Sigrid Buchner

A Cysteine-String Protein is Expressed in Retina and Brain of Drosophila

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine‐regulate neurotransmitter release by phosphorylation‐dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock‐out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock‐out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified ‘model’ synapse of the larval nerve muscle preparation revealed no difference between wild‐type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild‐type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

SummaryUsing a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.

Deoxyglucose mapping of nervous activity induced inDrosophila brain by visual movement

SummaryLocal metabolic activity was mapped in the brain ofDrosophila by the radioactive deoxyglucose technique. The distribution of label in serial autoradiographs allows us to draw the following conclusions concerning neuronal processing of visual movement information in the brain ofDrosophila.1.The visual stimuli used (homogeneous flicker, moving gratings, reversing contrast gratings) cause only a small increase in metabolic activity in the first visual neuropil (lamina).2.In the second visual neuropil (medulla) at least four layers respond to visual movement and reversing contrast gratings by increased metabolic activity; homogeneous flicker is less effective.3.With the current autoradiographic resolution (2—3 μm) no directional selectivity can be detected in the medulla.4.In the lobula, the anterior neuromere of the third visual neuropil, movement-specific activity is observed in three layers, two of which are more strongly labelled by ipsilateral front-to-back than by back-to-front movement.5.In its posterior counterpart, the lobula plate, four movement-sensitive layers can be identified in which label accumulation specifically depends on the direction of the movement: Ipsilateral front-to-back movement labels a superficial anterior layer, back-to-front movement labels an inner anterior layer, upward movement labels an inner posterior layer and downward movement labels a superficial posterior layer.6.A considerable portion of the stimulus-enhanced labelling of medulla and lobula complex is restricted to those columns which connect to the stimulated ommatidia. This retinotopic distribution of label suggests the involvement of movement-sensitive small-field neurons.7.Certain axonal profiles connecting the lobula plate and the lateral posterior protocerebrum are labelled by ipsilateral front-to-back movement. Presumably different structures in the same region are labelled by ipsilateral downward movement. Conspicuously labelled foci and commissures in the central brain cannot yet be associated with a particular stimulus. The results are discussed in the light of present anatomical and physiological knowledge of the visual movement detection system of flies.

Cell-specific immuno-probes for the brain of normal and mutant Drosophila melanogaster

SummaryWe have screened antibodies for immunocytochemical staining in the optic lobes of the brain of Drosophila melanogaster. Seven polyclonal antisera and five monoclonal antibodies are described that selectively and reproducibly stain individual cells and/or produce characteristic staining patterns in the neuropile. Such antisera are useful for the cellular characterization of molecular and structural brain defects in visual mutants. In the wildtype visual system we can at present separately stain the following: the entire complement of columnar “ T 1” neurons; a small set of presumptive serotonergic neurons; some 3000 cells that contain and synthesize γ-amino butyric acid (GABA); and three groups of cells that bind antibodies to Ca2+-binding proteins. In addition, small groups of hitherto unknown tangential cells that send fine arborizations into specific strata of the medulla, and two patterns of characteristic layers in the visual neuropile have been identified by use of monoclonal antibodies generated following immunization of mice with homogenates of the brain of Drosophila melanogaster.

Histamine is a major mechanosensory neurotransmitter candidate in Drosophila melanogaster.

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.

Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior

The role of histamine as a fast neuro-transmitter of imaginai insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdcJK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdcp211, hdcp217, and hdcp218, variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.

The Wuerzburg hybridoma library against Drosophila brain.

Abstract: This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach “from antibody to gene”, several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the “cysteine string protein” gene (Csp, mAb ab49), the “synapse-associated protein of 47 kDa” gene (Sap47, mAbs nc46 and nb200), and the “Bruchpilot” gene (brp, mAb nc82). By a “candidate” approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of “epidermal growth factor receptor pathway substrate clone 15” (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila

In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.

Mapping stimulus-induced nervous activity in small brains by [3H]2-deoxy-D-glucose

SummaryNervous activity may be localized in anatomical sections of brain tissue by the autoradiographic deoxyglucose technique. The method provides sufficient structural preservation and spatial resolution for detailed functional investigation of complex but small-sized nervous systems when the original technique is modified as follows: (i) use of 3H instead of 14C as radioactive label, (ii) application of labeled deoxyglucose in concentrations close to physiological glucose levels rather than in trace amounts, (iii) stimulation for 4–9 h after deoxyglucose application instead of 20–45 min, (iv) subsequent preparation avoiding aqueous phases at all stages from fixation to autoradiography, and (v) plastic embedding of the tissue such that serial semithin sections of good structural preservation may be routinely cut. Brief aqueous fixation and dehydration at room temperature as has been described for vertebrates apparently cannot preserve stimulus-induced distribution of radioactive label in the brain of the fly Drosophila melanogaster. Aspects of the results that illustrate the potential and some limitations of the present technique are discussed.