS, exhibited morphological defects in any ganglionic neuropile whose cells were mutant. The defects included reduction in ganglionic volume, a condensed appearance, and for a very large clone, degeneration. Examination of many such mosaics indicated that small clones restricted to one side of the CNS were not usually lethal. However, mosaics with large clones, with clones on either side of the posterior slope of the protocerebrum, or with clones encompassing symmetrical structures on both sides of the CNS rarely survived to adulthood. Mosaics with AChE-null tissue on either side of the optic lobes or the posterior-inferior protocerebrum had marked deficits in optomotor behavior, although they were outwardly normal in their movement and posture. Mosaics with Ace mutant tissue in the first-order optic lobe, the lamina, lacked a synaptic component of the electroretinogram, the "off" transient. Tests of courtship behavior revealed that AChE mosaics with mutant clones in the superior protocerebrum were often capable of demonstrating male courtship. However, their behavior was quantitatively and perhaps qualitatively deficient. In order to study critical periods for the effects of mutant AChE, temperature-sensitive mutations of the Ace gene were isolated. Flies bearing certain of these new mutations produced AchE activity that was thermolabile in vivo and in vitro. The critical period during which the mutants were most susceptible to conditional lethality was late in embryogenesis.