Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed

Z-projections of confocal stacks of whole-mount adult <i>Drosophila</i> brains (frontal view) stained with ten different monoclonal antibodies of the Würzburg hybridoma library.

<p>Note in (H) that the bright spot at the left rim of the projection is due to a staining artefact from outside the brain. Scale bar 100 µm.</p

Staining details for mAb nb169.

<p>(A-D) The horizontal cryo-sections stained with mAb nb169 and DAB development show the cell bodies in the lateral protocerebrum at the dorso-ventral level of the fan-shaped and ellipsoid bodies (A), the giant neurons in the caudal cellular cortex with their main neurite (in an adjacent section) at the level of the noduli (B), and the cell bodies innervating the accessory medulla, a horizontal layer of the medulla, and a fiber extending to and branching with varicosities in the lamina cortex (C, D). (E-G) Horizontal cryo-sections double-stained with nb169 (red) and anti-serotonin antiserum (green) demonstrate that serotonergic neurons and cells that contain the nb169 antigen are distinct but that their neurites in the lamina cortex are closely apposed. (H-J) GFP (green) expressed under the control of the Janelia Gal4 line R33G11 co-localizes with the nb169 antigen (red) in frontal confocal optical section (H, I1-3) and in horizontal cryo-section (J1-3). (K-M) In the larval nervous system the nb169 antigen (red) is localized in 10 cells (K, L) of which at least 8 also express GFP (green) under the control of R33G11-Gal4 (M, enlargement of K). Scale bars in A-F, H, K-M: 50 µm; in G, I, J: 10 µm.</p

Z-projections of confocal stacks of whole-mount brains double-stained with mAb nb168 and anti-PDH antiserum (A1-3) or mAb nb169 and anti-GFP in a fly expressing GFP driven by Janelia Gal4 line R49C04 (B1-3).

<p>The perfect co-localization in A3 indicates that nb168 could recognize <i>Drosophila</i> PDF or its precursor protein, whereas co-localization in B3 is restricted to the two very large cells stained by nb169 in the posterior protocerebrum. Scale bar 100 µm.</p

Examples for the co-localization of staining by mAbs ab47 (A-C) and nc24 (D-F) and GFP driven by selected Janelia Gal4 lines.

<p>Due to the variability of cell body positions in different individuals it is only suggestive that the Gal4 expression patterns overlap. Scale bar 100 µm.</p